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Symposium: Fibril Assembly and Structure: 935-Symp Fibril Formation by Amyloid-beta and by Low-complexity Sequences: In- Progress and Challenges sights from Solid State NMR Robert Tycko. 932-Symp Laboratory of Chemical Physics, NIDDK, National Institutes of Health, High Resolution Structure Determination of Amyloid Fibrils Bethesda, MD, USA. Robert G. Griffin. I will describe results from two projects in which my lab uses solid state NMR Chemistry/Francis Bitter Magnet Laboratory, Massachusetts Institute of methods (plus other techniques) to characterize the molecular structures of am- Technology, Cambridge, MA, USA. yloid fibrils. First, I will review our work on fibrils formed by amyloid-b pep- Many peptides and proteins form amyloid fibrils whose detailed molecular tides. This is a long-running project in which we have shown that amyloid-b structure is of considerable functional and/or pathological importance. fibrils are typically polymorphic at the molecular level, identified certain mo- In this presentation we review the macroscopic structural properties of lecular structural features that distinguish polymorphs from one another, devel- fibrils and outline approaches to determining the microscopic structure oped full molecular structural models for fibrils formed in vitro and fibrils of these systems to high resolution using magic angle spinning (MAS) derived from human brain tissue, and investigated correlations between varia- NMR techniques. In particular, we discuss a series of 2D and 3D heteronu- tions in amyloid-b fibril structure and variations in Alzheimer’s disease charac- clear and homonuclear dipole recoupling experiments involving spectral teristics. Second, I will discuss studies of fibrils formed by the low-complexity assignments and distance and torsion angle measurements aimed at accom- domain of the FUS protein (FUS-LC). This is a collaborative project with the plishing this goal. Key to obtaining high resolution is the ability to measure lab of Steven McKnight at the University of Texas Southwestern Medical Cen- a sufficient number of structural constraints per residue. We discuss the ter, in which we seek to understand the phenomenon of aggregation and fibril structures of three different systems determined using these approaches: formation by low-complexity sequences that are rich in glycine and serine res- (1) a fibril performed by an 11 amino acid peptide derived from transthyr- idues but nearly devoid of hydrophobic residues. From solid state NMR data, etin using a set of 70 torsion angle and distance constraints; (2) fibrils we find (surprisingly) that a specific 57-residue segment constitutes the struc- formed by A-beta (1-42), the toxic species in Alzheimers, using a set of turally ordered, immobilized core of fibrils formed by the 214-residue FUS- >500 distance constraints; and (3) a preliminary structure of fibrils forned LC domain, although the core-forming segment has nearly the same amino by beta-2-microglobulin, the 99 amino acid protein associated with dialysis acid composition as the full FUS-LC sequence. Experiments in the McKnight related amylosis. The spectra also provide information on the arrangement lab indicate a close connection between the FUS-LC fibril core structure and of the monomers in the strands that form sheets, and the sheets that ulti- intermolecular interactions in the phase-separated droplet state of FUS-LC mately form the fibrils. Contrary to conventional wisdom, the spectral solutions. data indicate that the molecules in the fibril are microscopically well ordered. Symposium: Biophysics of Lipid-modified 933-Symp GTPases Aggregation of the TAU Protein: Insights from Atomistic and Mesoscale Simulations 936-Symp Joan-Emma Shea. Ras-Membrane Interactions and their Modulation by Effector Proteins UCSB, Santa Barbara, CA, USA. Roland Winter. In this talk, I will discuss the self-assembly of peptides derived from the Tau TU Dortmund University, Dortmund, Germany. protein. Using atomistic simulations, I will present mechanistic insights into In a combined chemical-biological and biophysical approach we explored the the early stages of fibrillization, and the effect of external agents, such as os- free energy contributions to the membrane partitioning of Ras proteins, such as molytes, in either promoting or disfavoring aggregation. I will also present N-Ras and K-Ras4B, and compared the theoretical predictions with experi- field-theoretic simulation of the assembly of Tau peptides into coacervate mental data on the membrane insertion of Ras proteins of various anchor sys- structures. tems into rationally designed model and natural membrane systems. Various factors fostering or reducing the membrane partitioning properties are dis- 934-Symp cussed, including hydrophobic effects, lipid chain mismatch, electrostatic inter- b High Resolution Fibril Structure of Amyloid- (1-42) by Cryoelectron actions, membrane-mediated protein-protein interactions, and terms that Microscopy account for line tension effects between coexisting lipid domains and lipid sort- Dieter Willbold1,2. ing, and change the lateral organization of the lipid bilayer system. For hetero- 1 2 € ICS-6, Reserch Centre Juelich, Julich, Germany, Institut fur Physikalische geneous membranes, localization and sequestration at domain boundaries as € € € Biologie, Heinrich-Heine-Universitat Dusseldorf, Dusseldorf, Germany. well as formation of protein clusters and collective lateral organization via b We present the structure of an A (1-42) fibril composed of two intertwined an effective lipid sorting mechanism provide complementary ways of inducing protofilaments determined by cryoelectron microscopy (cryo-EM) to 4.0- membrane nanodomains that could potentially operate as effective signaling ˚ Angstrom resolution, complemented by solid-state nuclear magnetic reso- platforms. PDEd and the Ca-binding protein calmodulin (CaM) are known to nance experiments. The backbone of all 42 residues and nearly all side function as potential binding partners for farnesylated Ras proteins, leading chains are well resolved in the EM density map, including the entire N ter- to a modulation of the spatiotemporal organization of K-Ras4B, and hence b minus, which is part of the cross- structure resulting in an overall ‘‘LS’’- have also been studied. We showed that PDEd is not able to extract K- shaped topology of individual subunits. The dimer interface protects the hy- Ras4B from model membranes. Instead, an effective delivery of PDEd-solubi- drophobic C termini from the solvent. The characteristic staggering of the lized K-Ras4B to the plasma membrane was proposed. Compared to PDEd, nonplanar subunits results in markedly different fibril ends, termed CaM exhibits additional interaction sites to the G-domain of K-Ras4B and ‘‘groove’’ and ‘‘ridge,’’ leading to different binding pathways on both fibril was shown not to be required for the transport of K-Ras4B to the plasma mem- ends, which has implications for fibril growth. Each monomer that binds to a brane. The influence of CaM on the interaction of GDP- and GTP-loaded K- certain fibril end sees the same interface, in contrast to a true dimeric inter- Ras4B with anionic model raft membranes has been investigated by a combi- face (in the case of a C2 symmetry), where added monomers would alternat- nation of different spectroscopic and imaging techniques. The results suggest ingly see either two identical binding sites or a curb preformed by the that binding of the acidic CaM to the polybasic stretch of K-Ras4B reverses b preceding subunit. The strands are staggered with relation to one another its charge, leading to repulsion of the complex from anionic membranes. in a zipper-like manner. At both fibril ends, the binding site for the addition of subunit i contains contributions of subunits i-1, i-2, i-3, i-4, and i-5, or 937-Symp iþ1, iþ2, iþ3, iþ4, and iþ5, respectively. Therefore, five Ab(1-42) subunits Allosteric Regulation of Small GTPases on Membranes are required to provide the full interface for monomer addition. For a frag- Jacqueline Cherfils. ment of six subunits, the capping subunits would have the same full contact CNRS and ENS Paris-Saclay, Cachan, France. interface as those in an extended fibril. We define this structural element of Lipidated small GTPases and their regulators assemble at the surface of mem- six subunits as the minimal fibril unit. This minimal fibril unit may also be branes to propagate actions in the cell, but an integrated understanding of their the minimal seed size for nucleation as suggested by results from Ab aggre- interactions with the lipid bilayer is still lacking. We addressed this question for gation studies monitored by small angle neutron scattering and analytical ul- Arf GTPases, which orchestrate a variety of regulatory functions in lipid and tracentrifugation. membrane trafficking, and the guanine nucleotide exchange factors (GEF)

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Brag2, which controls integrin endocytosis and cell adhesion and is impaired in analyse DNA origami structural offset with angstrom-level precision (2 A˚ ) from cancer and developmental diseases. Brag2 activates Arf GTPases by stimu- single-molecule studies. By combining the approach with multiplexed Exchange- lating the replacement of GDP by GTP, leading to the active, membrane- PAINT imaging, we further demonstrated an optical nano-display with 55nm attached form of Arf which recruits downstream effectors. We showed previ- pixel size and three distinct colours, and with <1 nm cross-channel registration ously that, although Brag2 is highly active in solution, negatively charged accuracy. Combined with photo-activated crosslinker, we further demonstrated membranes stimulate its efficiency by up to 3 orders of magnitude. We selective labelling on single molecule targets with custom patterns and sub- analyzed the determinants of this spectacular contribution of membranes by diffraction (30 nm) resolution. These methods open up possibilities for direct combining crystallography, coarse-grained molecular dynamics and reconstitu- and quantitative optical observation and perturbation of individual biomolecular tion of Arf GTPases and Brag2 in artificial membranes. We found that the Arf/ features in crowded environments. Brag complex forms multiple interactions with charged lipids to organize a pre- cise orientation of the complex with respect to the membrane that determines its 941-Plat activation kinetics. We discovered that this determinant can be harnessed by a Measuring 3D Forces during Capillary Network Remodelling new type of cell-active, allosteric inhibitors, that impair the orientation of the Dobryna Zalvidea. complex by binding at the GEF/lipid interface. ICTD, IBEC, Barcelona, Spain. Patterning of capillary networks is essential for the proper systemic distribution 938-Symp of cells, nutrients, and oxygen in developing as well as adult healthy and un- Lipid Binding Specificity of the KRAS Membrane Anchor healthy tissues. Mechanical models describing the relation between blood John F. Hancock. flow and angiogenesis, vessel pruning, etc. had been introduced in biology at Integrative Biology and Pharmacology, University of Texas Health Science earlier times with great success [1]. Along with these studies, in vitro models Center, Houston, TX, USA. of sprouting angiogenesis have shown mechanical descriptions of the interac- The KRAS membrane anchor exhibits exquisite specificity for phosphatidylser- tion of endothelial cells/extracellular matrix (ECM) [2, 3]. Nevertheless, the ine (PtdSer) over other anionic phospholipids by virtue of defined structural dy- lack of appropriate methods for in vivo and in situ force measurement had namics of the C-terminal polybasic domain and farnesyl group. This specificity hampered the study of the forces that capillary wall components apply on the extends to PtdSer with specific combinations of lipid side chains. PtdSer bind- ECM during formation/pruning of capillaries. Here, we show that, by devel- ing is therefore essential for stable KRAS plasma membrane (PM) binding and oping a new method for imaging and measuring forces in vivo and in situ, the spatial organization of KRAS into nanoclusters that together are essential we can describe mechanically the remodelling of capillary networks. We intro- for signal transmission. In consequence depleting the PM of PtdSer would be duced a multiphoton imaging system that surpasses the simultaneous channel expected to abrogate KRAS function. Two inhibitors of SM metabolism acquisition problems that the present commercial systems have, but retains have recently been shown to reduce PM PtdSer levels and abrogate KRAS the ability of deep imaging with subcellular resolution. To quantify the forces signaling. We therefore used RNAis against a further 18 C.elegans SM biosyn- we introduced a reference system in the chorioallantoic membrane (CAM) of thetic enzyme orthologs in a worm model of activated KRAS signaling to more the chicken embryo by adding a tunable biocompatible fluorescent nanofibres broadly assess potential therapeutic possibilities. We found that knock down of matrix (electrospun polylactic acid fibres in a predesigned arrangement) that many of these enzymes strongly or moderately suppress the multi-vulva pheno- was minimally invasive and able to deform following the deformation of the type induced by an oncogenic mutant of LET60, a KRAS ortholog. Concor- CAM stroma. We numerically modelled the system as fibres on an elastic ma- dantly pharmacological agents that alter the function of these enzymes in terial (CAM) and, by characterising mechanically the system, we could retrieve mammalian cells also cause mislocalization of KRAS from the PM, regardless forces on the nN/nm scale. We also identified the formation of new functional of whether the net effect of the compound is to increase or decrease cellular connections and found an oscillatory behaviour of the forces that we interpret SM. The common mechanism of action was depletion of PM PtdSer content. with a simple biomechanical model of damping oscillations, where the period- These compounds also potently inhibit the proliferation of oncogenic KRAS- icity depends on the matrix structure. The damping term drives the system to a transformed pancreatic cancer cells. Thus normal SM metabolism is required quiescent state. to maintain PM PtdSer levels and hence KRAS function. Similar results were obtained by shRNA or CRIPSR knock down of components of the ER- 942-Plat PM PtdSer exchange machinery, which also abrogated KRAS PM binding Variable-Angle Total Internal Reflection Fluorescence Microscopy: To- and nanoclustering, again highlighting the key dependence of KRAS function wards a New Way to Probe Single Cell Adhesion Strength on PM PtdSer content. Dalia El Arawi, Cyrille Vezy, Rodolphe Jaffiol. 939-Symp LNIO, Institut Charles Delaunay, CNRS, France, Troyes, France. Role of the G-Domain in RAS Isoform Dependent Turmorigenisis Total Internal Reflection Fluorescence (TIRF) microscopy is a widespread Sharon L. Campbell. technique used to study cellular process occurring near the contact region University of North Carolina, Chapel Hill, NC, USA. with the glass substrate. In this framework, we have recently proposed a new strategy to investigate adhesion strength at the single cell level (Biophysical Platform: Optical Microscopy and Journal, 111, 2016, p1316-1327 and AIMS Biophysics, 4, 2017, p438-450). This approach involves variable-angle Total Internal Reflection Fluorescence Superresolution Imaging: Methods II Microscopy (vaTIRFM). vaTIRFM allows to map in real time, the cell topog- raphy (i.e. the membrane-substrate separation distance) with a nanometric axial 940-Plat resolution, and the effective refractive index of the cell cortex. Topography and Optical Imaging and Labelling of Individual Biomolecules in Dense refractive index are two key parameters allowing to observe close contacts Clusters related to focal adhesion process, and non-specific membrane/substrate interac- Mingjie Dai1,2, Ninning Liu2, Ralf Jungmann3, Peng Yin1,2. tions for example to highlight the crucial role of glycocalyx. vaTIRFM can also 1Department of Systems Biology, Harvard Medical School, Boston, MA, 2 3 be used to get the profile of the potential energy related to membrane/substrate USA, Wyss Institute, Harvard University, Boston, MA, USA, Max Planck interactions, from which it is possible to extract an energy of adhesion. We have Institute of Biochemistry and LMU, Munich, Germany. recently emphasized the benefits of vaTIRFM for different cancer cells in adhe- Recent advances in fluorescence super-resolution microscopy have allowed sub- sion on various substrates, or by blocking the adhesion and/or the migration cellular features and synthetic nanostructures down to 15 nm in size to be signaling pathways using integrin antagonists. imaged. However, direct optical observation of individual molecular targets (5 nm) in a densely packed biomolecular cluster remains a challenge. Here, 943-Plat we show that such discrete molecular imaging is possible using DNA-PAINT Biomolecular Interaction Studies using SEEC Technology (points accumulation for imaging in nanoscale topography) - a super-resolution Marie-Pierre Valignat. fluorescence microscopy technique that exploits programmable transient oligo- Laboratory of Adhesion and Inflammation, Inserm, Marseille Cedex 09, nucleotide hybridisation - on synthetic DNA nanostructures. We examined the ef- France. fects of high photon count, high blinking statistics, and appropriate blinking duty Surface Ellipsometric Enhanced Contrast (SEEC) technique coupled with op- cycle on imaging quality, and developed a software-based drift correction method tical microscopy, henceforth called SEEC technology, allows imaging with that achieves <1 nm residual drift (r.m.s.) over hours. This allowed us to image a microscopic planar resolution and measurement with orthoplanar nanoscopic densely packed triangular lattice pattern with 5 nm point-to-point distance, and resolution in the range of 1-500 nm at solid/liquid interfaces. The enhanced

BPJ 8590_8601 Monday, February 19, 2018 187a optical contrast offered by SEEC technology is attributed to specially designed Physiology, Perelman School of Medicine, University of Pennsylvania, substrate coatings in which multiple reflections and interferences yield dark Philadelphia, PA, USA. background and very high contrast sensitivity to thin surface deposits. Super-resolution microscopy, and in particular single molecule localization mi- Recently, we showed that Wet-SEEC technology (in liquid condition) is well croscopy, offers a unique opportunity for quantifying protein copy numbers suited for studies involving live imaging and characterization of bio-nano- with nanoscale resolution [1,2]. films such as Bacterial slime. In our present work, we move a step further to The development of methods able to access a precise molecular counting of demonstrate that SEEC technology coupled with automated microfluidic sys- protein copy numbers is essential, clearing the way to address several biolog- tem permits live, label-free and noninvasive study of molecular interactions ical questions using super-resolution techniques such as stochastic optical at nano, meso and microscale. In this present study, we demonstrate dynamic reconstruction microscopy (STORM). A suitable calibration method represents measurements of molecular monolayer such as Protein-protein interactions, the best way to address the challenges of molecular counting using super- polyelectrolyte multilayer construction and biomolecular interactions with resolution [3,4] since small organic fluorophores used in conjunction with living cells. Our study highlights the potential of SEEC technology to simulta- immunofluorescence need to be characterized for quantitative imaging. neously investigate different scales ranging from Nanoscale to microscopic Here we propose to use DNA origami as a standard and we show that this cali- level. bration method, besides quantifying the average protein copy number in a cell, allows determining the abundance of various oligomeric states [5]. In partic- 944-Plat ular, we show that quantitative approaches allow quantitative studies of the Strategies for High-Content Light Sheet Microscopy stoichiometry of membrane proteins, nucleoporins (NUP107) [6] and molecu- 1 2 1 Aaron Au , Christopher M.J. McFaul , Christopher M. Yip . lar motors [7]. 1Chemical Engineering, University of Toronto, Toronto, ON, Canada, 2 References: Institute of Biomaterials and Biomedical Engineering, University of 1.Durisic, N., et al., Single-molecule evaluation of fluorescent protein photoac- Toronto, Toronto, ON, Canada. tivation efficiency using an in vivo nanotemplate. Nat Methods, (2014) Light sheet microscopy has gained popularity in recent years due to its higher 2. Ulbrich, M.H. et al., Subunit counting in membrane-bound proteins. Nat throughput imaging speed at reduced sample phototoxicity compared to con- Methods, (2007) ventional confocal microscopy. However in practice, the arduous process of 3.Schmied, J.J., et al., DNA origami-based standards for quantitative fluores- sample mounting often hinders the high-throughput imaging pipeline to be effi- cence microscopy. Nat Protoc, (2014) cacious. In addition, a typical mounting method is restricted to small selections 4. Jungmann R. et al. Quantitative super resolution imaging with qPAINT Nat of biological system due to a large variation between sample shapes, mounting methods (2016) medium required, and the geometry of the light sheet platform. Inspired by the 5. Cella Zanacchi F. et al., DNA Origami: Versatile super-resolution calibration concept from Strnad et.al (Nat. Meth, 13(2), 139-142, 2016), we developed a standard for quantifying protein copy-number, Nat Methods, (2017) light sheet microscope that allows an adaptable open top sample chamber to 6. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by Super- be fitted on a confocal microscope base. This allows us to take advantage of Resolution Microscopy and Particle Averaging. Science, (2013) the existing confocal laser lines and galvo scanning mirrors to create the scan- 7.Derr, N.D., et al., Tug-of-war in motor protein ensembles revealed with a pro- ning light sheet. The additional optics can easily be removed to and the micro- grammable DNA origami scaffold. Science, (2012) scope can be converted back to a confocal microscope. The inverted light sheet configuration provides generous amount of space for large sample holders that 947-Plat can be adopted for a wide range of biological sample sizes and geometries. Us- High throughput Automated Multi Target Super-resolution Imaging ing rapid-prototyping approaches, the mounting space can be iteratively rede- Farzin Farzam1, Sheng Liu2, Cedric Cleyrat3, Keith A. Lidke1. signed and customized for a plethora of biological systems of interest. This has 1Physics and Astronomy, Univeristy of New Mexico, Albuquerque, NM, allowed us to prototype sample mounts to perform high-throughput imaging. USA, 2Biomedical Engineering, Purdue University, West Lafayette, IN, We demonstrate the versatility of our in house light sheet system with various USA, 3Pathology, Univeristy of New Mexico, Albuquerque, NM, USA. biological samples in their optimized sample mounts. Single molecule super-resolution of multiple targets is challenging due to limited availability of dyes with sufficient photophysical properties (i.e. good 945-Plat blinking behavior). An additional difficulty that naturally arises when using SideSPIM - A Flexible Multipurpose Platform for Light Sheet Microscopy multiple dyes of different emission wavelength is the need for different optical 1 1 2 2 Per Niklas Hedde , Leonel Malacrida , Siavash Ahrar , Albert Siryaporn , paths and thus multiple cameras (or different regions on one camera) causing 1 Enrico Gratton . differential aberrations that become important at the <100 nanometer scale. 1Biomedical Engineering, University of California Irvine, Irvine, CA, USA, 2 To avoid these problems, it is possible to image multiple targets using a single Department of Physics and Astronomy, University of California Irvine, optical path with a single dye and optimal buffer by using a sequential imaging Irvine, CA, USA. approach [1]. To facilitate higher throughput using sequential imaging we have We devised a flexible multipurpose platform for light sheet microscopy. Based automated several steps in the data collection and image processing pipeline on a regular inverted microscope, a custom designed chamber is used to allow and this improves reliability and reduces the user time required at the micro- selective plane illumination from the side (sideSPIM). This eliminates the need scope. After initial selection of cells and image planes, the microscope m of dipping into the sample container, hence small sample volumes ( 100 l) will collect super-resolution data unsupervised, which in practice is often over- can be realized and objective lenses can be interchanged quickly. We demon- night. For sequential imaging, the sample is removed from the microscope strate that this flexible multipurpose platform is suitable for a large variety of and re-labeled at the bench. The sample is re-mounted and after a quick, applications involving samples of substantially different sizes and geometries. user-verified re-alignment to saved bright-field image data, the microscope For example, we captured the microcirculation of erythrocytes in a zebrafish again collects data on every cell without supervision. We show the reliability embryo at 5 stacks/s (40 planes), studied the growth of bacteria biofilms under of the microscope by imaging alpha-tubulin and then beta-tubulin in dozens flow using a fluidic device, and probed the diffusion of EGFP tagged proteins in of cells. We have applied the microscope in the study of clathrin-dependent mammalian cells by acquiring fast time series (200 fps) and subjecting them to internalization of FcεRI (the high-affinity IgE receptor) by analyzing the fluorescence fluctuation based image mean square displacement (iMSD) and super-resolution images of clathrin and IgE in 10s of cells at many time points two-dimensional pair cross-correlation function (2D-pCF) analysis. We further after cross-linking IgE with a synthetic antigen. evaluated the potential of the sideSPIM system for high throughput screening [1] Christopher C. Valley, Sheng Liu, Diane S. Lidke, Keith A. Lidke, of droplets containing cells for the production of antigens. Finally, we demon- ‘‘Sequential Superresolution Imaging of Multiple Targets Using a Single Fluo- strate how spectral information can be obtained with this camera-based system rophore’’, PLoS ONE, 2015. 10(4) using the phasor approach. Work supported in part by NIH grants P50 GM076516 and P41 GM103540. Platform: Membrane Pumps, Transporters, and 946-Plat Exchangers Quantitative Super-Resolution Microscopy using DNA Origami Francesca Cella Zanacchi1,2, Raffaella Magrassi1,3, Alberto Diaspro1, 948-Plat Carlo Manzo2,4, Nathan D. Derr5, Melike Lakadamyali2,6. Is the Ton Transport System a Rotary Electromotor? 1Nanophysics Department, Italian Institute of Technology, Genoa, Italy, Wenchang Zhou, Jose Faraldo-Go´mez. 2ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 3National Research National Heart Lung and Blood Institute, Bethesda, MD, USA. Council (CNR), Biophysics Institute (IBF), Genoa, Italy, 4Universitat de Vic, To acquire scarcely available nutrients, gram-negative bacteria use Vic, Spain, 5Smith College, Northampton, MD, USA, 6Department of active-transport systems that span both their outer and inner membranes. The

BPJ 8590_8601 188a Monday, February 19, 2018 outer-membrane component is a passive high-affinity receptor that captures the the WT protein, despite similar levels of surface expression. While the substrate from the environment; the inner-membrane component energizes disease-causing mutation preserves the overall gating and stoichiometry of further uptake into the periplasm. In many pathogens, this component is the the transporter, the mutant has a notably faster activation rate and slower deac- Ton complex, which consists of three membrane proteins: TonB, ExbB, and tivation rate than the WT. By quantitatively measuring the lysosomal pH in fi- ExbD. In a breakthrough, subcomplexes of ExbB and ExbD, and of ExbB broblasts, we found that the patient’s lysosomes are hyperacidic, potentially alone, were recently crystallized and their atomic structures determined. The explaining much of the disease phenotype. Taken together, these results are structures show ExbB forms pentamers with a channel-like architecture, and consistent with the hypothesis that the mutation leads to a gain of function, that ExbD inserts into this pore as a single transmembrane helix. Here, we with increased acidification due to increased currents through hClC-7. discuss the potential mechanistic implications of these structures, in the context of established concepts in membrane transport and bioenergetics. Specifically, 951-Plat we hypothesize that ExbB-ExbD features a rotary alternating-access mecha- How GLUT1 Transporter Accompanies Glucose along Transport: A nism coupled to the transmembrane movement of protons. In this model, Detailed Atomistic View of the Mechanism ExbD undergoes reversible right- and left-handed screw motions within the Matthieu Ng Fuk Chong, Lylia Challali, Sonia Abbar, Catherine Etchebest. ExbB pore, as a result of which Asp25 in ExbD becomes alternatively exposed DSIMB, UMR-S1134, Inserm-University Paris Diderot, Paris, France. to the space on either side of the membrane. Owing to the ExbB symmetry, Glucose is an essential source of energy for the mammalian cells. Its transport there are five equivalent outward-facing and inward-facing states, each of is achieved by facilitative diffusion through the glucose transporter GLUT1 ex- which can protonate and deprotonate. A final and crucial feature of this model pressed into erythrocytes and endothelial cells of the blood brain barrier. A defi- is that the preferred handedness of the screw motion of ExbD at any given time ciency of GLUT1 or mutations inactivating this protein causes a drastic is determined by the protonation state of Asp25. Under a proton-motive-force, reduction in the amount of glucose passing through the brain, which causes cen- this dynamical asymmetry dictates that ExbD will rotate preferentially in one tral nervous system dysfunction. On the opposite, as a main energy source, direction, thereby converting electrochemical energy into mechanical energy. GLUT1 is over-expressed in tumors, which makes it an important therapeutic Through TonB, this energy would then be mechanically transduced to the target in hepatocellular carcinoma. GLUT1 belongs to the major facilitator su- outer-membrane receptors, to power substrate translocation into the periplasm. perfamily (MFS). MFS transporters allow the flow of solutes through the mem- The mechanism of ExbBD hypothesized here would be unprecedented among brane through a cycle of conformational changes. The different states adopted known active transporters, and provides a specific conceptual framework for by the protein during transport were deduced from biochemical studies and future experimental studies. partially detailed by solving X-ray structures of MFS members in multiple con- formations In the present work, starting from the X-ray structure of GluT1 949-Plat solved in an inward-open conformation, we have first completed the X-ray Depicting the Translocation Process of the Protein Antibiotic Colicin E9 structure (Deng 2014) for which an intracellular region was missing. Then, through OmpF based on a large set of molecular dynamics simulations (covering more than Patrice Rassam, Nicholas Housden, Colin Kleanthous. 5 microseconds) combined with docking calculations and efficient analysis Biochemistry, University of Oxford, Oxford, United Kingdom. tools, we were able to characterize the dynamics of the protein, the energetics Bacteriocins are potent narrow spectrum protein antibiotics, with specificity of the glucose pathway and the key structural elements of the transport. We dictated by their intricate protein-protein interactions within the Gram- show that, besides side chain motions that accompany glucose translocation, negative cell envelope. Translocation of group A bacteriocins into their target transmembrane helices motions are also required. We have identified a strong cell is facilitated through interaction with components of the Tol system. Inter- coupling between intracellular and extracellular domains that contributes to action with the Tol system is mediated through an intrinsically disordered facilitate the transport. We have also examined impacts of mutations on glucose N-terminus of the bacteriocin using non-specific porins to thread through to transport. Overall, for the first time, this study depicts the conformational the periplasm where Tol-binding epitopes can acquire their targets. Colicin changes that are required to allow glucose passive diffusion from the extracel- E9 is a nuclease targeted to the cytoplasm, including a TolB-binding epitope lular to the intracellular compartment. housed within the unstructured N-terminus. That sequence is flanked by two OmpF binding epitopes, which expedite passage of the TolB-binding epitope 952-Plat across the outer membrane. Dissecting Steps in ATP-Driven Protein Translocation through the SecY The relatively low affinity of these OmpF-binding epitopes for OmpF and sensi- Translocon on Single Molecule Level tivity of the complex to ionic strength and pH has limited the use of structural Tomas Fessl1,2. biology in dissecting these interactions. Through the use of chemical modifica- 1Faculty of Science, University of South Bohemia, University of South tion with TMEA to form trimeric OBS constructs, a high avidity interaction with Bohemia, Ceske Budejovice, Czech Republic, 2Astbury Centre for Structural the OmpF trimer has been engineered leading to 100-fold decrease in Kd. Molecular Biology, University of Leeds, Leeds, United Kingdom. Labelling of tridentate OBS constructs (for both OBS1 and OBS2 from the N Most outer and inner membrane proteins in bacteria are transported and in- and C-termini of the unstructured colicin N-terminus respectively) with TMR serted by the ubiquitous Sec machinery (SecYEG complex) in the plasma mem- has allowed the binding of each epitope to be investigated through fluorescence brane. Similar function is provided by Sec61 complex in the ER of eukaryotes. microscopy on both intact and membrane permeabilised E. coli cells. These ex- In prokaryotes, many proteins are transported post-translationally through the periments reveal that whilst OBS2 can bind OmpF from its extracellular, OBS1 SecYEG at the expense of ATP hydrolysis by the peripheral membrane protein can only bind OmpF from its periplasmic face. This supports a hook model for SecA and in concert with the trans-membrane proton motive force. Using a the interaction of the colicin with OmpF in which OBS1 passes through the combination of biochemical, computational and single molecule techniques pore of one OmpF subunit, with OBS2 then displacing OBS1 and OBS1 bind- we have discovered a two-way communication between SecA ATPase and ing to an adjacent OmpF subunit within the same OmpF trimer, thereby pre- SecYEG channel, and proposed a new Brownian motor model of translocation senting the TolB-binding epitope on the periplasmic face of OmpF. [1]. We have extended this approach to investigate initiation of translocation and to estimate the translocation rate. FRET based recurrence analysis of freely 950-Plat diffusing single proteoliposomes improved the time resolution (0.1-50 ms time Electrophysiological Characterization of a Disease-Causing Mutation in scale) and allowed to follow the initial unlocking and opening of the channel. Human CLC-7 The initiation is rate limiting and is driven by ATP hydrolysis and depends on Alissa J. Becerril, Joseph A. Mindell. the unlocking by the signal peptide. Total internal reflection microscopy on im- NINDS, NIH, Bethesda, MD, USA. mobilized proteoliposomes was used to observe individual translocation hClC-7 is a human chloride-proton antiporter that is thought to play a critical events, duration of which yielded an estimate of the translocation rate. Termi- role in the acidification of lysosomes. Mutations leading to loss of hClC-7 func- nation of translocation is fast (milliseconds) and does not require ATP tion have been shown to lead to osteopetrosis, an increase in bone calcification, hydrolysis. and to lysosomal storage disease in humans. However, we were recently pre- sented with a patient who carries a previously undescribed ClC-7 point muta- 953-Plat tion but without osteopetrosis. Instead, the patient manifests a novel disease Functional and Structural Studies of Interplay between an ABC Trans- pattern that includes albinism, lysosomal storage disease, and growth retarda- porter and its Surrounding Membrane Environment tion. To determine the effects of this mutation on transporter function, we intro- Su-Jin Paik1, Alicia Damm1, John Manzi1, Maxime Dahan1, duced this mutation into a plasma-membrane-targeted hClC-7 and expressed Patricia Bassereau1, Emmanuel Margeat2, Daniel Levy1. this protein in Xenopus oocytes to study its activity with electrophysiology. 1UMR 168, Institut Curie, Paris, France, 2Centre de Biochimie Struturale, Notably, the mutant hCLC-7 currents are significantly larger than those of Montpellier, France.

BPJ 8590_8601 Monday, February 19, 2018 189a

ABC (ATP Binding Cassette) transporter is one of the largest transmembrane by indirectly driving Kþ translocation through a separate subunit, KdpA, which protein families. ABCs hydrolyze ATP for the efflux of various types of sub- resembles a Kþ-selective channel and belongs to the Superfamily of Potassium strates including lipids, drugs and antibiotics. Several human ABCs are Transporters. Our recent X-ray crystal structure of the entire KdpFABC com- involved in a cellular multi-drug resistance phenotype against e.g. anticancer plex at 2.9A˚ resolution provided the first detailed view of its architecture. This agents, HIV protease inhibitors, immunosuppressive agents or analgesics. In structure has generated new hypotheses about the mechanisms of inter-subunit this study, we investigate a homologue of human ABC, BmrA, to understand coupling that convert a passive ion conduit into a pump that provides both alter- the feedback between the function, conformations and the physicochemical nating access and the Kþ-affinity changes that are required for active transport. properties of its surrounding membrane including lipid composition and mem- We have mutated residues implicated in gating and charge-transfer between brane curvature at both collective and single-molecule level. To do so, BmrA subunits and have explored the pump’s mechanism through enzyme-coupled was reconstituted in liposomes where protein density and membrane properties ATPase assays and Kþ-transport assays. Electrogenic transport of Kþ is de- were controlled. In order to obtain accurate quantitative measurement, the lipo- tected in proteoliposomes with a voltage-sensitive dye called DiSC3. These somes were well-characterized in terms of protein orientation, rate of incorpo- functional assays also allow us to analyze inhibition of the complex by phos- ration and lamellarity of liposomes. Our results show a synergistic effect of the phate analogs, nucleotide analogs, and channel blockers, which provide further negatively charged lipids and the lipid matrix on the ATPase activity of recon- insight into the transport mechanism and guide preparation of samples for stituted BmrA. The presence of 10% of negatively charged lipids (cardiolipin structural study by cryo-EM. In addition to a new EM structure of the wild- or phosphatidylglycerol) was indeed enough to stimulate the activity two- or type complex in the Kþ-bound E1 conformation, we are working on additional three-fold depending on lipid matrix (phosphatidylcholine or phosphatidyleth- structures of the phosphorylated E2 state in association with phosphate ana- anolamine, respectively). Furthermore, by modulating the size of liposomes logues such as BeF3, AlF4 and VO4. The combination of functional analysis from 30 to 150 nm of diameter, the activity was inhibited at least two-fold in of site mutants and structural analysis of global conformation will offer insight higher positive-curvature. To investigate whether the difference of activities into inter-subunit cooperation and will illustrate how ion channel architecture is related to apo-conformations affected by membrane properties, we per- can be adapted for ATP-driven active transport. formed single-molecule Fo¨rster resonance energy transfer in fluorescence cross-correlation spectroscopy with the reconstituted system. The native Platform: Protein Structure and Conformation II cysteine at nucleotide binding domains (NBDs) was labeled fluorescently to delineate the distance between two NBDs. The first results suggest that the 956-Plat apo-BmrA has large continuous conformational spectrum. In addition, no sig- Analysis of Protein Sequence and Structural Consequences of Amino Acid nificant difference of spectrum was observed between two lipid compositions Variants Associated with Autoimmune Inflammatory Bowel Disease while the activities were significantly different. Chang Chen1, Constance Jeffery2. 1Bioengineering, University of Illinois, Chicago, IL, USA, 2Biological 954-Plat Sciences, University of Illinois, Chicago, IL, USA. Electrophysiological Characterization of Hyperaldosteronism-Associated Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcer- Na/K Pump Mutations ative colitis (UC), comprise a group of immune disorders with symptoms that Dylan J. Meyer1, Craig Gatto2, Pablo Artigas1. can have significant negative impact on a patient’s quality of life. Over one 1Cell Physiology & Molecular Biophysics, Texas Tech University Health million people are affected in the USA alone. The causes are not understood, Sciences Center, Lubbock, TX, USA, 2School of Biological Sciences, Illinois but both genetic and environmental factors are involved. The results of studies State University, Normal, IL, USA. employing NGS (Next Generation Sequencing) and GWAS (Genome-Wide Primary aldosteronism, a form of hyperaldosteronism leading to hypertension, is Association Studies), published by several groups, have identified dozens of al- often initiated by an aldosterone-producing adenoma within the zona glomerulosa leles that vary between patients and healthy individuals and that might be of the adrenal cortex. Somatic mutations of ATP1A1, encoding for the Na/K pump involved in genetic predisposition to IBDs. We conducted a systematic in silico a1-subunit, have been found in these adenomas. It was proposed that a passive in- analysis to predict the impact of the corresponding single amino-acid polymor- ward current transported by several of these mutant pumps under physiological phisms (SAPs) on the sequences and structures of the proteins. The results of conditions is a ‘‘gain-of-function’’ activity, producing membrane depolarization the analysis identified SAPs that are likely to affect protein function, structure and concomitantly increasing aldosterone production. Here we sought to deter- & stability, and therefore might be involved in genetic predisposition to IBDs. mine whether inward currents through the hyperaldosteronism-associated Na/K The results suggest which SAPs may be of high priority for in vitro analysis of pump mutants L104R, V332G, delF100-L104, and EETA963S are large enough the purified mutant proteins to aid in further understanding of the molecular to induce cell depolarization of the cells that harbor them. By combining two- mechanisms of disease and for potential future development of therapeutics electrode voltage clamp with 3H-ouabain binding, we compared the turnover rates to alleviate the symptoms of IBD. of inward currents through each human mutant pump to the turnover rate of out- ward current by human wild-type pumps. The turnover rates of the inward currents 957-Plat at 50 mV were (s1) 22.8 5 1.9 for L104R, 63.4 5 6.2 for V332G, 522 5 120 Evolution of CDR H3 Flexibility at an Immunomic Scale 1 2 3 for delF100-L104, and 19.7 5 1.5 for EETA963S, while the wild-type pump’s Jeliazko R. Jeliazkov , Adnan Slojka , Daisuke Kuroda , 2 3 2 4 turnover rate of outward current was 32.0 5 1.5. Thus, the ‘‘leak’’currents through Nobuyuki Tsutchimura , Kouhei Tsumoto , Naoki Katoh , Jeffrey J. Gray . 1Program in Molecular Biophysics, Johns Hopkins University, Baltimore, EETA963S and L104R are likely too small to induce significant cell depolariza- 2 tion. Additionally, inward currents were absent from G99R under many tested con- MD, USA, Department of Informatics, Kwansei Gakuin University, Nishinomiya, Japan, 3Department of Bioengineering, The University of ditions, including at mammalian ion concentrations and body temperatures, and 4 when co-expressed with FXYD1. Instead, outward currents were observed, but Tokyo, Tokyo, Japan, Department of Chemical and Biomolecular þ Engineering, Johns Hopkins University, Baltimore, MD, USA. with significantly increased K0.5’s for intracellular Na (13.4 5 1.2 mM for wild-type and 32.5 5 3.7 mM for G99R, at 0 mV) and extracellular Kþ (1.1 5 Antibodies can evolve rapidly and specifically in response to antigens. Anti- 0.1 mM for wild-type and 4.6 5 0.2 mM for G99R, at 0 mV). Taken together, body evolution is driven by somatic hypermutation (SHM) that preferentially our results point to ‘‘loss-of-function’’ as the common mechanism for the contri- targets the complementarity determining regions (CDRs), particularly the third bution of these Na/K pump mutants to hyperaldosteronism. heavy chain loop (H3). Elucidating the interplay between SHM and antigen- binding activity is fundamental to understanding adaptive immunity. In recent 955-Plat years, experimental and computational studies have collectively led to a hy- Mechanistic Studies of the KDP Potassium Transport Complex pothesis that SHM rigidifies the bound state of the antibody, minimizing the Marie E. Sweet1, Paula Upla1, Xihui Zhang1, Bjørn P. Pedersen2, entropic cost of antigen binding and thereby improving affinity. However, David L. Stokes1. the individual studies are limited, with no single rigidity study analyzing 1Structural Biology, NYU School of Medicine, New York, NY, USA, more than four unique antibodies. Here, we report for the first time the rigidity 2Aarhus Institute of Advanced Studies, Aarhus University, Aarhus, Denmark. analysis of the CDR H3 loops of hundreds of human and mouse antibody crys- The bacterial potassium pump Kdp is a unique pump-channel hybrid that res- tal structures. Our rigidity theory analysis is verified by experimentally deter- cues cells in low Kþ conditions by pumping against 10,000-fold gradients. The mined B-factors and prior rigidity studies. We find rigidity increases to be largest subunit of the KdpFABC complex, KdpB, resembles a minimal P-type less drastic than previously reported. Finally, we expand our analysis to Roset- ATPase pump, whose well-studied members like the Ca-ATPase (SERCA) taAntibody models of the two thousand most frequently occurring antibodies in employ a mechanism wherein large-scale conformational changes directly peripheral-blood antibody repertoires and find that the difference in rigidity couple ATP hydrolysis to ion translocation. Kdp diverges from this family between the naı¨ve and antigen-experienced repertoires is minimal. This

BPJ 8590_8601 190a Monday, February 19, 2018 large-scale rigidity analysis suggests that SHM may not modulate structural ri- 1iTHES Research Group, and Theoretical Molecular Science Laboratory, gidity or facilitate binding activity as previously thought. Riken, Wako City, Japan, 2Theoretical Molecular Science Laboratory, Riken, Wako City, Japan, 3Quantitative Biology Center, Riken, Kobe, Japan, 958-Plat 4Advanced Institute for Computational Science, Riken, Kobe, Japan, Analyzing the Structure and Symmetry of Membrane Proteins through the 5Department of Biochemistry and Molecular Biology, Michigan State Systematic Online Database EncoMPASS University, East Lansing, MI, USA. Edoardo Sarti, Antoniya Aleksandrova, Lucy R. Forrest. Inside of a cell is highly crowded with a large number of macromolecules Computational Structural Biology, NINDS (NIH), Bethesda, MD, USA. together with solvents and metabolites. How variable interactions within dense Integral membrane proteins represent 30% of the genome of each living or- cellular environments may affect the structure and dynamics, and ultimately ganism, and constitute the target of 50% FDA-approved drugs. They also pre- function of biomolecules is one of the most fundamental questions in life sci- sent distinctive structural features, such as the predisposition for internal and ence. Recently, we constructed full atomistic models of the cytoplasm of bac- quaternary symmetries that are often involved in the protein’s transport mech- teria [1]. Using these model, we performed all-atom molecular dynamics (MD) anism. Several databases of structures of membrane proteins exist, but none of simulation with the highly parallelized MD simulator GENESIS [2]. Influence them classify the proteins or assign relationships between the proteins that they of crowding and non-specific interaction between macromolecules on the trans- enumerate. Moreover, available annotations of symmetries of membrane pro- lational/rotational motion of proteins are analyzed. Anomalous diffusion and teins appear to be incomplete. To address these issues, we present here the the localization of metabolites, such as ATP and amino acids, on the macromol- novel Encyclopedia of Membrane Proteins Analyzed by Structure and Symme- ecule surface are also investigated. Furthermore, destabilization/stabilization try (EncoMPASS), a fully-automated online database through which we aim to mechanism of protein structure via non-specific interaction with macromole- introduce a more flexible representation of the structural relationships between cules and metabolites were also investigated at the molecular level. These find- experimentally-determined membrane protein structures. EncoMPASS pro- ings contribute to improve the understanding of cellular crowding effect, and vides a structure- and symmetry-oriented analysis of over 2000 high- provides important pictures about the microscopic behavior of drug molecules resolution structures, incorporating more than 90% of all membrane protein co- in vivo. ordinate files deposited in the PDB. Analyses of sequence, structure, and sym- Reference metry are reported for each entry. A set of graphical tools helps the user to [1] Michael Feig, Ryuhei Harada, Takaharu Mori, Isseki Yu, Koichi Takahashi, investigate how other structures relate to the structure of interest and to each Yuji Sugita Complete atomistic model of a bacterial cytoplasm for integrating- other in terms of sequence and structure similarity, and how frequently each physics, biochemistry, and systems biology Journal of Molecular Graphics and residue of each single-chain subunit superposes with other topologically similar Modelling, 58, 1–9, (2015) [2] Isseki Yu, Takaharu Mori, Tadashi Ando, Ryu- chains. A complete analysis of quaternary and internal symmetries is also avail- hei Harada, Jaewoon Jung, Michael Feig, Yuji Sugita Biomolecular interac- able, and each symmetric region is illustrated and associated with the relevant tions modulate macromolecular structure and dynamics in atomistic model of symmetry axis and symmetry transformation. The database is updated monthly a bacterial cytoplasm eLife 2016;5:e19274. and its underlying code is freely available. Thanks to these characteristics, EncoMPASS can be used for organizing resources for protein structure deter- 961-Plat mination, benchmarking sequence alignment tools, and inferring membrane Protein Fold Switching is Widespread protein functionalities via comparative studies. Lauren Porter, Loren Looger. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, 959-Plat USA. Inactivation Mechanism of Protein Kinase a Revealed by Markov Model A central tenet of biology is that globular proteins have a unique three- Yingjie Wang1,2, Jonggul Kim1,2, Cristina Olivieri2, Jiali Gao1, dimensional structure under physiological conditions. Recent work has chal- Gianluigi Veglia1,2. lenged this dogma by demonstrating that some proteins switch folds, a process 1Department of Chemistry, University of Minnesota, Minneapolis, MN, that can involve changes as drastic as a-helical bundles morphing into USA, 2Department of Biochemistry, Molecular Biology and Biophysics, b-sheets. To date, fold switches have been viewed as isolated examples of a University of Minnesota, Minneapolis, MN, USA. peripheral phenomenon, but their frequency has been neither quantified nor Eukaryotic protein kinases (EPK) serve as on-and-off switches for regulating estimated. Here we quantify the frequency of protein fold switches by system- various signaling cascades. Their aberrant activation is implicated in various hu- atically and exhaustively searching the PDB. We found that 100 proteins man diseases such as cancer and heart failure. Unlike many other members in the switch folds, and dozens of these proteins undergo large-scale changes in EPK family, the catalytic subunit of protein kinase A (PKA-C) is constitutively secondary and tertiary structure. Furthermore, we gathered multiple lines of active. Recent NMR relaxation dispersion experiment, however, suggest that evidence suggesting that protein fold switching is a significant biological phe- sparsely-populated, conformationally excited states in PKA-C exist, revealing nomenon. Firstly, protein fold switches occur in all kingdoms of life. Sec- an equilibrium between different conformations in the ms to ms timescale. ondly, they foster a highly diverse range of biological functions. Thirdly, Here we employed unbiased adaptive sampling and Markov model to describe half of the structures indicating that a given protein switches folds have the conformational transitions of PKA-C in the apo, binary (with ATP), and been solved in the past 5 years, and >90% have been solved in the past 12 ternary (with ATP and pseudosubstrate, PKI5-24) forms. Detailed conformational years. Fourthly, expressing, purifying and/or solving the structures of these landscape from a total of 250 ms MD trajectories revealed multiple high energy proteins is often difficult. Based on these lines of evidence, we hypothesized states in the apo and binary forms. Importantly, our simulations identify distinct that protein fold switching may be significantly more common than currently inactive conformations, whose population is modulated upon ligand binding. In anticipated. Thus we sought to identify other protein fold switches with only the apo PKA-C, we found two orthogonal inactivation paths that correspond to one solved structural state in the PDB. To do this, we identified some charac- an interruption of interior hydrophobic packing around the R-spine, and the teristic features of the 100 identified fold switches—low sequence destabilization of the aC helix by the C-terminal tail, which both contribute to complexity and likely independent folding cooperativity—and searched the disrupt the catalytic machinery. Notably, the conformational transition along PDB for other proteins with similar features. From this, we identified a signif- the first inactivation path, which may be initiated by conformational rearrange- icant number of both definite protein fold switches and likely candidates. ment of the aC-b4 loop, is not captured by the currently available X-ray struc- These results demonstrate that protein fold switching is likely more common tures, but it recapitulates the active-to-inactive conformational equilibrium than currently appreciated, which has implications for cell biology, genomics, probed by NMR experiments. Nucleotide binding with shifts the population of and human health. the kinase conformers toward the inactive state that is characterized by an out- ward movement of the aC helix. The PKI binding traps the kinase in the active 962-Plat state. We envision that characterization of metastable states that are inactive may Automatic Building of Protein Atomic Models from Cryo-EM Maps facilitate the design allosteric inhibitors that deactivate oncogenic EPK mutants. Guillaume Bouvier, Benjamin Bardiaux, Michael Nilges. Structural Biology and Chemistry, Institut Pasteur, Paris, France. 960-Plat Cryo-electron microscopy (Cryo-EM) has emerged as a powerful method to Dynamics and Interactions of Proteins and Metabolites in Cellular Crowding obtain electron density maps of protein assemblies. Environments: All-Atom Molecular Dynamics Study of Proteins and However, reconstruction of atomic models from near atomic resolution (3-5 A) Metabolites in Cellular Crowding Environments: All-atom Molecular Dy- cryo-EM maps is still challenging. Several tools developed for X-ray crystal- namics Study lography are widely used to interpret high resolution EM maps (around 3 A), Isseki Yu1, Takaharu Mori2, Tadashi Ando3, Ryuhei Harada4, but poorly resolved side-chain densities hamper sequence attribution by auto- Jaewoon Jung4, Yuji Sugita2, Michael Feig5. matic procedures at lower resolution.

BPJ 8590_8601 Monday, February 19, 2018 191a

Furthermore, segmentation of EM maps into subunits remains a difficult prob- biochemical/biophysical methods and direct visualization by cryo-electron mi- lem when no structures of these subunits exist, or when conformational changes croscopy. In the first approach, we introduced stiff, bivalent DNA-PNA hetero- occur between the isolated and complexed form of the subunits. duplexes, presenting 6’-sialyl-LacNAc with distinct distances, to identify an To tackle these issues, we have developed a graph-based method to thread optimal, bivalent receptor spacing. Upon a multivalent presentation of these most of the C-a trace of the protein backbone into the EM map. The EM den- bivalent clusters on long DNA strands, impressive enhancement factors sity is described as a weighted graph such as the resulting minimum spanning regarding a viral binding inhibition could be obtained. In a second approach, tree encompasses the high density region of the map. A pruning algorithm is the highly ordered arrangement of rigid Qb phage capsids provide a scaffold then applied to clean the tree and find the most probable positions of the C-a for optimally spaced trivalent SA cluster matching geometrically the HA atoms, using sidechain density when available, in the form of C-a trace arrangement on the virus surface. The promising potential for application of fragments. functionalized multivalent rigid bioscaffolds was validated in ex vivo human By complementing experimental EM maps with contact predictions from lung tissues and a mouse model. Thus, we could demonstrate that our strategy sequence co-evolutionary information, we demonstrate that our approach is ranging from in silico molecular modeling studies to in vivo inhibition of viral able to correctly segment EM maps into individual subunits and recover infections allow new insights on the principles governing multivalent interac- most of the sequence attribution to generate full-atom models. tions and is straight forward in order to develop new antivirals. 963-Plat 965-Plat Quantitative Characterization of the Conformational Dynamics of the Un- Imparting Nonnative Gold Binding Functionality by Bioengineering bound Lipase-specific Foldase Lif by MD Simulations and Fluorescence Bacterial Fimbrial TIP Protein FimH with Gold Binding Peptides Spectroscopy Meagan C. Small1, Jessica L. Terrell2, Dimitra N. Stratis-Cullum2, Neha Verma1, Jakub Kubiak2, Peter Dollinger3, Filip Kovacic3, Margaret M. Hurley1. Holger Gohlke1, Claus Seidel2, Karl-Erich Jaeger3. 1Sensors and Electronic Devices Directorate, U.S. Army Research 1Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Laboratory, Aberdeen Proving Ground, MD, USA, 2Sensors and Electronic Dusseldorf,€ Dusseldorf,€ Germany, 2Molecular Physical Chemistry, Heinrich Devices Directorate, U.S. Army Research Laboratory, Adelphi Laboratory Heine University Dusseldorf,€ Dusseldorf,€ Germany, 3Molecular Enzyme Center, MD, USA. Technology, Heinrich Heine University Dusseldorf,€ Julich,€ Germany. Bio-inorganic hybrid materials have many potential applications in medicine, Highly flexible or intrinsically disordered proteins may access an astronomical electronics, and nanotechnology. Accordingly, biomolecules with predictable number of conformations [1]. The lipase-specific foldase (Lif) from Pseudo- and controllable binding to inorganic surfaces are of great interest. Peptides monas aeruginosais such an example of a highly flexible protein with intrin- and proteins are ideal candidates for hybrid materials because they are either sically disordered regions in the unbound state. Lif is a steric chaperone, 1) easy to synthesize as is the case with peptides in which metal binding resi- which acts as a conformational switch for the activation of lipase A (LipA). dues can be incorporated or 2) relatively easy to engineer due to their robust- The crystal structure of the Burkholderia glumae LipA-Lif complex has ness as with proteins. When combined with the ability to genetically modify been solved, in which Lif forms an a-helical scaffold embracing LipA in a organisms, bioengineered proteins can be introduced into bacterial cells to headphone-like structure. Yet, the exact mechanism of action of Lif remains confer nonnative functionality, and thereby engineer whole-cell behavior. enigmatic [2,3]. Due to the intrinsic disorder of Lif, the characterization of Imperative to this is an understanding of protein dynamics to predict the impact typical conformations by means of molecular dynamics (MD) simulations is of a modification on native functionality and, further, verify its conformational challenging. To quantitatively characterize the conformational dynamics of accessibility. In this work, we present our combined experimental and compu- Lif, we performed MD simulations, starting from the open, headphone-like tational efforts in bioengineering E. coli fimbrial tip protein FimH so that it structure of Lif to generate a conformational ensemble, and refined the result- binds to Au(111) surfaces. First, we demonstrate that integration of a series ing ensemble against high precision Fo¨rster resonance energy transfer of gold binding peptides (GBPs) into the FimH lectin domain at I52 disrupts (hpFRET) measurements by using the maximum entropy method. We identi- bacterial cell binding to mannose, the native ligand, while imparting nonnative fied 35 Lif conformations ranging from partially closed to entirely closed. gold-binding capability. Second, we show from computational models and sim- These structures provide the basis for a detailed atomistic model of Lif dy- ulations of select mannose-bound engineered FimH variants that the inserted namics in the unbound state. We hypothesize how the multitude of states in GBPs perturb the secondary structure around the mannose binding site, thereby fast exchange provide the basis for LipA binding despite the fickleness of disrupting pivotal hydrogen bonds between mannose and the lectin domain the structure. leading to mannose disassociation. Third, we provide insights from simulations Platform: Bioengineering and Biomaterials of the full series of engineered FimH variants into structural features that enhance binding to Au(111). To conclude, we will discuss these findings in 964-Plat the context of our work in designer biofilms and seeding engineered consortia Tailored Multivalent Biomolecules for an Optimal Interaction with Influ- onto Army-relevant materials. enza a Virus Hemagglutinin - From In Silico Modeling to In Vivo Viral Infection Inhibition 966-Plat Daniel Lauster1, Victor Bandlow2, Susanne Liese3, Kai Ludwig4, High throughput Electroporation System for Bio-Molecule Delivery into Marlena Stadtmuller€ 5, Simon Klenk6, Sandra S€anger5, Christoph Bo¨ttcher4, Zebrafish Follicle Thorsten Wolff5, Christian Hackenberger6, Oliver Seitz2, Roland Netz3, Tayyebeh Saberbaghi1, Ebrahim Ghafar-zadeh2, Chun Peng2. Andreas Herrmann1. 1York University, Richmond Hill, ON, Canada, 2York University, Toronto, 1Department of Biology, Humboldt-Universit€at zu Berlin, Berlin, Germany, ON, Canada. 2Department of Chemistry, Humboldt-Universit€at zu Berlin, Berlin, This study presents a new approach for drug delivery into the cell layers of Ze- Germany, 3Department of Physics, Freie Universit€at Berlin, Berlin, brafish Follicle. Zebrafish follicle is used as a model of ovarian development. Germany, 4Department of Biology, Chemistry, and Pharmacy, Freie Zebrafish follicle consists of an oocyte surrounded by two thin layers of cells Universit€at Berlin, Berlin, Germany, 5Robert-Koch-Institut, Berlin, (theca and granulosa cell). The delivery of biomolecules in to the cells in these Germany, 6Leibniz Forschungsinstitut fur€ Molekulare Pharmakologie (FMP), layers using electroporation is an unmet challenge. Electroporation is a non- Berlin, Germany. invasive method widely used for transferring molecules into cells for various The influenza A virus surface is characterized by a densely packed layer of applications including stem cell based tissue construction and gene therapy. spike proteins, containing hemagglutinin (HA) as the major component. The Despite broad advantages of electroporation, no reports have been released trimeric HA is essential for attachment to host cells. Abundant sialic acid for application of drug delivery into Zebrafish follicle cells. Herein we reports (SA) containing biomolecules serve as natural receptors for HA allowing a the design, implementation of a new high throughput electroporation system for multivalent interaction with the viral surface. To prevent such interactions, a delivery of bio-molecules into zebrafish follicle cells of (Danio rerio)ina multitude of biomimetic nanoparticles displaying SA in a multivalent fashion controllable manner. This platform consists of an array of 32 microfluidic have been described so far. However, all of these binding inhibitors display chambers incorporated with the same number of addressable microelectrodes the HA targeting receptor in a structurally undefined arrangement. We pursued interconnected to a computer for the controlled electrical stimulation. We a new strategy with rigid bioscaffolds presenting SA in an adjusted spatial demonstrate and discussed the advantage of proposed method for gene delivery arrangement for an optimal interaction with HA. Development of inhibitors into the cells using PI and DAPI. This study will take us a step closer to deliver is based on a synergistic concept of complementary theoretical, experimental other types of genes into Zebrafish follicle for ovarian treatment purposes.

BPJ 8590_8601 192a Monday, February 19, 2018

967-Plat show an innovative 3D material to serve as a platform for controlling mecha- Deformability of Individual Cells Probed by Electrical and Optical Signals notransduction by mimicking natural 3D cellular environments. Our material Zuzanna S. Siwy1, Preston Hinkle1, Trisha M. Westerhof2, Yinghua Qiu1, contains a novel form of microporous structures represented by micron-sized David J. Mallin1, Matthew L. Wallace1, Chih-Yuan Lin1, Edward L. Nelson2, channels embedded in a hydrogel matrix of a well-defined stiffness and conduc- Peter Taborek1. tivity. The material guarantees pore interconnectivity independently of pore 1Dept. of Physics and Astronomy, University of California, Irvine, Irvine, density and size, and different types of surface functionalization are possible. CA, USA, 2Department of Medicine, University of California, Irvine, Irvine, Furthermore, the material also provides a large and spatially controlled cell- CA, USA. surface contact area through the specific architecture of its pores, such that Deformability, the extent to which a particle changes shape under applied the mechanical properties of the environment have large impact on the cells. stresses, has recently gained attention as an important mechanical marker for We here show data on how different cell types grow in the microporous mate- cells. Cell deformability is determined by physiological properties such as cyto- rials and future applications where cellular mechanotransduction can be skeletal structure, and can be related to the cell’s phase in its mitotic cycle and exploited. can be used as a marker to differentiate cancerous and non-cancerous cells. Given the wide range of information gained from deformability measurements, 970-Plat a deformability-based cell detector is highly desirable. One recent method for An EEL-Inspired Artificial Electric Organ: 110 Volts from Water and Salt measuring cell deformability is flow deformability cytometry, where cells are Anirvan Guha1, Thomas B.H. Schroeder1,2, Aaron Lamoureux3, driven through microconstrictions at fast velocities and deform under the large Gloria VanRenterghem3, David Sept4,5, Max Shtein3, Jerry Yang6, hydrodynamic loads. Cell deformation is usually measured optically using a Michael Mayer1,4. microscope and high-speed camera; however, while optical detection is accu- 1Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland, rate, it is financially and computationally expensive. Here we report deform- 2Department of Chemical Engineering, University of Michigan, Ann Arbor, ability cytometry experiments performed with simultaneous optical and MI, USA, 3Department of Materials Science and Engineering, University of electrical detection using the resistive pulse technique. By synchronizing the Michigan, Ann Arbor, MI, USA, 4Department of Biomedical Engineering, optical and electrical signals in time, both recorded with 200,000 Hz, we are University of Michigan, Ann Arbor, MI, USA, 5Center for Computational able to directly observe the effect of deformation of the cells on the resistive Medicine and Biology, University of Michigan, Ann Arbor, MI, USA, pulse signal, leading to the eventual possibility for replacing optical detection 6Department of Chemistry and Biochemistry, University of California San with resistive pulse entirely. The experiments were performed using a non- Diego, San Diego, CA, USA. traditional channel design characterized by narrow entrances along with a rela- The rapidly growing interest in electronic devices which interface with and tively larger central cavity. The channel design induces bidirectional deforma- exist inside the human body has brought with it a call for power generation con- tion as the cells pass through the channel, which was confirmed through cepts which are biocompatible, mechanically robust, and able to convert chem- experimental observation and finite element analysis modeling. ical energy within the body to electricity. While traditional batteries are not designed with these characteristics in mind, inspiration for such an energy 968-Plat scheme can be found in nature: the electric organ within the electric eel (Elec- Fluorescent Nanosensors for Two-Photon Infrared Imaging of Dopamine trophorus electricus) is able to generate peak voltages of 600 V and currents of Release in Brain Tissue up to 1 A using repeating sequences of ionic gradients across ion-selective cell Jackson T. Del Bonis-O’Donnell, Ralph Page, Abraham Beyene, membranes. Here we present an eel-inspired power concept employing a Eric Tindall, Ian McFarlane, Markita Landry. repeating tetrameric sequence of hydrogels which positions high- and low- Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA, USA. salinity aqueous reservoirs across cation- and anion-selective hydrogel Non-invasive methods for direct imaging of neurotransmission at a molecular membranes. Upon mechanical activation, these hydrogels are brought into level will transform our ability to study and understand the brain and mental self-registered contact, producing a voltage which scales indefinitely with the health. Recently, we have developed near-infrared (NIR) fluorescent nanosen- number of units stacked in series and a current which scales with the number sors for the sensitive and rapid detection of the neuromodulatory neurotrans- of units in parallel. The use of hydrogels allowed us to investigate numerous mitter dopamine. These nanosensors emit fluorescence in the NIR-II window strategies for patterning series and parallel arrangements of tetramers, (1000-1700 nm) where absorption and scattering of light by brain tissue is min- including fluidics, printing, and folding. With various implementations of imal, making them optimally suited for in vivo imaging deep within the brain. this concept, we were able to reach a peak voltage of 110 V and a power density The fluorescence response of dopamine nanosensors is reversible and exhibits a of up to 27 mW/m2. These systems are soft, transparent, flexible, and poten- rapid response to dopamine with 10 nM sensitivity. Conventional approaches tially biocompatible, making them an appealing alternative to traditional batte- to imaging in brain tissue rely on strongly scattering visible light wavelengths ries for powering wearable and implantable electronics. for either excitation, emission, or both, which impose a limit on imaging qual- ity. To overcome this drawback for in vivo imaging of dopamine, we demon- 971-Plat strate non-linear photoexcitation of nanosensors using a 1560 nm femtosecond Wet Adhesive Nanomaterials Inspired by the Barnacle Adhesive pulsed laser, a technique we call dual near-infrared excitation-emission micro- Christopher R. So1, Elizabeth Yates2, Luis Estrella3, Ashley Schenck2, Catherine Yip4, Kathryn J. Wahl3. scopy (NIR-EE). We show improved dopamine nanosensor imaging quality 1 2 with two-photon excitation at a depth of 500 microns in optically opaque tissue US Naval Research Laboratory, Washington, DC, USA, Chemistry, United States Naval Academy, Annapolis, MD, USA, 3Chemistry Division, code phantoms. As we construct the first NIR-EE laser scanning microscope, we 4 validate our nanosensors for in vivo neurotransmitter imaging using larval ze- 6176, US Naval Research Laboratory, Washington, DC, USA, United States brafish. We deliver nanosensors into dopaminergic clusters of the diencephalon Naval Academy, Annapolis, MD, USA. using a glass capillary and observe local spikes in fluorescence intensity which Barnacles produce a micron-thick layer of ordered amyloid-like nanofibers we attribute to catecholamine release into the extracellular space occupied by from proteins that function as a permanent wet adhesive. Recent proteomic the nanosensors. Future work aims to correlate neurotransmitter detection with work from our lab shows that barnacles achieve this largely through display optogenetic stimulation of dopaminergic neurons expressing ChR2, comple- of complex charged chemistries using small and flexible side-chains, folded menting our previous work in acute rodent brain slices. Ultimately, these in a manner similar to adhesive silks used by spiders and insects. Their well- methods will allow for imaging of both synaptic and volume transmission of defined, modular, nature result in novel biomaterials that serve many purposes: dopamine with unprecedented spatial and temporal resolution to better under- adhesion, durability, bacterial resistance, and even potent enzymatic activity. stand the effects of drugs, psychiatric disorders, and neuroplasticity. Fibers are shaped by a highly conserved domain alternating between short 20-residue low complexity sequences (Gly/Ser/Thr/Ala residues) and regions 969-Plat with charged and aromatic side chains, with more than 80 such domains in Interconnected Microchannels in Hydrogels to Control Cell Adhesion and just five proteins. The adhesive properties of these unique sequences and their Mechanotransduction function in an amyloid-like structure remain unclear. To study this, we produce Mohammadreza Taale, Christine Arndt, Christine Selhuber-Unkel. miniaturized synthetic peptides from consensus barnacle cement sequences and Biocompatible Nanomaterials, University of Kiel, Kiel, Germany. also insert sequences into a host amyloid system of bacterial fimbriae to pro- Cells respond to external mechanical stimuli through the activation of mecha- duce abundant and engineered wet adhesive mimics. Short synthetic peptides notransduction, which influences cell proliferation, migration and differentia- demonstrate that certain cement sequences specifically recognize and activate tion, and adhesion. Likewise, diseases such as cancer and cardiac cement polymerization to form bioinspired nanomaterials. Further, bacterial dysfunctions are strongly related to cellular mechanotransduction. Here we biofilms are demonstrated to be a viable route for the growth of recombinant

BPJ 8590_8601 Monday, February 19, 2018 193a cement fibrils. These materials are characterized by AFM-based nanomechan- For tumor suppression, it is fundamental that p53 binds to target sites of DNA ical measurements and compared to the Wild-Type barnacle adhesive. Syn- correctly. How does p53 search for and recognize the target sites among a huge thetic and recombinant adhesive materials provide a route to scale up and DNA within a physiological time? Here, we developed a new method for pro- study a scarce but potent class of multifunctional adhesive nanostructures pro- ducing the aligned DNA array, DNA garden (Bull. Chem. Soc. Jpn. 2017), and duced by one of the most tenacious marine fouling organisms in the ocean. investigated the target search dynamics of p53 using a single-molecule fluores- cence microscopy. p53 possessed two 1D-sliding modes along DNA, and the Platform: Protein-Nucleic Acid Interactions sliding distance was maintained under change of [Mg2þ] and [Ca2þ] (J. Mol. Biol. 2015). The modes and sliding distance were regulated by disor- 972-Plat dered C-terminal domain and linker (Biophys. J. 2017; Biochemistry 2017). Nanopore Tweezers Reveal Detailed RNA Polymerase Dynamics at a Target recognition probability of p53 was 7% and was regulated by the activa- Sequence-Specific Pause Element tion and inactivation of p53 (J. Mol. Biol. 2016). Target recognition of p53 was Ian C. Nova1, Abhishek Mazumder2, Jonathan M. Craig3, achieved by 1D sliding along DNA, rather than 3D encounter. Furthermore, we Andrew H. Laszlo3, Ian M. Derrington3, Matthew T. Noakes3, developed high-speed single-molecule imaging system and applied to visualize Henry Brinkerhoff3, Jasmine Bowman3, Jonathan W. Mount3, the movement of p53 along DNA. Results showed that p53 can pass over ob- Benjamin I. Tickman1, Richard H. Ebright2, Jens H. Gundlach3. stacles on DNA by using intersegmental transfer or hopping/jumping. Overall, 1Molecular Engineering, University of Washington, Seattle, WA, USA, we will present how p53 behaves on DNA and how the observed dynamics of 2Rutgers University, New Brunswick, NJ, USA, 3Physics, University of p53 are relevant to the biological functions. Washington, Seattle, WA, USA. 975-Plat We applied a new single-molecule technique named SPRNT (Single-molecule Structural Characterization of the HIV-1 Reverse Transcriptase Initiation Picometer Resolution Nanopore Tweezers) to analyze the translocation of sin- Complex gle E. coli RNA polymerase (RNAP) core complexes during transcription. Kevin Larsen1, Yamuna Mathiharan2, Kalli Kappel1, Aaron Coey1, Inspired by nanopore DNA sequencing with biological porin MspA, SPRNT Dong-Hua Chen2, Lauren Madigan2, Georgios Skiniotis2, Joseph Puglisi2, enables observation of motor protein movement along nucleic acids with Elisabetta Viani Puglisi2. sub-nucleotide spatial resolution, sub-millisecond temporal resolution, and 1Biophysics, Stanford University, Stanford, CA, USA, 2Structural Biology, the ability to apply assisting and opposing forces. In addition, the sequence Stanford University, Stanford, CA, USA. in the RNAP complex is provided by nanopore sequencing, enabling the study Reverse transcription of the HIV-1 RNA genome into double-stranded DNA is a of sequence-dependent translocation behaviors. Using SPRNT, we monitored central step in infection and a common target of anti-retrovirals. The reaction is RNAP dynamics at the sequence-specific pause element yrbL. By tracking hun- catalyzed by viral enzyme reverse transcriptase (RT) that is packaged in an infec- dreds of RNA polymerase molecules while varying applied force and [NTPs] Lys tious virion along with 2 copies of dimeric viral genomic RNA and host tRNA 3, over three orders of magnitude, we reveal, in unprecedented detail, the various which acts as a primer for initiation of reverse transcription. Upon viral entry, initi- enzyme states and complex kinetics during this pause element. ation is slow and non-processive compared to elongation. A rich body of structural data on RT, a heterodimer of p51 and p66 subunits, have shown how its polymer- 973-Plat ase and RNase H domains interact with DNA/DNA and DNA/RNA duplexes in Sliding of Lac Repressor along DNA is Sequence-Dependent and Allosteri- the absence and presence of antiviral drugs. Critical elements in the viral RNA cally Regulated and host tRNA that are necessary for efficient initiation have been identified Alessia Tempestini1, Carina Monico1, Lucia Gardini1,2, Francesco Vanzi1,3, through biochemical and biophysical data. However, we lack insight into the Francesco S. Pavone1,4, Marco Capitanio1,4. structural rearrangements that occur during initiation, whereby RT binds to a large 1 2 LENS, Sesto Fiorentino, Italy, National Institute of Optics–National bimolecular tRNA-viral RNA complex. Here we apply several biophysical tech- 3 Research Council, Florence, Italy, Department of Biology, University of niques, including cryo-electron microscopy and single-molecule fluorescence, to 4 Florence, Firenze, Italy, Department of Physics and Astronomy, University characterize the three-dimensional structure of the HIV RT initiation complex. of Florence, Sesto Fiorentino, Italy. Our results illustrate how RNA structure in the initiation complex alters RT Transcription factors and DNA-binding proteins bind their specific target se- conformation and serves as a barrier for efficient initiation. quences with rates higher than allowed by 3D diffusion alone. Generally accepted models predict a combination of free 3D diffusion and 1D sliding 976-Plat along non-specific DNA [1]. One important issue in the field of protein-DNA CRISPR Cas9 Mediated DNA Unwinding Detected using Site-Directed interaction is the understanding of how proteins interact with non-cognate Spin Labeling DNA sequences and how they find the sequence of interest along the DNA. Narin S. Tangprasertchai1, Rosa Di Felice1, Xiaojun Zhang1, Here, we study the mechanism of DNA target search by a single lac repressor Ian M. Slaymaker2, Carolina Vazquez Reyes1, Wei Jiang1, Remo Rohs1, protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and Peter Qin1. few base-pair resolution technique based on laser tweezers [2]. The improved 1Univ. of Southern California, Los Angeles, CA, USA, 2Broad Institute of resolution of our method allows measurements at physiological salt concentra- MIT and Harvard, Cambridge, MA, USA. tions, contrary to previous single-molecule in vitro measurements, and reveals In a type II Clustered-Regularly-Interspersed-Short-Palindromic-Repeats important novel aspects of LacI target search mechanism. Remarkably, we (CRISPR) system, RNAs derived from the CRISPR locus complex with the measure 1D-diffusion with 20 base-pair resolution and find that sliding of CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that LacI along DNA is sequence-dependent. Moreover, by controlling the struc- cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR- tural state of a single LacI molecule through inducer molecules, we demon- Cas9 system has been successfully adapted for genome engineering in a wide strate that only the active (inducer-free) LacI conformation slides on DNA range of organisms, leading to a revolution in genome engineering that is still during target search, whereas inactive (inducer-bound) LacI weakly interacts rapidly unfolding. Studies have indicated that a series of conformational changes with DNA and does not show sliding. This might be an important regulatory in Cas9, coordinated by the RNA and the target DNA, direct the protein into its mechanism in vivo to switch off sliding and minimize DNA crowding when active conformation, yet, details on these conformational changes, as well as the repressor is inactive. Last, the transition from sliding to strong binding to their roles in the mechanism of function of Cas9, remain to be elucidated. Build- the target sequence requires an additional load-dependent conformational ing on our recent work demonstrating the use of site-directed spin labeling to change of LacI. Our data reveal how LacI target search process is regulated monitor nucleic-acid dependent conformational changes in Streptococcus pyo- by molecular switching and DNA sequence and provide a comprehensive genes Cas9, we reported here direct detection of Cas9-mediated DNA unwind- model of LacI facilitated diffusion mechanism. ing, which is a crucial step in Cas9 target recognition, by a combination of site- [1] Monico, C. et al., Int. J. Mol. Sci. 14, 3961-3992 (2013). directed spin labeling and Molecular Dynamics simulations. Our results support [2] Capitanio, M. et al., Nature Methods, 9, 1013–1019 (2012). a model in which the unwound non-target strand is stabilized by a positively- charged patch located between the two nuclease domains of Cas9, and reveal un- 974-Plat even increases in flexibility along the unwound non-target strand upon scissions Single-Molecule Characterization of P53 on DNA using DNA Array ‘‘DNA of the DNA backbone. This study establishes the synergistic combination of Garden’’ spin-labeling and Molecular Dynamics to directly monitor Cas9-mediated Kiyoto Kamagata. DNA conformational changes, and yields information on the target DNA in Institute of Multidisciplinary Research for Advanced Materials, Tohoku different stages of Cas9 function, thus advancing mechanistic understanding University, Sendai, Japan. of CRISPR-Cas9 and aiding future technological development.

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977-Plat the cleaved strand. The DNA cleavage mechanism is similar to type IIA topoi- Enhanced Proofreading Governs CRISPR-Cas9 Targeting Accuracy somerases, which cleave both strands of dsDNA through 5’ phosphotyrosine in- Janice S. Chen1, Yavuz S. Dagdas2, Benjamin P. Kleinstiver3, termediates to form a protein-DNA gate. In type IIA topoisomerases duplex Moira M. Welch4, Alexander A. Sousa4, Lucas B. Harrington1, DNA is passed through this gate. Although it is assumed type IA topoisomerases Samuel H. Sternberg5, Keith J. Joung3, Ahmet Yildiz1, Jennifer A. Doudna1. also pass the intact DNA strand through the cleaved strand via a protein gate, this 1Department of Molecular and Cell Biology, University of California, gate-opening has never been directly measured. We developed a single- Berkeley, Berkeley, CA, USA, 2Biophysics Graduate Group, University of molecule magnetic tweezers assay to directly detect gate opening of type IA top- California, Berkeley, Berkeley, CA, USA, 3Department of Pathology, oisomerases. In parallel, we have probed the conformational changes associated Harvard Medical School, Boston, MA, USA, 4Center for Computational and with gate-opening via molecular dynamics simulations. With the single- Integrative Biology, Massachusetts General Hospital, Charlestown, MA, molecule assay, we measured the force-dependent kinetics of gate opening USA, 5Department of Chemistry, University of California, Berkeley, and closing as well as cleavage and ligation kinetics of E. coli topoisomerase I Berkeley, CA, USA. and III. By varying the magnesium concentration, we determined the The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes magnesium-dependent cleavage and religation kinetics. Furthermore, we show (SpCas9) has been widely repurposed for genome editing. DNA cleavage of that type IA topoisomerases can close and re-ligate DNA against high forces. Cas9 is controlled by the conformational state of the HNH nuclease domain, This ability to work against high force suggests a fail-safe, spring-loaded mech- but the mechanism that governs HNH activation at on-target DNA while anism by which type IA topoisomerases can operate under conditions of extreme reducing cleavage activity at off-target sites remains poorly understood. Using tension, such as might be found at converging replication forks or sites of inter- single-molecule Fo¨rster resonance energy transfer (smFRET), we identified an chromosomal interactions. To our knowledge this is the first time such a spring- intermediate state of Streptococcus pyogenes Cas9, representing a conforma- like conformational change has been connected to enzymatic activity. tional checkpoint between DNA binding and cleavage. Upon DNA binding, the HNH domain transitions between multiple conformations before docking Platform: Microtubules and Associated Motors into its active state. We showed that the high-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) Cas9 variants are trapped in an inactive 980-Plat state when bound to mismatched targets. We find that a non-catalytic domain Cryo-Electron Tomography Reveals that Dynactin Recruits a Team of within Cas9, REC3, recognizes target complementarity and governs the Dyneins for Processive Motility HNH nuclease to regulate overall catalytic competence. Exploiting this obser- Danielle A. Grotjahn1, Saikat Chowdhury1, Yiru Xu1, vation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that dem- McKenney J. Richard2, Trina Schroer3, Gabriel C. Lander1. onstrates high genome-wide specificity without compromising on-target 1The Scripps Research Institute, La Jolla, CA, USA, 2University of activity in human cells. These results offer a more comprehensive model to California-Davis, Davis, CA, USA, 3John Hopkins University, Baltimore, rationalize and modify the balance between target recognition and nuclease MD, USA. activation for precision genome editing. A key player in the intracellular trafficking network is cytoplasmic dynein, a protein complex that transports molecular cargo along microtubule tracks. It 978-Plat has been shown that vertebrate dynein’s movement becomes strikingly Investigating and Modelling the Target Recognition Dynamics of the enhanced upon interacting with a cofactor named dynactin and one of several CRISPR-Cas Surveillance Complex Cascade cargo-adapters, such as BicaudalD2. However, the mechanisms responsible for Marius Rutkauskas1, Tomas Sinkunas 2, Inga Songailiene2, this increase in transport efficiency are not well understood, largely due to a Virginijus Siksnys2, Ralf Seidel1. lack of structural information. We used cryo-electron tomography to visualize 1Peter Debye Institute for Soft Matter Physics, Leipzig University, Leipzig, the first 3-dimensional structure of the dynein-dynactin complex bound to mi- Germany, 2Institute of Biotechnology, Vilnius University, Vilnius, Lithuania. crotubules. Our structure reveals that the dynactin-cargo-adapter complex re- Type I and Type II CRISPR-Cas systems are core elements of bacterial and cruits and binds to two dimeric cytoplasmic dyneins. Interestingly, the archaeal adaptive immunity and tools for the emerging genome editing and dynein motor organization closely resembles that of axonemal dynein, suggest- gene silencing technologies. Molecular recognition by these systems is achieved ing that cytoplasmic dynein and axonemal dyneins may utilize similar mecha- by directional base-pairing between the crRNA-component of a specific surveil- nisms to coordinate multiple motors. We propose that grouping dyneins onto a lance complex and the target DNA. During this process a triple-strand RNA- single dynactin scaffold promotes collective force production as well as unidi- DNA hybrid called R-loop is formed. Full zipping between the crRNA along rectional processive motility. These findings provide a structural platform that the DNA target causes a conformational change in the surveillance complex facilitates a deeper biochemical and biophysical understanding of dynein regu- that finally promotes DNA destruction. This recognition is highly promiscuous lation and cellular transport. and tolerates multiple mismatches between crRNA and the target DNA. This is advantageous for bacterial immunity but not at all desired in genome engineering 981-Plat applications. The effects of mismatches on the target recognition vary strongly, Structural Analysis of a Human Mitotic Kinesin and Its Potential Binding depending on the type and the position of the mismatch. Currently there is no uni- Site for a Small Molecule Inhibitor 1,2 3 1 4 fying model that can predict the impact of a particular mismatch or a combina- Hee-Won Park , Zhujun Ma , Haizhong Zhu , Shimin Jiang , Robert C. Robinson4, Sharyn A. Endow3,5. tion of mismatches. Here we employed single-molecule supercoiling 1 experiments to quantify the dynamics of R-loop formation by the surveillance Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada, 2Department of Biochemistry and Molecular Biology, Tulane complex Cascade from St. thermophilus. Specifically, resolve short-lived 3 R-loop intermediates that occur on single and double mismatched targets and University School of Medicine, New Orleans, LA, USA, Neuroscience & Behavioural Disorders Programme, Cancer & Stem Cell Biology quantify the transition rates between the different states. R-loop formation is 4 zipper-like process in which the crRNA sequentially displaces the non-target Programme, Duke-NUS Medical School, Singapore, Singapore, Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and DNA strand. Based on this we developed a model containing only few basic pa- 5 rameters that accurately describes observed R-loop dynamics, such as the life- Research), Biopolis, Singapore, Department of Cell Biology, Duke times of R-loops of different length, the impact of mismatches in the seed University Medical Center, Durham, NC, USA. region and the supercoil dependence of R-loop formation. Most importantly Kinesin motors are potential targets for anti-cancer drug therapy because of the model describes how the impact of two mismatches adds up in a nontrivial their essential roles in mitosis. Kinesin-14 KIFC1 is an unusually favorable manner. This provides a fundamental basis to describe the behavior of drug target, because it is required for viable division in many cancer cell lines, CRISPR-Cas surveillance/effector complexes on targets with arbitrary but it is nonessential in almost all human cells. The most potent of the KIFC1 sequence. inhibitors that have been reported to date is AZ82, which has been shown to inhibit nucleotide binding and release from KIFC1-MTs, although little is 979-Plat known about the binding site of AZ82 in KIFC1. Structural studies have iden- Direct Observation of Type IA Topoisomerase Gate Opening tified two inhibitor binding sites in the kinesin motors, but kinesin-5 is the only Maria Mills1, Yuk-Ching Tse-Dinh2, Keir C. Neuman1. kinesin motor crystallized to date bound to an inhibitor. Here we report three 1Laboratory of Molecular Biophysics, NHLBI, NIH, Bethesda, MD, USA, new crystal structures of kinesin-14 KIFC1 and two closely related homo- 2Florida International University, Miami, FL, USA. logues, KIFC3 and Ncd, and address the question of whether the AZ82 inhibitor Type IA topoisomerases cleave single-stranded DNA and relieve torsional strain binds to a previously identified kinesin inhibitor binding site in KIFC1. We in discrete steps corresponding to the passage of the intact DNA strand through further show that AZ82 has no observable inhibitory effects on Ncd-MTs

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ATPase activity under our assay conditions, paralleling the lack of inhibition how kinesin-8 depolymerizes microtubules is unclear. Here, we tracked the reported previously for KIFC3 and kinesins other than KIFC1. Structural anal- microtubule end-binding activity of yeast kinesin-8s, Kip3, with high precision ysis of our new crystal structures shows that one of the previously identified in- under varying loads using optical tweezers. We observed that single Kip3 motors hibitor binding sites does not favor AZ82 binding in KIFC1 because of stayed dynamically attached to the microtubule end for up to 200 s, making properties that include its size and loop conformation, but the other site is suf- several 8-nm forward and backward steps. Interestingly, while small hindering ficiently large in all three kinesin-14 motors for AZ82 to bind. Structural fea- loads led to backward slipping away from the end, assisting loads suppress tures of AZ82 binding to this site in KIFC1 could explain its ATP such diffusive stepping and decrease the end-residence time of Kip3 exponen- competitive effects. The specificity and stability of drug binding are most likely tially. We hypothesize that Kip3 is searching for the weakest bound tubulin dimer achieved by AZ82 interactions with residues of the KIFC1 binding site that are and that force either applied by consecutive motors or external load accelerates unique compared to the other two kinesin-14 motors. tubulin removal. Understanding the mechanics of kinesin-8’s end activity will provide important insight for cell division with implications for cancer research. 982-Plat Native Kinesin-1 does not Preferentially Bind to GTP-Rich Microtubules 985-Plat in vitro Hook1 Induces Superprocessive Motility of Dynein and is Required for Qiaochu Li1, Stephen J. King2, Jing Xu1. 1 2 Trafficking of Signaling Endosomes in Neurons Physics, University of California, Merced, Merced, CA, USA, University of Mara A. Olenick1, Erika L.F. Holzbaur2. Central Florida, Orlando, FL, USA. 1Biochemistry and Molecular Biophysics Graduate Group, University of Molecular motors such as kinesin-1 work in small teams to actively shuttle Pennsylvania, Philadelphia, PA, USA, 2Dept. of Physiology, University of cargos in cells, for example in polarized transport in axons. Here, we exam- Pennsylvania, Philadelphia, PA, USA. ined the potential regulatory role of the nucleotide state of tubulin on the run In neurons, transport of signaling endosomes by cytoplasmic dynein is vital for length of cargos carried by multiple kinesin motors, using an optical trapping- neuronal development and survival, yet it remains unclear how dynein is spatially based in vitro assay. Based on a previous report that kinesin binds preferen- and temporally regulated in this context. We hypothesize that adaptor proteins tially to GTP-tubulin-rich microtubules, we anticipated that multiple-kinesin mediate precise cargo transport by differentially regulating dynein associated cargos would run substantially greater distances along GMPCPP microtubules with distinct cargos. To assess this model, we focused on Hook proteins, Hook1 than along GDP microtubules. Surprisingly, we did not uncover any signifi- and Hook3. Using single molecule TIRF assays and inducible dimerization assays, cant differences in run length between microtubule types. A combination of we found that Hook proteins act as dynein adaptors that induce superprocessive single-molecule experiments, comparison with previous theory, and classic motility. Hook proteins induced both longer run lengths and higher velocities microtubule affinity pulldown assays revealed that native kinesin-1 does not than the previously characterized dynein activator, BICD2. These results indicate bind preferentially to GTP-tubulin-rich microtubules. The apparent discrep- that dynein adaptors can differentially regulate dynein to allow for organelle- ancy between our observations and the previous report likely reflects differ- specific tuning of the motor. Next, we used live cell imaging to investigate the ences in post-translational modifications between the native motors used role of Hook proteins in dynein-driven transport in primary neurons. We found here and the recombinant motors examined previously. Future investigations that Hook1 co-migrates with a subpopulation of Rab5-endosomes, which display will help shed light on the interplay between the motor’s post-translational retrograde-directed motility and faster velocities than Hook1-negative endosomes. modification and the microtubule’s nucleotide-binding state for transport Knockdown of Hook1 produced modest effects on the overall motility of Rab5- regulation in vivo. endosomes, but significantly reduced the motility of BDNF-signaling endosomes. 983-Plat Using microfluidic chambers with BDNF-Quantum dots to model signaling endo- Differential Phosphorylation in the Motor Domain of Mitotic Kinesin-5 some transport from the axon terminal, we found Hook1 knockdown significantly Cin8 Regulates its Functions In Vivo decreased in the frequency of BDNF-endosomal motility along the mid-axon sug- Nurit Siegler1, Alina Goldstein1, Ofer Shapira1, Darya Goldman1, gesting that Hook1 may affect both the generation and motility of signaling endo- Ervin Valk2, Mardo Ko˜ivom€agi2, Mart Loog2, Larisa Gheber1. somes. This inhibition of motility was specific to the depletion of Hook1 but not 1Chemistry Department, and Ilse Katz Institute for Nanoscale Science and Hook3 indicating signaling endosomes are a distinct Hook1-dynein cargo. Technology, Ben Gurion University of the Negev, Be’er Sheba, Israel, Together, this work suggests that Hook1 acts as a specific dynein adaptor for 2Institute of Technology, University of Tartu, Tartu, Estonia. BDNF uptake and signaling endosome transport from the distal axon back to the Cin8, a kinesin-5 mitotic motor protein in S. cerevisiae, moves directionally soma, and supports the model that differential association with cargo-specific along microtubules (MTs) by hydrolyzing ATP. Kinesin-5 mitotic motors adaptors efficiently regulates dynein function in neurons. exhibit tetrameric structure that enables them to crosslink two MTs, thus per- forming their essential roles in mitotic spindle formation and elongation. It 986-Plat was previously demonstrated in our lab, that Cin8 is differentially phosphory- Microtubule Associated Proteins and Bundling Regulate Kinesin and Dynein lated during anaphase in one or more of the three cyclin-dependent kinase 1 Processivity and Force Generation to Direct Intracellular Trafficking (Cdk1) sites located in its catalytic domain: S277, T285 and S493. It was Abdullah R. Chaudhary1, Linda Balabanian1, Florian Berger2, also demonstrated that this phosphorylation affects Cin8 localization to the Christopher L. Berger3, Adam G. Hendricks1. 1 2 mitotic spindle, and mitotic spindle elongation. Bioengineering, McGill University, Montreal, QC, Canada, Laboratory of Here we studied the effect of each phosphorylation site on the regulation of Sensory Neuroscience, The Rockefeller University, New York, NY, USA, 3 Cin8 activity during mitosis. For this purpose, we quantitatively characterized Department of Molecular Physiology and Biophysics, University of the effect of phosphorylation of Cin8 at the three Cdk1 sites using in vitro phos- Vermont, Burlington, VT, USA. phorylation, in vivo fluorescence intensity distribution analysis, viability test The motor proteins kinesin and dynein transport organelles, mRNA, proteins, and and immunostaining. We found that in vivo the Cdk1 sites differ in the timing signaling molecules along the microtubule cytoskeleton. Motor proteins are regu- of their phosphorylation, and the strength of their effect. Thus they exhibit dif- lated to direct cargoes to specific locations in the cell. One primary pathway of ferentialregulation of Cin8 activity during anaphase, with the S277 site being regulation is through the polymer tracks that motors move along, where tubulin most important in regulating Cin8 localization to the mitotic spindle, while post-translational modifications, microtubule-associated proteins (MAPs), and the S493 site affecting the rate of spindle elongation. the organization of the polymer network alter motor motility. To systematically examine the factors that contribute to cytoskeleton-based regulation of motor pro- 984-Plat tein motility in a simple in vitro system, we extracted intact, native microtubule Kinesin-8 Depolymerizes Microtubules with a Force-Dependent Mechanism networks from cells and tracked the motility of single fluorescently-labeled motor Anita Jannasch, Michael Bugiel, Erik Sch€affer. proteins using TIRF microscopy. We find that microtubule acetylation does not ZMBP, Cellular Nanoscience, University of Tuebingen, Tuebingen, directly alter kinesin-1 motility. However, acetylated microtubules are often Germany. bundled, and kinesin-1 association rates and run lengths are enhanced along Microtubules are highly dynamic cellular filaments with dramatic structural rear- bundled microtubules. We next examined motility in the presence of the neuronal rangements and length changes during the cell cycle. Therefore, accurate control MAP tau that stabilizes axonal microtubules and crosslinks them into bundles. of their length is key to many essential processes like cell division. Apart from the Dysregulation of tau leads to a range of neurodegenerative diseases known as intrinsic dynamic instability of microtubules, one destabilizing, active factor are tauopathies including Alzheimer’s disease (AD). To investigate the role of tau motor proteins of the kinesin-8 family. They can depolymerize microtubules in a in regulating bidirectional transport, we isolated phagosomes driven by collective and length-dependent manner by interacting with their ends. However, kinesin-1, kinesin-2, and dynein and reconstituted their motility along

BPJ 8590_8601 196a Monday, February 19, 2018 microtubules. We find that tau biases bidirectional motility by tuning the balance apses: the parallel fiber - Purkinje cell synapse in the cerebellar cortex, the ca- of forces exerted by teams of kinesin and dynein motors, such that the processivity lyx of Held in the auditory brainstem, and the CA3 - CA1 synapse in the and forces exerted by opposing dynein motors are enhanced. Comparison of tra- hippocampus. I will introduce a conditional knock-in approach to selectively jectories to a mathematical model suggests that tau biases bidirectional motility mutate all PKC phosphorylation sites in the prominent PKC phosphorylation towards the minus end by increasing the unbinding rate of kinesin-1. Taken substrate Munc18-1, an SM family protein that is essential for release. Finally, together, these results demonstrate that the microtubule cytoskeleton directs intra- I will describe initial efforts using this new mouse model to delineate the mo- cellular trafficking through its network organization and associated proteins. lecular events underlying enhanced neurotransmission during PTP.

987-Plat 990-Symp The Interplay of Diffusion, Motor-Driven Walks, and Tethering in Intra- Trans-Synaptic Protein Organization Aligning Vesicle Fusion with Receptors cellular Transport Thomas Blanpied. Saurabh Mogre, Elena Koslover. Physiology, University of Maryland School of Medicine, Baltimore, MD, University of California, San Diego, La Jolla, CA, USA. USA. Intracellular components move around in the cytoplasm via a combination of Synapse strength and reliability are dependent on nanometer-scale architecture passive diffusion and motor-driven transport through direct coupling to molec- within each of the two connected cells: in the active zone where the Ca channel- ular motors orhitchhiking’ on other motile organelles. The balance between vesicle relationship is tightly controlled, and in the postsynaptic density where these modes of transport is regulated by a variety of adaptors, linkers, and teth- AMPA and NMDA receptors are clustered within small subdomains. Several ering proteins that modulate the interaction of organelles with motors and the key questions remain open, however, including the mechanisms establishing microtubule tracks on which they move. We utilize analytical techniques and these functional molecular assemblies, how their organization is maintained af- numerical simulations to investigate the efficiency of these transport modes, ter synaptogenesis, and which aspects of nanostructure contribute to plasticity focusing on narrow tubular projections such as fungal hyphae and neuronal of synaptic function. To delineate these nanoscale structures and test their axons. Our model investigates the effects of physical transport parameters on impact to receptor activation and synaptic signaling will require live-cell anal- achieving cellular objectives such as uniform dispersion of particles or delivery ysis. To pursue this, we are developing functional approaches for concurrent of organelles to specific targets. We characterize the length and time scales over use with single-molecule localization microscopy. We find that RIM and other which different transport modes dominate organelle motion, and show that both key proteins regulating vesicle fusion are mutually co-enriched within diffusion and bidirectional motor-driven transport are needed for efficient nanometer-scale subregions of the AZ. This organization impacts synapse func- target capture and exploration of intracellular space in biologically relevant re- tion in many ways. First, by mapping sites of single-vesicle fusion events gimes. In certain cellular systems, organelles tether to microtubules when not within individual AZs, we revealed that evoked fusion occurs in a confined sub- actively moving. Proximity to microtubule tracks while tethered increases region of the AZ where RIM density is highest. Second, the distributions of the rate of active transport while rendering the organelles immobile in the pas- RIM and receptors are tightly co-aligned across the synaptic cleft. Third, con- sive state. We quantify the competition between these effects and derive current PALM and GCaMP6f imaging in single synapses allows us to examine parameter-dependent conditions under which tethering enhances long range how postsynaptic nanostructure controls NMDA receptor activation. Finally, transport and improves the target capture time. These results shed light on within the PSD, single-molecule tracking and FRAP reveal that the pattern the optimization of transport machinery in various cells and provide experi- of receptor positions is controlled by a combination of receptor binding and mentally testable predictions for the effects of modulating transport through macromolecular crowding. Together, combining functional and super- physical mechanisms like tethering. resolution imaging reveals that the nanoarchitecture of the active zone directs vesicle fusion to occur preferentially at sites nearest postsynaptic receptor en- Symposium: Synaptic Vesicle Fusion and sembles. This provides a simple organizational principle of CNS synapses to maintain and control synaptic efficiency. Potentially both direct and indirect Retrieval trans-synaptic interactions may link dynamic regulation of presynaptic vesicle cycling and postsynaptic receptor trafficking. 988-Symp Molecular Mechanisms of Synaptic Vesicle Priming 991-Symp Axel Brunger. Visualizing Membrane Structural Remodeling during Fusion and Fission HHMI/Molecular and Cellular Physiology, Stanford University School of in Live Cells Medicine, Stanford, CA, USA. Ling-Gang Wu. The central nervous system relies on electrical signals traveling along neurons NINDS/NIH, Bethesda, MD, USA. at high speeds. Signals are also transmitted between two neurons, or from a Fusion and fission mediate many biological processes, such as exocytosis, endo- neuron to a muscle fiber through synaptic junctions. Synaptic transmission cytosis, intracellular trafficking, and viral entry. Despite intense studies, mem- relies on the release of neurotransmitter molecules into the synaptic cleft. brane structural remodeling mediating fusion and fission had not been real- This release in turn depends on a process called membrane fusion to ensure time observed, and is thus poorly understood. With super-resolution STED that the neurotransmitter molecules that are contained in synaptic vesicles microscopy, we observed the hemi-fused U-shaped structure for the first time are released into the synaptic cleft as quickly as possible. Membrane fusion in live cells, the neuroendocrine chromaffin cells and pancreatic b-cells. This is an important process in many areas of biology, including intracellular trans- structure was generated from fusion pore opening or closure (fission) at the port and hormone release, but it occurs much faster (< 1 millisecond) for syn- plasma membrane. Unexpectedly, its transition to full fusion or fission was deter- aptic vesicle fusion than for these other processes. Moreover, it is precisely mined by competition between fusion and calcium/dynamin-dependent fission calcium regulated. Recent structural and biophysical studies of priming and cal- mechanisms, and was surprisingly slow (seconds to tens of seconds) in a signif- cium triggering of synaptic vesicle fusion will be presented. icant fraction of the events. These results provide key missing evidence over the past three decades proving the hemi-fusion and hemi-fission hypothesis in live 989-Symp cells, and reveal the hemi-fused intermediate as a key structure controlling Translating Neuronal Activity at the Synapse: The Role of the Protein fusion/fission, as fusion and fission mechanisms compete to determine its tran- Kinase C Cascade in Short-Term Plasticity sition to fusion or fission. Passing through hemi-fusion, vesicle fusion at the Diasynou Fioravante. plasma membrane generates an U-shape membrane profile, which may enlarge University of California, Davis, Davis, CA, USA. or shrink while maintaining vesicular membrane proteins. Closure of fusion- Neurons dynamically regulate neurotransmitter release through many pro- generated U-profiles, which produces various sizes of vesicles, is a major mech- cesses known collectively as synaptic plasticity. Post-tetanic potentiation anism mediating rapid and slow endocytosis. Strong calcium influx triggers (PTP) is a widespread form of synaptic plasticity that lasts for tens of seconds, dynamin-mediated closure, whereas weak calcium influx facilitates the merging and which may have important computational roles and contribute to short- of U-profiles with the plasma membrane via shrinking rather than full-collapse as term memory. According to a leading mechanism, the high-frequency stimula- generally believed. U-profile shrinking and merging are mediated by strong tion that is necessary to induce PTP increases presynaptic calcium, which ac- membrane tension provided by dynamic assembly of filamentous actin, tivates calcium sensors to regulate the amplitude and time-course of involving ATP hydrolysis, N-WASP and formin. In summary, we have visual- increased release. At the ‘‘Synaptic Vesicle Fusion and Retrieval’’ symposium, ized a series of membrane structural changes that mediate vesicle fusion, merg- I will discuss our investigations into the role of the calcium-sensitive protein ing with the plasma have membrane, fission, their crucial function in exo- and kinase C (PKC) as an essential regulator of PTP at 3 mammalian excitatory syn- endocytosis, and a part of their underlying molecular mechanisms.

BPJ 8590_8601 Monday, February 19, 2018 197a

degree of interaction was shown to be correlated with the SANC spontaneous Symposium: Cardiac Contractility þ beating rate. Specifically, Ca2 has been shown to behave—directly and indi- 992-Symp rectly through activation of post-translational modification signaling—as a Elucidating the Molecular Mechanisms for Activation of the L-Type Cal- node signaling mechanism that interacts with both membranal and internal molecules. Fresh and cultured rabbit SANCs and a coupled-clock computa- cium Channel in the Fight or Flight Response 2þ Livia C. Hool. tional model were used here to ascertain how Ca , directly and indirectly M311 Anatomy, Physiology and Human Biology, The University of Western through PKA signaling, determines and is determined by SANC bioelectrical, biophysical and bioenergetic SANC function. Computational modeling pre- Australia, Perth, Australia. 2þ In cardiac muscle sympathetic stimulation leads to an increase in heart rate and dicts that cAMP and Ca activated PKA signaling controls the SANC sponta- contraction as a result of increased calcium influx through the L-type Ca2þ neous beating rate. Specifically, the interaction of PKA with phospholamban molecules has been shown to be the dominant factor that affects the sponta- channel (Cav1.2). This involves activation of the b-adrenergic receptor and an increase in cAMP dependent protein kinase A (PKA). It is unknown whether neous beating rate in the entire physiological range. Moreover, the level of PKA activity determines the amount of Ca2þ released from the SR and the direct modification of Cav1.2 is responsible for altered channel function and as 2þ a result the mechanism has remained controversial. We searched for the func- ATP production rate. However, while reducing the amount of Ca release tionally relevant site for PKA phosphorylation on the human cardiac L-type by interfering with any coupled-clock mechanism affects PKA signaling, Ca2þ channel pore forming a1 subunit using a novel approach. Functional only a reduction in the ATP level below a certain threshold leads to a deterio- ration in PKA dynamics. Experimental results show differences in the spatial studies were performed on proteoliposomes containing human Cav1.2 channel protein and phosphatidylcholine lipid only. Using this approach channel func- and temporal kinetics of PKA signaling for different regimes in the cell tion is studied in isolation from the auxiliary subunits of the channel complex at different steady state levels of PKA. Thus, feedback between bioenergetic and the regulatory pathways present in the cell. We confirm a direct effect of and biophysical mechanisms affects and is affected by pacemaker function, and similar signaling cascades control pacemaker bioelectric, bio- PKA on Cav1.2 channel function. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal physical and bioenergetic properties. isoforms and a protein kinase A anchoring protein (AKAP) was not required. Mutation of Ser1928 and potential PKA substrate serines in the cytoplasmic Platform: Protein-Lipid Interactions II Repeat I-II and II-III linker regions did not attenuate the effect nor truncation of the C terminus. We find that direct phosphorylation of a single serine that lies 996-Plat Molecular Recognition at the Membrane Interface: Protein-Membrane in close proximity to the Repeat IV S6 region on Cav1.2 alters posttranslational folding and is necessary and sufficient for an increase in function responsible Electrostatic Interactions Modulate the Biological Function of Anti-HIV for the ‘‘Fight or Flight’’ response. Antibodies Jose L. Nieva1, Edurne Rujas1, Sara Insausti1, Daniel P. Leaman2, 1 1 2 3 993-Symp Beatriz Apellaniz , Johana Torralba , Lei Zhang , Jose M. Caaveiro , 2 Is the Heart Druggable? Development of a Calcium Sensitizer Michael B. Zwick . 1University of the Basque Country, Bilbao, Spain, 2The Scripps Research Brian Sykes. 3 University of Alberta, Canada, Edmonton, AB, Canada. Institute, La Jolla, CA, USA, Kyushu University, Fukuoka, Japan. Cardiovascular Disease is a major killer of men and women worldwide, but few Binding of antibodies 4E10 or 10E8 to the helical juncture that connects the drugs are available to treat cardiac disease or arrest. Drugs that mobilize calcium membrane-proximal external region (MPER) and the transmembrane domain are useful, but not without risk. The development of a calcium sensitizer that (TMD) of the Envelope glycoprotein (Env) gp41 subunit, translates into one directly modulates the response of the sarcomere, but does not perturb calcium of the broadest neutralizing activities against HIV-1 described to date. Accord- homeostasis, could be an important approach for treatment of HF. Drugging the ingly, these antibodies and their mechanisms of action provide timely models in heart is difficult since we want to make small changes, not to stop the heart the development of effective vaccination and immunotherapeutic strategies. completely, and need to not affect skeletal or smooth muscle. The question in Both, 4E10 and 10E8, exhibit unusual adaptations to attain specific, high- this presentation is whether the thin filament of striated muscle is an attractive affinity binding to the helical MPER epitope anchored into the viral membrane target - increasing calcium sensitivity by direct modification of the calcium bind- interface. We report here that manipulation of the paratope surface through ing properties of Troponin-C and the TnC:TnI interface which propagates the mutagenesis can be used to adjust the electrostatic interactions of 4E10 and calcium signal. Targeting of this protein-protein interface has been difficult 10E8 with the membrane, and modulate their biological activity (neutralization and the hydrophobic cleft - helical target motif is common. We have developed potency and polyreactivity). a cardiac TnC-TnI chimera as a target for drug screening, and determined it’s 997-Plat structure in the presence of 3-methyl-diphenylamine. The potential of covalent Cholesterol and Neurosteroids Bind Common Sites but Assume Different drugs has been demonstrated and targeting the unique C84 of cardiac troponin-C Orientations in a Pentameric Ligand Gated Ion Channel could be a good way to target the heart. The drug Levosimendan has been the sub- Melissa M. Budelier, Wayland W.L. Cheng, John R. Bracamontes, ject of a number of clinical trials is thought to act at least in part as a calcium Zi-Wei Chen, Krishnan Kathiresan, Laurel Mydock-McGrane, sensitizer. We have recently shown that Levosimendan forms a thioimidate Douglas F. Covey, Alex S. Evers. bond with C84 in vitro and have also demonstrated the efficacy of covalent Washington University in St. Louis, St. Louis, MO, USA. bond formation in situ using rat trabeculae. The results suggest that a reversible Cholesterol is an essential component of cell membranes. In addition to acting covalent allosteric compound could be a good way to drug the heart. We present a as a solvent for intrinsic membrane proteins (IMPs), it has been shown to bind more complete characterization of the in situ action of Levosimendan and other specifically within the extracellular and transmembrane domains (TMDs) of compounds which act via a reversible covalent mechanism. some IMPs. We have developed methods, based on photoaffinity labeling 994-Symp and mass spectrometry, to identify cholesterol binding sites in IMPs and eluci- Spectroscopic Probes of Cardiac Contractility and Therapeutic Discovery date the orientation of cholesterol within these sites. In this study, we used two David Dale Thomas. cholesterol analogue photolabeling reagents, with the photoreactive moiety on Biochem/Molec Biol/Biophys, University of Minnesota, Minneapolis, MN, opposite ends of the sterol, to map cholesterol binding in Gloeobacter ligand- USA. gated ion channel (GLIC). We then compared this binding to that of the choles- terol metabolite, allopregnanolone, an endogenous neurosteroid and allosteric 995-Symp modulator of pentameric ligand gated ion channels (pLGICs). In GLIC, we Post-translational Modification Signaling and Bioelectrical, Biophysical identified multiple labeled peptides that mapped two cholesterol binding sites: and Bioenergetic Pacemaker Function one site between the intracellular side of TM3 and TM1 of adjacent subunits Yael Yaniv. and one intrasubunit site at the extracellular side of the TMDs between TM1 Technion, Haifa, Israel. and TM4. Neurosteroids also bind to both of these sites. In the intersubunit Sinoatrial node cells (SANCs) that reside in the heart’s primary pacemaker con- site, cholesterol is oriented such that the 3-OH group points extracellular, trol the heart rate and rhythm. Experimental and computational evidence show whereas in the intrasubunit site, the 3-OH group points intracellular. This is that SANC function is controlled both by mechanisms on the cell membrane in contrast to neurosteroids, which photolabel with the opposite orientation. and by internal mechanisms of the sarcoplasmic reticulum (SR) and mitochon- We also found that cholesterol increases, whereas allopregnanolone decreases dria. These mechanisms on the cell membrane and in the cell interact, and the the thermal stability of GLIC. These data indicate that cholesterol and

BPJ 8590_8601 198a Monday, February 19, 2018 neurosteroids can bind to common hydrophobic pockets in pLGICs, but that results from a single protein tracking study of fluorescently labeled CPR and their orientation and function are determined by the molecular interactions of cytochrome P450 2C9 (CYP2C9) molecules in which stochastic analysis was their unique functional groups. used to determine the dissociation constants of CPR/CYP2C9 complexes in a lipid bilayer membrane for the first time. Single protein trajectories demon- 998-Plat strate the transient nature of these CPR-CYP2C9 interactions and the measured Protein-Lipid Interactions on the HIV Membrane Defined by EPR Spec- ox Kd’s are highly dependent on the redox state of CPR. It is shown that CPR / troscopy CYP2C9 complexes have a much higher dissociation constant than CPR2-/ 1 1 1 2 2 Likai Song , Zahra Hayati , Mengtian Liu , Mikyung Kim , Ellis Reinherz . CYP2C9 or CPR4-/CYP2C9 complexes and a model is presented to account 1National High Magnetic Field Laboratory, Florida State University, 2 for these results. An Arrhenius analysis of diffusion constants was also carried Tallahassee, FL, USA, Dana-Farber Cancer Institute, Harvard Medical out, demonstrating that the reduced forms of CPR and CYP2C9 interact differ- School, Boston, MA, USA. ently with the biomimetic ER and may, in addition to protein conformational HIV enters human T cells through the fusion of viral and host-cell membranes. changes, contribute to the observed NADPH-dependent shift in Kd. This fusion process is mediated by a surface protein, gp41, and the platform provided by the cholesterol-rich viral membrane. The membrane-bound region 1001-Plat of gp41 plays critical roles in this fusion process and is a major target of anti- Caveolin-Assisted Sphingolipid Transport to the Plasma Membrane gp41 antibodies and vaccine design. Here, EPR and spin-labeling techniques Joanna Podkalicka1, Manuela Dezi1, John Manzi1, Aurelie Di Cicco1, were used to dissect the interactions between the viral membrane and the Daniel Levy1, Christophe Lamaze2, Patricia Bassereau1. membrane-bound region of gp41, including the membrane proximal ectodo- 1PhysicoChimie Curie (UMR CNRS 168), Institut Curie, Paris, France, main region (MPER) and the transmembrane domain (TM). The analyses re- 2Chemical Biology of Membranes and Therapeutic Delivery (INSERM vealed a helix-hinge-helix structure of the MPER sitting on the membrane U1143 - CNRS UMR 3666), Institut Curie, Paris, France. headgroup region in a manner impacted by para-hinge and other residues. Lipid and protein sorting are crucial processes that maintain unique biophysical The MPER exerts its fusogenic activity by perturbing membrane properties and biological properties of different organelles. Nevertheless, little is known and by inducing significant lipid order and membrane permeability changes. about mechanisms behind lipid sorting. Sphingomyelin (SM) is synthesized The MPER-induced membrane property changes are modulated by cholesterol at the trans Golgi network (TGN) and transported to the plasma membrane content and the TM. In addition, anti-gp41 neutralizing antibodies disrupt (PM) via an uncharacterized pathway. SM enrichment in transport carriers MPER-lipid interactions by altering MPER conformation on the membrane cannot be explained by a curvature-based mechanism of lipid sorting due to and by abolishing the membrane-perturbing activity of the MPER. These find- its property to form stiff membranes. A mechanism of protein-mediated lipid ings suggest that the membrane-bound region of gp41 facilitates HIV infection sorting has been proposed, taking advantage of proteins’ affinity for curved through its interaction with the cholesterol-rich viral membrane, and this inter- membranes and specific lipids. One of the proteins enriched in the same secre- action is disrupted by anti-gp41 neutralizing antibodies. tory pathway is caveolin, which shuttles between the Golgi and the PM where it 999-Plat forms cup-shape, SM-cholesterol-enriched domains called caveolae. The as- Structural Plasticity of the Pivotal Cytochrome C/Cardiolipin Complex in sembly process is initiated by the export of caveolin-enriched vesicles from Mitochondrial Apoptosis the Golgi but little is known about this first step except from a critical role Mingyue Li1, Abhishek Mandal1, Maria DeLucia1, Jinwoo Ahn1, of lipids in caveolin oligomerization. The goal of this project is to determine Vladimir A. Tyurin1, Valerian E. Kagan2, Patrick C.A. van der Wel1. the role of caveolin in SM trafficking from the TGN to the PM and to decipher 1Department of Structural Biology, University of Pittsburgh School of the molecular mechanisms behind caveolin assembly using a bottom-up Medicine, Pittsburgh, PA, USA, 2Departments of Environmental and approach. We purified caveolin 1 (Cav1) and reconstituted into small unilamel- Occupational Health, Pharmacology, and Chemical Biology, University of lar vesicles, what was shown by density centrifugation and visualisation of Pittsburgh, Pittsburgh, PA, USA. proteo-liposomes by cryo-electron microscopy. Additionally, we analysed The interaction of cytochrome c (cytc) and cardiolipin (CL), a mitochondrial Cav1 reconstitution in presence of cavtratin - peptide disrupting Cav1 oligo- specific anionic lipid, has been identified as critical to initialize mitochondrial merization. Without cavtratin Cav1 creates multilamellar, undulated structures, apoptosis. To fulfill this function, cytc and CL form a complex with significant while in its presence unstructured aggregates are observed. In parallel our in CL peroxidase activity. Understanding the structural specificity of the cytc/CL cellulo studies showed differential SM localization in MLEC Cav1 KO cells, complex is crucial to drug development targeting early apoptosis in cancer and indicating that Cav1 play a role in SM trafficking. Our ongoing studies aim cardiovascular diseases. In our studies, we constitute an in vitro functional to reconstitute Cav1 into giant unilamellar vesicles, where the recruitment of assay designed to mimic cytc and CL in the proapoptotic stage and are able Cav1 and SM to curved membranes will be investigated, complemented by to quantify oxygenated molecular species of CL by lipidomics analysis using quantitative measurements of membrane mechanics. mass spectroscopy. Moreover, the structure and dynamics of peroxidase- active cytc bound to CL are investigated by both through-space and through- 1002-Plat bond correlation experiments of magic angle spinning (MAS) nuclear magnetic Gramicidin Increases Lipid Flip-Flop in Symmetric and Asymmetric Lipid resonance (NMR) spectroscopy. Residues showing strong structural perturba- Vesicles 1,2 3 4 tions upon binding to CL are identified. Remarkably, cytc in complex with Milka Doktorova , Frederick A. Heberle , Drew Marquardt , 2 2 2 3 CL is associated with elaborate dynamics that indicate structural plasticity. Radda Rusinova , Lea Sanford , Thasin Peyear , John Katsaras , Gerald Feigenson5, Olaf S. Andersen2. A specific region of cytc is found to exhibit both significant structural and 1 dynamics perturbations. In general, our results support a model of interaction Tri-Institutional PhD Program in Computational Biology and Medicine, Weill Cornell Medical College, New York, NY, USA, 2Physiology and consisting of a clustering of CL molecules while cytc retains much of its 3 native fold and is bound to the membrane surface. The cytc dynamic changes Biophysics, Weill Cornell Medical College, New York, NY, USA, Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, TN, and structural plasticity may have a strong implication for its proapoptotic 4 activity. USA, Chemistry and Biochemistry, University of Windsor, Windsor, ON, Canada, 5Molecular Biology and Genetics, Cornell University, Ithaca, NY, 1000-Plat USA. Cytochrome P450 / Cytochrome P450 Reductase Complex Formation Unlike transmembrane proteins, phospholipids can reorient within the mem- Depends on NADPH: A Single Protein Tracking Study brane by moving from one leaflet to the other. Spontaneous lipid translocation James A. Brozik, Carlo Barnaba, Evan Taylor. tends to be very slow, however, and cells facilitate the process with enzymes Chemistry, Washington State University, Pullman, WA, USA. that catalyze the transmembrane lipid movement and thereby regulate the trans- Cytochrome P450-Reductase (CPR) is a versatile NADPH-dependent electron bilayer lipid distribution. It has been proposed that non-enzymatic membrane- donor located in the cytoplasmic side of the endoplasmic reticulum. It is an spanning proteins may also accelerate lipid flip-flop in a non-specific manner electron transferase that is able to deliver electrons to a variety of by introducing bilayer defects, thus easing the translocation of polar lipid membrane-bound oxidative partners, including the drug-metabolizing enzymes head groups across the bilayer by allowing them to move along the protein- of the cytochrome P450s (P450). CPR is also stoichiometrically limited membrane interface [BBA, 1978, 509, 537; Biophys J, 1981, 33, 373; Biochem, compared to its oxidative counterparts and hypotheses have arisen about 1994, 33, 6721; Biochem, 2001, 40, 10500]. We examined this possibility using possible models that can overcome the stochiometric imbalance, including qua- deuterated phospholipids and the gramicidin channels, which have well- ternary organization of P450 and diffusion-limited models. Described here are defined structure and function that make them ideal candidates for probing

BPJ 8590_8601 Monday, February 19, 2018 199a protein-membrane interactions. Making use of recently developed protocol and 1005-Plat assays for the preparation and characterization of asymmetric lipid vesicles, we Towards Improved Biophysical Calculations to Identify Disease-Causing studied compositionally and isotopically asymmetric proteoliposomes contain- Mutations ing gramicidin. Protein incorporation, conformation and function were exam- Kresten Lindorff-Larsen1, Amelie Stein1, Kaare Teilum1, ined with small-angle x-ray scattering, circular dichroism and a stopped-flow Alex Toftgaard Nielsen2, Rasmus Hartmann-Petersen1. spectrofluorometric assay. Differential scanning calorimetry revealed the effect 1Department of Biology, University of Copenhagen, Copenhagen, Denmark, of the protein on the melting transition temperatures of the two bilayer leaflets, 2Novo Nordisk Foundation Center for Biosustainability, Technical which over time merged into a single peak indicating lipid scrambling. Using University of Denmark, Lyngby, Denmark. proton NMR, we monitored the transbilayer lipid distribution in both symmet- To leverage fully the possibilities of genome sequencing in diagnosis, we ric POPC and asymmetric POPC/DMPC vesicles with and without the protein. need accurate methods to assess the pathogenicity of mutations. Despite Our results show that gramicidin increases lipid flip-flop in a concentration- the increasing number of human genomes being sequenced, about half of dependent manner. the variants found in each newly sequenced genome are novel. Thus, we sug- gest that data-driven methods should be complemented by hypothesis-driven 1003-Plat approaches. In particular, biophysical calculations are applicable to a wide Mapping the Interactions of Alpha-Synuclein to Lipid Membranes in the range of proteins for which 3D-structures or models are available. We Physiological Limit have recently demonstrated that in silico saturation mutagenesis, biophysical Peter J. Chung1, Hyeondo Hwang2, Alessandra Leong2, Erin J. Adams3, modelling and covariation-based calculations can predict the metabolic sta- Ka Yee C. Lee1. bility and thus functional levels of protein in cells (Nielsen, et al., PLoS Ge- 1Department of Chemistry and the James Franck Institute, University of netics 2017). Specifically, we showed that missense variants in MSH2, Chicago, Chicago, IL, USA, 2Department of Chemistry, University of known to cause hereditary cancer predisposition, lead to folding defects Chicago, Chicago, IL, USA, 3Biochemistry and Molecular Biology, and degradation. These are accurately identified by our biophysical calcula- University of Chicago, Chicago, IL, USA. tions and methods that examine sequence conservation and co-evolution, The discovery that alpha-synuclein, an intrinsically disordered protein, was outperforming traditional predictors. To assess the predictive power of bio- linked to a multitude of neurodegenerative diseases (‘‘synucleinopathies’’) physical and co-variation modelling more systematically, we have extended prompted intense efforts to understand the function of alpha-synuclein. While these analyses to a wider spectrum of diseases, from metabolic disorders, to many hypotheses centrally involve the ability of alpha-synuclein to bind to cancer predisposition and diseases of the nervous system. Our findings show highly-curved lipid membranes, it is difficult to reconcile the diversity of that both approaches correctly identify many disease-causing missense mu- sub-neuronal organelles (and corresponding lipid membranes) with which tations across many different proteins. An additional advantage of biophys- alpha-synuclein is purported to interact. To that end, we have pioneered a ical stability calculations is that they help identify the molecular mechanism, generalizable technique to encapsulate uniform spherical-nanoparticles with loss of functional protein levels via degradation, leading also to putative av- a single membrane (with lipid composition of our choosing), creating an in vi- enues for developing treatments. At the same time, our calculations highlight tro probe with precise membrane curvature in a previously un-realizable bio- the need for developing and benchmarking improved predictors of protein logical limit (35-50 nm). Using isothermal titration calorimetry we report stability that are more broadly applicable—including to mutations that the binding affinities of alpha-synuclein variants as a function of increasing involve readjustments of the protein structure—and can deliver accurate re- membrane curvature and lipid composition. While previous measurements sults also from homology models. We have therefore also benchmarked ex- required an un-physiological high mol% of anionic lipids to recapitulate strong isting methods for these scenarios, and developed novel experimental alpha-synuclein binding, we demonstrate the complex interplay between lipid approaches to study protein stability at high throughput using deep muta- charge, lipid intrinsic curvature, and membrane curvature to tease out the tional scanning. biochemical and biophysical parameters that drive alpha-synuclein binding. These parameters will be correlated to states in the dynamic lipid composition of sub-neuronal organelles, providing the data necessary to develop hypotheses 1006-Plat on the molecular action and dysfunction of alpha-synuclein within the neuron. Mutant Phenotype Prediction and Protein Model Discrimination using Deep Sequencing Data Platform: Protein Folding, Stability, and Shruti Khare, Kritika Gupta, Arti Tripathi. Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India. Evolution Studies on fitness landscapes of proteins using single-site saturation mutagen- esis libraries aid in understanding the effect of mutations on protein stability 1004-Plat and function. Deep sequencing of a single-site saturation library is often em- Prediction of Changes in Protein Folding Stability Upon Single Residue ployed to exhaustively analyse mutational effects. We analysed a single-site Mutations saturation mutagenesis library of the 101 residue E. coli cytotoxin, CcdB. 1,2 3 2 Carlos A. Bueno , Davit A. Potoyan , Ryan R. Cheng , Two mutational sensitivity parameters namely, MSseq and RankScore were Peter G. Wolynes2,4. derived from the mutational data by considering the phenotype information 1 Systems, Synthetic and Physical Biology, Rice University, Houston, TX, at multiple expression levels. The MSseq represents the functional effect for in- USA, 2Center for Theoretical Biological Physics, Rice University, Houston, dividual mutations whereas RankScore denotes overall mutational tolerance of TX, USA, 3Department of Chemistry, Iowa State University, Houston, IA, a residue position. 4 USA, Department of Chemistry, Rice University, Houston, TX, USA. The MSseq data was analysed to investigate the effects of substitutions on Point mutations that preserve the overall fold of a protein can change the sta- mutant phenotypes. Active site residues could be identified based on the bility. Designing enzymes that remain active at severe conditions, such as sequencing information alone. Among aromatic and charged categories, high temperature or low pH, is just one of the many possible applications of phenotype was observed to be differentially dependent on residue size. a method that can reliably predict stability changes upon mutation. Experi- Remarkably similar results were observed for two other small proteins, mental measurements of stability changes upon mutation have been collected PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). A in databases, and, in this work, we use two different models to predict these model was designed for predicting functional effects of mutations. The per- changes. The first method is Direct Coupling Analysis (DCA), which takes formance of the model compared favourably with other widely used compu- as input a multiple sequence alignment of proteins that share the same fold tational predictors, SNAP2 and SuSPect as well as to the ProTherm and yields a probability model for sequences in that family. This probability database. model can be used to obtain an Energy function based on the sequence. The sec- The RankScore information was employed in an attempt to solve the decoy ond method is the Associative Memory, Water Mediated, Structure and Energy discrimination problem. The property of RankScores to correlate well with res- Model, which is a coarse-grained protein folding model that has been used for idue depths in the protein was exploited for this purpose. Predicted secondary structure prediction and mechanistic studies of proteins. We show that both structure and model quality information was incorporated into the RankScore models can present good correlations with experimental data in several protein parameter to further enhance the performance. Our method selects the families. native-like models in 80% of the cases in a dataset comprising of models This work is partially supported by the Center for Theoretical Biological Phys- for 163 proteins. We also show that considering this additional information im- ics sponsored by the NSF (PHY-1427654 ) and by the National Institute of Gen- proves model discrimination by mutational sensitivity parameters other than eral Medical Sciences (R01-GM44557). RankScore.

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1007-Plat principle, proteostasis is affected in any disease that involves misfolded or The Physical Origins of Enzyme Evolution: Correlating the Active Site mutant proteins that do not fold with normal efficiencies; and hence overdraw Electric Fields of Antibiotic Resistance along Evolutionary Trajectories on the finite proteostasis resources of the cell. Tracking the proteostasis capac- in TEM b-Lactamases ity of cells has the generic potential to track neurodegenerative diseases with Samuel H. Schneider, Jacek A. Kozuch, Steven G. Boxer. diverse specific molecular origins. Thus building new approaches to identify Chemistry, Stanford University, Stanford, CA, USA. the efficiency of proteostasis is highly desired in order to track the risk of cells Unraveling the physical origins of enzyme evolution and catalysis remains a succumbing to damage from protein misfolding and aggregation. In this proj- long sought after goal in the biophysical community though progress has ect, a cysteine-reactive aggregation-induced emission (AIE) fluorogen was been often limited to indirect correlations between structure and function. To- built for measuring the unfolded cell load and proteostasis capacity in cells. wards that goal previous work in our lab has used the vibrational Stark effect This fluorogen, TPE-MI, can selectively detect solvent-exposed cysteines on (VSE) to quantify electric fields at enzyme active sites, finding a direct corre- intracellular proteins. Upon proteome folding stresses incurred by heat shock lation with the reaction’s free energy barrier for wild-type along with both con- or tunicamycin, TPE-MI reactivity increased in accordance with an unfolded ventional and subtle mutations using site-directed mutagenesis and Amber proteome. In cells expressing mutant aggregation-prone Huntington protein suppression.1 The VSE provides a quantitative framework for interpreting (associated with Huntington’s disease) there was a large increase in TPE-MI vibrational frequency shifts in proteins in terms of electric fields. In this fluorescence prior to inclusion formation, suggesting that the collapse of pro- work we address whether this framework can be utilized to probe the evolu- teostasis is one of the features for Huntington’s diseases and such an effect tionary trajectory of an enzyme towards new function. As an ideal model sys- emerges in cells prior to the formation of visible aggregates. The formation tem for exploring the physical origins of enzyme evolution, b-lactamases have of protein aggregates, which is a known common feature of neurodegenerative been extensively studied in terms of both natural and directed evolution from diseases, actually alleviates proteostasis stress. Our new approach to measure their primary antibiotic targets (e.g. penicillins) towards extended-spectrum unfolded load provides a new capacity to probe proteostasis capacity in cells activity against cephalosporins and other b-lactams. Here, we focused on the and mechanisms related to protein quality control which are of increasing evolution of TEM-1 which represents one of the most commonly encountered importance in the research as well as for biomarker utility in early stage diag- b-lactamases and the origin of over 170 clinically observed mutants. nosis of neurodegenerative diseases. Combining isotopic labelling, vibrational spectroscopy, MD simulation, and ki- netic analysis, we correlate the free energy of activation and electric fields 1010-Plat along the enzyme’s evolutionary trajectory from the perspective of several Folding Proteins From One End to the Other b-lactam antibiotics, highlighting the general importance of electrostatics in Micayla A. Bowman, Patricia L. Clark. enzyme catalysis and evolution. Combining VSE measurements with kinetic Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, and structural analysis provides a new physical observable to rationalize the USA. complexity of enzyme evolution with implications for antibiotic resistance, Proper protein folding is essential for all biological processes. Although the directed evolution, and protein design. cellular environment can support the folding of an enormously diverse range (1) Fried, S. D.; Boxer, S. G. Electric Fields and Enzyme Catalysis. Annu. Rev. of protein structures, our ability to refold proteins in vitro is limited to small, Biochem., 2017, 86, 387-415. simple folds. Molecular chaperones can increase the yield of correctly folded protein for a diverse range of substrates, but a chaperone interaction is required 1008-Plat for only 20% of the proteome under normal growth conditions. This suggests Funnel GAS Model for Protein Many-Body Systems under the Crowded that other features of the cellular environment are responsible for increasing Environment folding yield in vivo. One such difference is vectorial folding of the polypeptide Macoto Kikuchi1, Yoshikatsu Tada1, Nobu C. Shirai2. chain during its synthesis from N- to C-terminus by the ribosome. Co- 1Cybermedia Center, Osaka University, Toyonaka, Japan, 2Center for translational folding can alter protein folding pathways and even the final Information Technologies and Networks, Mie University, Tsu, Japan. folded structure. However, current in vitro protein refolding techniques cannot In order to investigate thermodynamic properties of protein many-body systems, recapitulate vectorial protein appearance or folding. We have developed a it is desired to construct an appropriately coarse-grained model. One of the impor- novel assay to test the effect of vectorial chain appearance on protein refolding tant features of proteins are their funnel-like free-energy landscapes, which make in vitro. This assay uses an ATP hydrolysis-driven protein unfoldase/translo- the proteins fold properly to their native states. Effects of crowding by other mac- case to drive vectorial appearance of the polypeptide chain and enable folding romolecules are also important in a inner-cell environment. The aim of this study to begin from either the N- or C-terminus of a substrate. We are using this assay is to propose a simple model of protein many-body systems based on the lattice to directly test the impact of vectorial appearance of a polypeptide chain on its gas model taking the above two factors into account. A protein is expressed as final native fold, folding kinetics, and refolding yield. a particle on a lattice and it has several internal states of different sizes and free energy; the folded state has the lowest free energy and occupies a single site, while in the unfolded state the proteintakes one of a few possible states of different sizes. 1011-Plat Some intermediate states are also defined if necessary. It will be appropriate to call The Effect of the Ribosome on Nascent Chain Dynamics 1 1 2 this model as ‘‘funnel gas model’’. In addition, we introduce the ‘‘crowders’’,each Madeleine K. Jensen , Samuel Itskanov , Avi J. Samelson , Susan Marqusee1. of which occupies a single site. We consider only the on-site excluded-volume ef- 1 2 fect between the crowders and the proteins. By considering the crowders by the UC Berkeley, Berkeley, CA, USA, UC San Francisco, San Francisco, CA, grand-canonical ensemble, they are exactly eliminated and their effects are USA. included in properly modified free energy of each state of the protein. This modi- Protein folding on the ribosome is fundamentally different from full-length fication of the free energy indicates that the effect of crowder on each state is deter- protein refolding. Constrictions presented by the structure of the ribosome mined by the volume of the state and the density of the crowders. Then it is clearly exit tunnel, the large size of the ribosome, and the negative charge of the understood that the existence of the crowders makes the expanded states of the ribosome surface, among other factors, have been suggested to modulate pro- unfolded state unstable and thus enhances the folding. We discuss the crowding tein folding on the ribosome, and the ribosome has been shown to thermody- effects more deeply based on the combination of the funnel-gas model and the namically destabilize nascent chains (Samelson et al., PNAS 2016). In realistic free-energy landscapes obtained by the Go-like model. contrast to the energy landscape for a full-length protein, translation poses a situation in which the nascent chain can access conformations before the 1009-Plat entire amino acid sequence is present. Elongation of the nascent chain grad- Lighting Up Protein Misfolding and Aggregates by Chemical Probes ually increases the conformational space available to the protein until it can Yuning Hong. access its native state. We are interested in how the energy landscape of a Department of Chemistry and Physics, La Trobe University, Melbourne, protein changes during translation and how the conformations a nascent Australia. chain samples differ from those a full-length protein can occupy. Here, we Maintaining proteostasis is an essential housekeeping function for cell survival. measure protein unfolding kinetics and assess the dynamics of a ribosome- It involves chaperones and degradative pathways to ensure proteins fold stalled nascent chain in comparison with off-the-ribosome proteins using correctly and to remove those that are misfolded, damaged or aggregated. In pulse proteolysis and smFRET.

BPJ 8590_8601 Monday, February 19, 2018 201a

Platform: Membrane Receptors and Signal 1014-Plat Biophysical Features of the abTCR Mechanome that Drive High Avidity Transduction T-Cell Recognition Yinnian Feng1, Kristine N. Brazin2,3, Eiji Kobayashi2,3, Robert J. Mallis4, 1012-Plat Ellis L. Reinherz2,5, Matthew J. Lang1,6. Differential LAT Microcluster Composition and ACTIN-Dependent 1Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Movement at the Immunological Synapse Center TN, USA, 2Laboratory of Immunobiology, Dana-Farber Cancer Institute, Anthony Vega1, Jonathon Ditlev1, Darius Koster2, Xiaolei Su3, Ron Vale3, Boston, MA, USA, 3Department of Medical Oncology, Dana-Farber Cancer Satyajit Mayor4, Michael K. Rosen1, Khuloud Jaqaman1. Institute, Boston, MA, USA, 4Department of Biological Chemistry and 1 Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, Molecular Pharmacology, Harvard Medical School, Boston, MA, USA, 2 3 USA, The University of Warwick, Coventry, United Kingdom, University 5Department of Medicine, Harvard Medical School, Boston, MA, USA, 4 of California San Francisco, San Francisco, CA, USA, National Centre for 6Department of Molecular Physiology and Biophysics, Vanderbilt University Biological Sciences, Bangalore, India. School of Medicine, Nashville, TN, USA. During T cell activation, LAT (Linker for activation of T cells) T-lymphocytes use surface T-cell receptors (abTCRs) to recognize sparse forms phase-separated clusters that move from the periphery to the antigenic peptides bound to MHC molecules (pMHC) on antigen presenting center of the immunological synapse (IS). Recent work has described cells. Such sensitivity permits the activation of immune response at single two distinct actin networks within the IS, an Arp2/3 mediated network molecule level. Physically, T-cell patrolling and cytoskeleton rearrangement at the periphery and a formin-mediated network proximal to the both create biophysical forces, by which peptide discrimination, bond life- center. While LAT cluster velocityhasbeenshowntocorrelatewith time and signaling are precisely tuned. However, how these mechanical sig- that of actin movement, the mechanism that regulates cluster movement nals are transduced to initiate in-vivo cellular events is obscure. Meanwhile, through these networks remains unknown. Our own observations of fluo- the cooperation of chemical and physical aspects of T-cell activation has yet rescently labeled cluster components in Jurkat T cells indicated that to be found. Using optical traps, a new TCR triggering model is elaborated cluster composition changes as they move to the IS center. We hypothe- based on its biophysical features. We have shown that the physicochemical sized that this change in composition may alter cluster-actin interactions, triggering thresholds manifest themselves in a load and force direction necessary for efficient cluster movement. To test this hypothesis, first we dependent manner. Without external load, T-cell can only be activated under studied in vitro the clustering and mobility of reconstituted LAT clusters higher pMHC density but not physiological conditions. Under external load, in a lipid bilayer in the presence of active actomyosin networks. At immunosurveillance like shear force (10 pN per TCR) triggers T-cell Ca2þ steady-state, single-particle tracking and diffusion analysis revealed that flux with as few as 2 interfacial pMHCs synchronized with rapid position actomyosin promoted the mobility of LAT clusters in a composition- relaxation whereas normal force only triggers transient Ca2þ flux with 2 dependent manner. In the presence of a contracting actomyosin network, interfacial molecules. In-depth studies have revealed that such relaxation is Spatiotemporal Image Correlation Spectroscopy (STICS) analysis of actin mainly associated with intracellular actomyosin dependent transport via flow and cluster movement revealed that cluster composition dictated the 8-nm steps, behaviors implying the dispensability of TCR serial engage- degree of cluster co-movement with actin. Motivated by these observa- ment during the initial triggering. By considering molecular features of tions, we identified the molecular clutch linking cluster and actin move- abTCR mechanome, physicochemical threshold, and synergy of internal ment, and then studied the movement of LAT clusters in Jurkat T cells and external forces, this model provides significant implications for person- during IS formation after mutating the molecular clutch. These studies re- alized T-cell therapy and a paradigm shift relative to earlier activation vealed that change in cluster composition, specifically loss of the molec- models. ular clutch, is necessary for efficient cluster mobility. Interestingly, this change in composition seems to dependontheintegrityoftheformin- 1015-Plat mediated actin network. These results suggest that actin networks in the Fluorescence Localisation Imaging with Photobleaching at 5 nm Resolu- IS may facilitate a change in cluster composition necessary for efficient tion Reveals the Architecture of Basal EGFR Complexes and Mechanisms movement. of Autoinhibition and Activation Marisa L. Martin-Fernandez1, Laura C. Zanetti-Domingues1, 1013-Plat Dimitrios Korovesis1, Sarah R. Needham1, Christopher J. Tynan1, DNA Origami as a Nanoscale Platform for T-Cell Activation Selene K. Roberts1, David T. Clarke1, Daniel J. Rolfe1, Peter J. Parker2. Viktoria Motsch, Joschka Hellmeier, Gerhard J. Schutz,€ Eva Sevcsik. 1Research Complex at Harwell, STFC Central Laser Facility, Didcot, United Institute of Applied Physics, TU Wien, Vienna, Austria. Kingdom, 2Protein Phosphorylation Laboratory, Francis Crick Institute, In the human immune system, the recognition of an antigen by the T-cell recep- London, United Kingdom. tor (TCR) takes place within the contact area between the T-cell and the Epidermal growth factor (EGF) receptor (EGFR, or HER1/ErbB1) is a cell- antigen-presenting cell (APC). It is thought that the nanoscale spatial distribu- surface receptor tyrosine kinase that plays a fundamental signalling role in tion of proteins within this contact zone plays an essential role in the initiation regulation of cellular metabolism, growth and differentiation. We have devel- of an immune response. Despite extensive studies, the molecular details of this oped a super-resolution method based on fluorescence localisation imaging process, in particular the structural requirements for TCR triggering and how with photobleaching (FLImP) that probes in detail the architecture of dimers nanoscale events are translated into T-cell activation, are still poorly and oligomers of the EGFR with a 5 nm resolution. Using a fluorescent de- understood. rivative of EGF, we showed that growth factor-induced EGFR oligomerization Here, we use DNA origami decorated with TCR ligands anchored to is essential to signal regulation of signalling, mediated extracellularly by unoc- a planar glass-supported lipid bilayer to assess the effects of local cupied ligand-binding sites, and that it organizes kinase-active dimers in ways ligand density and arrangement on T-cell activation. Thus, our experi- optimal for auto-phosphorylation in trans between neighbouring dimers. Un- mental setup allows for the precise nanoscale arrangement of TCR derstanding of the mechanisms by which EGF induces formation of EGFR olig- ligands on the DNA origami scaffold, while at the same time permitting omers lags well behind. Currently founded on ligand-induced interactions the re-organization of ligand and TCR during T-cell activation. We between inactive monomers, mounting evidence suggests that besides a mono- used either fluorescently labeled CD3 antibody or recombinant TCRb- mer cohort, ligand-free EGFRs forms dimers and possibly oligomers whose reactive single chain antibody fragment (scFv) as stimulatory ligands autoinhibition mechanisms and signalling role are unclear. A key obstacle is that were placed on the DNA origami at one to 20 engineered that the nature of the ligand-free EGFR species populating the basal state is capture sites in different layouts and densities. The actual number of not well understood. Replacing EGF with antagonist anti-EGFR Affibody or ligands per origami was determined using several single molecule fluores- nanobody and combining FLImP with fluorescent resonance energy transfer cence microcopy methods and atomic force microscopy. The activation and single particle tracking we have found two multimeric EGFR basal states of T-cells interfaced with the APC-mimicking surfaces was measured that exploit distinct means of suppressing kinase domain function. Ligand-free using a calcium-sensitive fluorescent dye and the effects of local ligand EGFRs form dimers with tethered ectodomains structurally coupled across the density, nanoscale ligand arrangement as well as the nature of the plasma membrane to asymmetric tyrosine kinase domain dimers, autoinhibited ligand were assessed. Further, the rearrangement of TCR and ligands when forming part of linear tethered polymer structures. Ligand-free EGFRs in the process of T-cell activation was monitored by single molecule also form extended dimers coupled to inactive symmetric kinase dimers. microscopy. Upon ligand-binding, the ectodomains of the ligand-free tethered polymers

BPJ 8602_8612 202a Monday, February 19, 2018 become extended and singly-bound dimers sandwich ligand-free ones. The two 1018-Plat dynamically linked mechanisms allow face-to-face interactions to mount sig- An Efficient Molecular Dynamics Simulation Strategy to Investigate the nals reactive to stimulus strength. Mechanistic Basis for Biased Agonism at G Protein-Coupled Receptors Derya Meral, Davide Provasi, Marta Filizola. Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New 1016-Plat York, NY, USA. Glycoprotein Crowding Affects Cell Membrane Signalling A substantial complexity in G Protein-Coupled Receptor (GPCR) drug research Hao Pan1, Matthew Paszek1,2. stems from the receptor ability to couple to several effector pathways and from 1Field of Biophysics, Cornell University, Ithaca, NY, USA, 2Robert the differential activation of these pathways by so-called biased agonists. While Frederick Smith School of Chemical and Biomolecular Engineering, Cornell pharmacological data suggest that different receptor conformations are at the University, Ithaca, NY, USA. basis of this functional selectivity, characterization of its mechanistic details re- Proteincrowdingonbiologicalmembranes can limit membrane protein quires a thorough understanding of the thermodynamic and kinetic properties diffusion and influence on protein aggregation, conformation, and biomol- that control receptor activation. To start shedding light on these properties, ecular interactions. Cancer cells overexpress large glycosaminoglycans we have recently reported the analysis of extensive molecular dynamics simu- and mucin glycoproteins, including MUC1, which may contribute to lations of m-opioid receptor (MOR) bound to a classical opioid drug or a potent crowding on the cell surface. While altered Receptor Tyrosine Kinase G protein-biased agonist. Here, we apply an enhanced sampling strategy to effi- (RTK) activity is strongly associated with many types of cancer, our un- ciently investigate ligand-induced allosteric communication across MOR, us- derstanding of how membrane protein crowding impacts RTK activation ing order parameters describing the activation pathway derived from inactive remains limited. Current work focuses on direct interactions among and activated crystal structures of the receptor. Specifically, we employ a RTK and other membrane proteins but overlooks the significant effect metadynamics-based strategy that uses path variables to examine the conforma- of the crowded condition itself. The central hypothesis under our pro- tional sampling induced by opioid agonists with different bias profiles (e.g., posed work is that crowding of mucins on the cell membrane can slow morphine vs. oliceridine), as well as by inverse agonists (e.g., naltrexone). down RTK lateral diffusion, prolong RTK dimerization, and thus enhance Rigorous information theory analysis of the results of these simulations pro- RTK activation. To engineer specific states of macromolecular crowding vides a characterization of the information flow from the ligand-binding pocket on the plasma membrane, we established a modular library of semi- to the intracellular region of the receptor that is in general agreement with that synthetic mucins of varying length and extent of glycosylation. Fusion obtained from more extensive simulations. Furthermore, we show that a com- of mucin ectodomains to suitable membrane anchors and adoption of bination of metadynamics sampling and maximum caliber principles can yield inducible expression systems permit the control of the density and thick- valuable estimates of kinetic properties that control receptor activation by ness of the mucin-rich glycocalyx. Taking Epithermal Growth Factor Re- different ligands at an affordable computational cost. ceptor (EGFR) as an example, we found that diffusivity of EGFR decreases upon increased MUC1 crowding. Single molecule behavior 1019-Plat analysis indicated that on the crowded membrane surface, EGFR diffu- Molecular Timing of Membrane Signaling Reactions 1 1 1 sion is slower and exhibits correlations in velocities consistent with William Y.C. Huang , Steven Alvarez , Young Kwang Lee , 2 1 1 1,2 crowding effects. Furthermore, MUC1 crowding leads to a prolonged Yasushi Kondo , Jean K. Chung , Hiu Yue Monatrice Lam , John Kuriyan , Jay T. Groves1. EGFR phosphorylation. Together, our results establish a new physical 1 role for the glycocalyx in receptor activation. Department of Chemistry, University of California, Berkeley, CA, USA, 2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. 1017-Plat Timing is an integral part of signal transduction in living systems encoded in Transient Hetero-Dimerization of Opioid Receptors (GPCRS) Revealed by the underlying molecular mechanism. Many signaling proteins are autoinhi- Single-Molecule Tracking bited in the cytosol and only activate upon membrane recruitment. Release Peng Zhou1, Rinshi S. Kasai2, Koichiro M. Hirosawa3, Alexey Yudin1, of autoinhibition generally involves structural rearrangements of the protein Yuki M. Shirai2, Takahiro K. Fujiwara3, Akihiro Kusumi1,3. at the membrane surfaces that introduce a time lag between initial recruitment 1Membrane Cooperativity Unit, Okinawa Institute of Science and and activation. Both the mean and distribution of the activation lag times play Technology Graduate University (OIST), Onna-son, Japan, 2Institute for critical roles in enabling regulatory processes such as kinetic proofreading. We Integrated Cell-Material Sciences, Kyoto University Institute for Advanced develop a single-molecule assay to temporally resolve the activation process of Study, Kyoto, Japan, 3Institute for Frontier Life and Medical Sciences, Kyoto the Ras guanine nucleotide exchange factor SOS on membrane surfaces. Simul- University, Kyoto, Japan. taneous imaging of individual SOS molecules and localized Ras activation on Opioid receptors (OR) belong to the superfamily of G protein-coupled re- supported membrane microarrays maps the activation timing resulting from ceptors (GPCRs). Nearly all vertebrate species express four opioid recep- receptor-mediated membrane recruitment of SOS. The gamma-like shape of tors. Among them, three OR species, m (MOR), d (DOR), and (KOR), the activation time distribution reveals rate-limiting kinetic intermediates in exhibit 60% amino-acid identity to one another. These OR species the release of autoinhibition and establishes a basis for kinetic proofreading have been reported to form heterodimers, which exhibit pharmacologic in the activation of Ras. Once activated, a single SOS molecule is highly proc- profiles distinct from monomers and homodimers, revealing a new level essive, capable of activating hundreds of Ras molecules. Together, this sug- of molecular cross-talk between signaling pathways. In fact, various gests a relationship between the signaling timescale of protein copy number GPCRs have been proposed to form heterodimers, and thus the hetero- and the activation time of individual SOS molecule. dimers of these three OR species would provide an interesting paradigm Platform: Ion Channels, Pharmacology, and to study GPCR heterodimer formation and function. However, in living cells, obtaining evidence for the existence of OR heterodimers, character- Disease izing OR heterodimers, and finding the molecular mechanism for hetero- dimerization have proven to be difficult. Here, using single-molecule 1020-Plat imaging and tracking, we found that MOR and DOR as well as DOR Dissecting Function and Distribution of Sodium Channels and GAP Junc- and KOR formed transient heterodimers, with lifetimes of 50 ms, tional Proteins using Super-Resolution Patch-Clamp whereas KOR and MOR did not. We identified the domains responsible Anita Alvarez-Laviada1, Rengasayee Veeraraghavan2, Vania Braga3, for heterodimerization of MOR and DOR near the N-termini of both mol- Robert Gourdie4, Julia Gorelik1. ecules. A six amino-acid peptide with a key sequence in the domain of 1Dept. of Myocardial Function, National Heart and Lung Institute, Imperial MOR responsible for heterodimerization blocked MOR-DOR heterodime- College London, London, United Kingdom, 2Dept. of Biomedical rization, suggesting a possibility that this peptide could be used as a drug Engineering, The Ohio State University, Columbus, OH, USA, 3Molecular to modulate the MOR-DOR heterodimerization, thereby regulating the Medicine, National Heart and Lung Institute, Imperial College London, downstream signaling pathways that are under the control of OR crosstalk. London, United Kingdom, 4Virginia Tech Carilion Research Institute, Thus, such knowledge would be useful for developing new types of drugs Virginia Polytechnic University, Roanoke, VA, USA. that work by blocking the crosstalk between signaling pathways triggered Cardiac impulse propagation occurs via gap junction (GJ) and sodium channels by two or more GPCRs. Similar domains may mediate heterodimerization (Nav1.5) which provide excitatory current. Their function is essential for heart of other mammalian GPCRs. We will also report the DOR-KOR heterodi- contractility and is impaired in arrhythmia and heart failure. We demonstrate merization and homodimerization of the ORs. super-resolution patch-clamp to capture sodium currents associated with GJ

BPJ 8602_8612 Monday, February 19, 2018 203a and perinexal regions. We correlated nano-structural organisation of junctions contribution and to treat diseases associated with Piezo channel dysfunctions. to their function, as niches for Nav1.5. The small molecule Yoda1, which activates Piezo1 but does not affect Piezo2, Scanning ion conductance microscopy resolved junctional topography in is the only known molecular modulator exhibiting selectivity among mamma- neonatal rat ventricular cardiomyocyte. This was combined with cell- lian Piezo channels. To date, the molecular basis for this selectivity is unknown. attached Nav1.5 recordings from cardiomyocyte-cardiomyocyte and Here, a library of chimeras was created by replacing various protein regions cardiomyocyte-fibroblasts junctions. Functional and dominant-negative from mouse Piezo1 by their homologous counterpart from mouse Piezo2. Cx43EGFP adenoviruses were used to probe active/inactive junctions. The The characterization of these chimeras by calcium imaging and pressure- Navb1 adhesion inhibitor peptide (badp1) was tested. Whole-cell Nav-currents clamp electrophysiology led to the identification of a minimal Piezo1 region were recorded to separate non-junctional badp1 effects. To assess trans- responsible for the chemical activation of Piezo1 by Yoda1. This work paves depolarisation, current-clamp and optical measurements were combined. the way to elucidate the mechanisms underlying chemical activation of Piezo Plasma membranes were analysed for junctional protein density. channels and will help expand the list of selective small molecule modulators EGFP Exogenously-delivered Cx43 trafficked to regions where Nav activity and targeting the small Piezo channel family. cluster size were increased relative to native Cx43-junctions (p=0.015). Smaller 1023-Plat Nav-clusters (5-10) were abundant at native junctions, larger clusters (20-30) predominated at Cx43EGFP-junctions. Acute badp1 peptide treatment caused Knotbodies: A New Generation of Ion Channel Therapeutic Biologics EGFP Created by Fusing Knottin Toxins into Antibodies Nav downregulation at native (p<0.01) and Cx43 -rich junctions 1 1 1 2 (p<0.001). Lack of badp1 effect on the whole-cell current supports a role for Damian C. Bell , Aneesh Karratt-Vellatt , Sachin Surade , Tim Luetkens , b Edward W. Masters1, Naja M. Sørensen3, Neil Butt1, John McCafferty1. 1 as a trans-cellular adhesion molecule. Blockade of cardiomyocyte trans- 1 2 activation using optical/electrical recordings was demonstrated with badp1 IONTAS, Cambridge, United Kingdom, School of Medicine, University of Utah, Salt Lake City, UT, USA, 3Sophion, Copenhagen, Denmark. pre-treatment. Reduced Nav-current and smaller channel clusters were found at cardiomyocyte-fibroblast versus cardiomyocyte-cardiomyocytes junctions. Ion channels are key therapeutic targets; however, existing therapeutic classes Membranes from junction-remote regions showed similar currents, but have developmental disadvantages. Small molecule drug discovery has often struggled to deliver both potency and specificity of the desired ion channel different Nav-cluster size. target. Biologics (e.g. antibodies) have been investigated, aiming to improve Strength of intercellular junctions depend on Nav-current, intact Navb1 and the amount of functional Cx43. Cardiac junction type hetero- vs. homo-cellular potency and specificity. However, antibodies targeting ion channels face signif- icant challenges since externally accessible binding epitopes are limited. Using governs Nav activity and distribution. Novel badp1 peptide establishes impor- tance of perinexal regions for signal propagation. Our findings and biophysical the evolutionary toolbox of animal venoms, researchers have turned to mini- approach elucidate molecular origin of arrhythmogenic activity within/around protein toxins (knottins) that have potent ion channel modulatory activity. These offer potential as therapeutic agents but suffer from manufacturing dif- GJ, revealing rationale for the anti-arrhythmic effects of Navb1-mediated adhesion. ficulties, short in-vivo half-lives and often lack specificity. Addressing these shortfalls, IONTAS have developed a technology platform to 1021-Plat create a new therapeutic class: by fusing a knottin into an antibody, the result- Inhibition of the Potassium Channel Kv1.3 Reduces Infarction and Inflam- ing ‘‘KnotBody’’ retains the ion channel blocking activity from the knottin mation in Ischemic Stroke whilst gaining the extended half-life and additional specificity conferred by Heike Wulff1, Yi-Je Chen1, Hai M. Nguyen1, Izumi Maezawa2, multiple contact surfaces of the antibody. Lee-Way Jin2. We have made a series of proof-of-concept molecules that highlight the advan- 1Pharmacology, University of California, Davis, CA, USA, 2Pathology and tages of KnotBodies. Using phage display we have demonstrated that a Laboratory Medicine, University of California, Davis, CA, USA. ‘‘model’’ knottin (trypsin-blocking EETI-II) can be inserted via short linker Inhibitors of the voltage-gated Kþ channel Kv1.3 are currently in development peptides into peripheral diversity loops (CDRs) of antibodies. X-ray crystallog- as immunomodulators for the treatment of autoimmune diseases. Since Kv1.3 raphy and biochemical assays confirms that both knottin and antibody are is also expressed on microglia and has been shown to be specifically up- correctly folded and bind targets. Furthermore, we also demonstrate that bind- regulated on ‘‘M1-like’’ microglia we here tested the therapeutic hypothesis ing specificity of both antibody and peptide can be engineered. Finally, we that the brain penetrant small-molecule Kv1.3-inhibitor PAP-1 reduces second- demonstrate blocking of Kv1.3 and ASIC1a ion channels by substituting alter- ary inflammatory damage after ischemia/reperfusion. We studied microglial native knottins. Kv1.3 expression using electrophysiology and immunohistochemistry, and These proof-of-concept molecules highlight the advantages of the KnotBodyTM evaluated PAP-1 in hypoxia exposed organotypic hippocampal slices and in generating platform: thus, the modular nature of the KnotBodyTM binding sur- middle cerebral artery occlusion (MCAO) with 8-days of reperfusion in both face and the amenability of this format to phage display technology facilitates adult male C57/BL6 mice (60 min MCAO) and adult male Wistar rats (90 further optimisation of potency and selectivity of ion channel blockade by en- min MCAO). In both models PAP-1 administration was started 12 hours after gineering both knottin and antibody loop sequences. reperfusion. We observed Kv1.3 staining on activated microglia in ischemic in- farcts in mice, rats and humans and found higher Kv1.3 current densities in 1024-Plat acutely isolated microglia from the infarcted hemisphere than in microglia iso- Differential Metabolic and Nucleotide Sensitivity of Beta-Cell and Cardiac lated from the contralateral hemisphere of MCAO mice. PAP-1 reduced micro- KATP Channels glia activation and increased neuronal survival in hypoxia exposed Natascia Vedovato, Peter Proks, Olof H. Rorsman, Kostantin Hennis, hippocampal slices as effectively as minocycline. In mouse MCAO, PAP-1 Frances M. Ashcroft. dose-dependently reduced infarct area, improved neurological deficit score Physiology, Anatomy and Genetics, University of Oxford, Oxford, United and reduced brain levels of IL-1b and IFN-g without affecting IL-10 and Kingdom. brain-derived nerve growth factor (BDNF) levels or inhibiting ongoing phago- ATP-sensitive potassium (KATP) channels couple the metabolic state of a cell to cytosis. The beneficial effects on infarct area and neurological deficit score its electrical activity and play important physiological roles in many tissues. In were reproduced in rats providing confirmation in a second species. Our find- contrast to beta-cell (Kir6.2/SUR1) channels, which open when extracellular ings suggest that Kv1.3 constitutes a promising therapeutic target for preferen- glucose levels fall, cardiac (Kir6.2/SUR2A) channels remain closed. We inves- tially inhibiting ‘‘M1-like’’ inflammatory microglia/macrophage functions in tigated the molecular basis of this difference by two-electrode voltage clamp ischemic stroke. (TEVC) and patch clamp of Kir6.2/SUR1 and Kir6.2/SUR2A channels heter- Supported by R56NS098328, NS100294 and AG043788 from NIH. ologously expressed in Xenopus oocytes. Differential sensitivity to metabolic inhibition (azide) was observed in TEVC 1022-Plat of intact oocytes, indicating it results from differences in SUR, and not differ- A Minimal Protein Region Required for the Chemical Activation of the ences in beta-cell and cardiac metabolism. In inside-out patches, both channel Mechanosensitive Channel Piezo1 types were inhibited by ATP and activated by MgADP to similar extents. How- Jerome J. Lacroix. ever, when both nucleotides were present, the results were strikingly different. Graduate College of Biomedical Sciences, Western University of Health Thus, 100mM MgADP reduced MgATP block of Kir6.2/SUR1 from 24mMto Sciences, Pomona, CA, USA. 504mM (when measured immediately after patch excision), but had little effect The homologous Piezo1 and Piezo2 mechanosensitive ion channels are central on MgATP inhibition of Kir6.2/SUR2A. The difference in ATP block in the to many important mechanotransduction processes in both normal and patho- presence of 100mM MgADP was sufficient to explain why beta-cell KATP chan- logical states. Selectively targeting each Piezo homolog with pharmacological nels open in the absence of glucose and cardiac channels do not. Deleting the agents represents a promising avenue to probe their individual biological last 42 amino acids ‘the tail’ of SUR abolished MgADP activation of both

BPJ 8602_8612 204a Monday, February 19, 2018 channels. Replacing the tail of SUR2A with that of SUR1 switched the ATP combining a light-sensitive azobenzene group with a known Hv1 inhibitor, sensitivity in the presence of MgADP to that of SUR1 (and v.v.). These data 2-guanidinobenzimidazole (2GBI). The azobenzene group isomerizes from suggest that the ‘tail’ of SUR plays a critical role in coupling nucleotide bind- trans to cis under blue light illumination and thermally relaxes back from cis ing/hydrolysis to channel activation, and that this differs between cardiac and to trans in the dark. Four analogs (photoGBI1-4) were characterized on Hv1 beta-cell KATP channels. channels heterologously expressed in X. laevis oocytes using the excised patch-clamp technique. Light-induced conformational changes of the photo- 1025-Plat GBIs changed their efficacy of inhibition: in the dark, photoGBIs inhibit Structural Details of an Allosteric Mechanism for Bimodal Anesthetic Hv1, and under blue light, inhibition is released. PhotoGBI4 modulates proton Modulation of Pentameric Ligand-Gated Ion Channels currents of human sperm and macrophages and holds the promise to further our 1 2 1 2 Rebecca J. Howard , Zaineb Fourati , Stephanie A. Heusser , Haidai Hu , understanding of Hv1-mediated pH regulation. Reinis R. Ruza2, Ludovic Sauguet2, Erik Lindahl1,3, Marc Delarue2. 1Science for Life Laboratory, Department of Biochemistry & Biophysics, Stockholm University, Solna, Sweden, 2Unit of Structural Dynamics of Platform: Systems Biophysics Macromolecules, Institut Pasteur, Paris, France, 3Theoretical and 1028-Plat Computational Biophysics, KTH Royal Institute of Technology, Stockholm, Biophysical Techniques for the Study of Phase Transitions in Protein Sweden. Droplets and Cells Ion channel modulation by general anesthetics is a vital pharmacological pro- Raimund Schlussler€ 1, Shada Abuhattum1, Gheorghe Cojoc1, Timon Beck1, cess with implications for receptor biophysics and drug development. Although Felix Reichel1, Kyoohyun Kim1, Mirjam Schurmann€ 1, Paul Muller€ 1, functional studies have identified conserved sites of both positive and negative Jurgen€ Czarske2, Vasily Zaburdaev3, Titus Franzmann4, Simon Alberti4, anesthetic modulation in pentameric ligand-gated ion channels, a structural un- Jochen Guck1. derstanding of these bimodal effects is lacking. The prokaryotic model protein 1 Biotechnology Center, Technische Universit€at Dresden, Dresden, Germany, GLIC recapitulates anesthetic modulation of human ion channels, and is acces- 2 Electrical Engineering, Technische Universit€at Dresden, Dresden, Germany, sible to structure determination in both apparent open and closed states. Here, 3 Max Planck Institute for the Physics of Complex Systems, Dresden, we report new mutagenesis, electrophysiology, and crystallography data on 4 Germany, Max Planck Institute of Molecular Cell Biology and Genetics, GLIC variants in the presence and absence of general anesthetics, mapping a Dresden, Germany. mechanism for allosteric modulation. In particular, we document the crystallo- Recently, phase transitions of protein-RNA droplets in cells, and of the cyto- graphic conversion of two GLIC variants from apparent closed to open states, plasm of entire cells, have been shown to play important roles in physiological solely attributable to anesthetic binding within the transmembrane domain of and pathological processes in biology. Their molecular control is still unclear at each receptor subunit. These and other targeted mutations alter anethestic ef- this point. We have developed and are applying a range of biophysical tech- fects by shifting the relative stability of closed and open, apo and holo states, niques for the specific study and quantitative characterization of such phase involving at least three binding sites in the transmembrane domain. Our results transitions. We use a dual-beam laser trap, real-time deformability cytometry, support an integrated, multi-site model for allosteric modulation, and provide and atomic force microscopy for the viscoelastic characterization of cells and atomic details of both positive and negative modulation by one of the most protein-RNA granules in vitro. We combine opto-fluidic object rotation with common general anesthetics, providing potentially fruitful templates for drug quantitative phase microscopy to obtain phase images from multiple angles, development. which in turn are tomographically reconstructed by a back-propagation algo- 1026-Plat rithm to obtain 3D distributions of refractive index and mass density inside X-Ray Crystal Structures of the Influenza a M2 Proton Channel Bound to trapped objects. Finally, we have also established Brillouin microscopy for Amantadine, Rimantadine, and Inhibiting Compounds the 3D mapping of mechanical properties inside cells with diffraction-limited Jessica L. Thomaston, William F. DeGrado. resolution. I will present and discuss our findings obtained on phase- University of California, San Francisco, San Francisco, CA, USA. separated FUS protein droplets and on phase transitions in yeast cells under Inhibition of the M2 proton channel in the influenza A virus prevents viral repli- different kinds of stress with this unique toolset. cation from occurring. Two of the four FDA-approved drugs for the treatment of influenza infections, amantadine and rimantadine, target the M2 channel. 1029-Plat However, because M2 is a membrane protein, structural studies of drug binding Analysis of Apoptotic Event Time Correlations in Single Cells to the channel using x-ray crystallography have been limited because of the Alexandra Murschhauser1, Peter Ro¨ttgermann1, Daniel Woschee1, challenging nature of the target, with the one previously solved crystal structure David Garry2, Martina Ober1, Kenneth Dawson2, Joachim O. R€adler1. 1 limited to 3.5 A˚ resolution. Here, we have obtained multiple crystal structures Faculty of Physics and Center for NanoSciene (CeNS), Ludwig-Maximilians 2 of M2 in the presence of drugs and inhibiting compounds using lipidic University (LMU) Munich, Munich, Germany, Centre for BioNano cubic phase (LCP) crystallization techniques. We present the first crystal struc- Interactions, School of Chemistry and Chemical Biology, University College tures of rimantadine bound to M2 in both the Inwardclosed and Inwardopen con- Dublin, Dublin, Ireland. formations of the channel (2.0 A˚ , 2.5 A˚ ), as well as amantadine bound to the Signaling cascades in apoptosis are known to be highly heterogeneous at ˚ Inwardclosed conformation (2.0 A). At this resolution range the orientation of the single-cell level both in effect and dynamics. Cell fate decisions are the bound drug is unambiguous, and the ammonium group of the adamantane the final result of various possible pathways, each showing organelle- drugs can be seen interacting with ordered water molecules present within the specific signals as key players. Lysosomes as well as mitochondria are channel. Additionally, we report the binding of a dual-inhibiting compound to believed to be central actors in apoptosis, but also the impact of reactive both the wild type channel (2.6 A˚ ) and the drug-resistant V27A mutant (2.5 A˚ ). oxygen species (ROS) production is known to be involved in cell death. The position of the bound inhibitor within the channel shifts in the presence of The timing of cell death events and their sequence, however, are not fully the V27A mutation. These structures further our understanding of drug binding explored yet. Here, we establish a method to analyze fluorescence time and inhibition within the M2 proton channel and will help guide the design of courses of single cells during apoptosis induced by amino-functionalized compounds for the inhibition of drug-resistant mutants of the influenza M2 polystyrene nanoparticles (PS-NH2) and the protein kinase c inhibitor channel. staurosporine with automated time-lapse microscopy on micro-structured arrays. We monitor chronological order of cell death events with high 1027-Plat temporal resolution using time-correlation analysis by the use of two Azobenzene-Based Photoswitches for the Control of the Voltage-Gated different fluorescence markers per cell. In particular, we studied: lyso- Proton Channel Hv1 somal membrane permeabilization (LMP), mitochondrial outer membrane Andreas Rennhack, Elena Grahn, U. Benjamin Kaupp, Thomas K. Berger. permeabilization (MOMP), increase of ROS level and oxidative burst (Ox- Molecular Sensory Systems, Research Center Caesar, Bonn, Germany. Burst), caspase 3 activation (CASP-3), exposure of phosphatidylserine to The voltage-gated proton channel Hv1 is expressed in the plasma membrane of the outer membrane (PhS-FLIP) and loss of plasma membrane integrity various human cells, including macrophages, lung epithelial cells, and sperm. with the subsequent nucleus staining (PMP). We find distinct timing in Hv1 opening leads to an efflux of protons and thereby alkalizes the cell. In mac- the signal pathway and variance in event correlations indicative of rophages, Hv1 counteracts cytosolic acidification caused by NADPH oxidase cross-talk in the event cascade of apoptosis. Our results indicate that activity during the respiratory burst. In sperm, Hv1 might be involved in matu- both the mitochondria and the lysosomes catalyze separately the subse- ration. To better understand the physiological role of Hv1, we developed a quent processes. In summary, multi-dimensional time-correlation provides light-controlled inhibitor that allows us to control Hv1 with spatiotemporal pre- a dynamic fingerprint of signal progression that is capable to identify ef- cision. We synthesized photoisomerizable Hv1 inhibitors (photoGBIs) by fects at the single-cell level.

BPJ 8602_8612 Monday, February 19, 2018 205a

1030-Plat shape, function, and form. But how does this happen? How do we coarse grain A Study of Transcriptional Activation by the Transcription Factor GAL4 from -omics regulation to quasi-pre-programmed anatomical patterns? Here, in Saccharomyces Cerevisiae by 3D Orbital Tracking and In Vivo RNA we use embryonic mouse epidermis as a specific test case to address these ques- Labelling tions. Empirical measurements are acquired using the Molecular Atlas Platform Anh Huynh1, Micah Buckmiller2, Daniel R. Larson3, Tineke Lenstra4, - a multiscale imaging technology for visualizing proteomic data across many Matthew L. Ferguson1,5. orders of magnitude in scale - and DNA-PAINT for highly multiplexed super- 1Department of Physics, Boise State University, Boise, ID, USA, resolution imaging of immunofluorescent stained molecular targets. We focus 2Department of Chemistry, Boise State University, Boise, ID, USA, on studying the biomechanics of pattern formation, and the sequence of events 3Laboratory of Receptor Biology and Gene Expression, National Cancer leading to stem cell fate specification. Our results lead us to a head-on collision Institute, Bethesda, MD, USA, 4Division Gene Regulation (B4), Netherlands with a fundamental chick-and-egg question: Does genetic programming pattern Cancer Institute, Amsterdam, Netherlands, 5Biomolecular Sciences Program, morphology, or do the material effects of morphology pattern gene expression? Boise State University, Boise, ID, USA. Understanding what is going on at the molecular level within individual cells is 1033-Plat challenging. But deciphering stochastic biomolecular processes is crucial for A Physical Mechanism for Micro-vascular Adaptability our understanding of gene transcription and the intricacies of cellular meta- Shyr-Shea Chang1, Kyung In Baek2, Chih-Chiang Chang2, bolism. With 3D orbital tracking, we are able to visualize and monitor pre- Andrew Pietersen2, Tzung K. Hsiai3, Marcus Roper4. 1Mathematics, University of California, Los Angeles, Los Angeles, CA, mRNA and transcription factors in real-time using fluorescent tagging within 2 yeast cells at a high sampling rate. Our study demonstrates that we can track USA, Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA, 3Bioengineering/Medicine, University of California, Los Angeles, these molecules of interest, fluorescent-labeled GFP (pre-mRNA) and JF 646 4 dye (transcription factors), during the process of transcribing a gene that codes Los Angeles, CA, USA, Mathematics/Biomathematics, University of for metabolizing galactose. This method allows us to directly observe the California, Los Angeles, Los Angeles, CA, USA. movement of a single molecule and determine the time lag between the Techniques like MOST allow microvascular networks to be mapped with high GAL4 transcription factor binding to DNA and the activation of the mRNA (and increasing) resolution. However, despite an abundance of new data, little synthesis. The data we collected improves our knowledge of the details of tran- remains known about the physical principles that underlie network wiring. Here scriptional kinetics and how single celled eukaryotic organisms regulate tran- we use the zebrafish trunk microvasculature as a model to study the adaptability scription. This will expand our research on the transcription processes in of microvascular networks to damage. In particular, we use imaging of real em- similar genes and in multicellular organisms like humans. 3D orbital tracking bryonic zebrafish and mathematical modeling to show that following amputa- opens up a new window for exploring fundamental biochemical processes tion of the zebrafish tail, the microvascular network is adapted to locally reroute through a dynamic view of single fluorescent molecules in living systems at blood flow through the nearest collateral vessels, increasing both hematocrit high speed (Redman, Hayden, and Ferguson 2016). and fluid shear stress in those vessels. Shear stress is known to be an effector Our research is funded by Research Corporation for Science Advancement and for vessel growth and repair, and this adaptation has the benefit of also the Gordon and Betty Moore Foundation through Grant GBMF5263.10 and by increasing blood supply to regrowing tissues. We go on to look for similar mo- National Institute of General Medical Sciences through Grant 1R15GM123446. tifs for adaptability in a data set of cortical blood vessels. In addition to Redman, Nathan A., Eric J. Hayden, and Matthew L. Ferguson. 2016. ‘‘Char- revealing the exquisite hydraulic engineering of the zebrafish microvascular acterizing Transcription and Splicing Kinetics by 3D Orbital Tracking.’’ Bio- network, our results suggest a general organizing principle for microvascular physical Journal 110 (3, Supplement 1): 66a. networks. 1034-Plat 1031-Plat Tissue-Specific Interactions and Functional Versatility of Drug Targets Designing Single-Cell Experiments with Discrete Stochastic Models Characterize Adverse Effects of the Drugs Zachary Fox, Brian Munsky. Jihye Hwang1,2. Colorado State University, Ft Collins, CO, USA. 1Pharmacy, Keimyung University, Daegu, Republic of Korea, 2IT Modern experimental advances have made it possible to measure the single-cell Convergence and Engineering, POSTECH, Pohang, Republic of Korea. and single-molecule heterogeneity of DNA, RNA and protein species. These There have been many efforts to reduce adverse effects of drugs. Nonetheless, discrete, stochastic data can be highly informative, especially when they are the relation between drug targets in terms of localization and their associated combined with the chemical master equation, or its direct solution using the adverse effects is still unclear. Here, we characterized a mechanism of drug finite state projection (FSP) approach. We hypothesize that FSP models may adverse effects by investigating drug targets within the framework of spatial di- be used to design even better single-cell-single-molecule experiments. To versity of a network (among tissues). Tissue-specific gene expression patterns accomplish this goal, we derive the FSP based Fisher information matrix were applied to measure the rearrangements of protein-protein interactions (FSP-FIM), which estimates the information a new experiment may reveal (PPIs) around target proteins. We hypothesized that undesirable side effects when analyzed with a discrete stochastic model. Unlike previous FIM analyses, would emerge from differences in PPIs of target proteins among multiple tis- the FSP-FIM makes no assumptions on the distribution shape of the single-cell sues. Functions of proteins are mediated by PPIs; therefore, dissimilarities in data. We verify the FSP-FIM using analytical results for the-well characterized interacting partners of proteins among tissues may lead to different functional gene expression model of mRNA production and degradation. We also demon- outcomes. We found a strong positive correlation between rewiring of interact- strate how the FSP-FIM may be used to design single-cell experiments, such as ing partners of targets and the frequency of the associated adverse effects of single-molecule fluorescent in-situ hybridization (smFISH) analyses. We illus- drugs. Furthermore, we observed that the rewiring rate strongly correlates trate this capability on a model of bursting gene expression, in which a gene with the functional versatility of target proteins. Particularly, membrane targets stochastically transitions between active and inactive states, and produces frequently use rewiring of PPIs and induce different functional outcomes in non-Gaussian mRNA fluctuations. We computationally test hundreds of poten- different tissues. Our analysis revealed for the first time that tissue-specific dif- tial experiments, and we determine the optimal temporal measurement period ferences in interactions are among key mechanisms behind the functional (i.e., the ideal time between smFISH experiments). We compare our results versatility of drug targets, and network rewiring could be an underlying mech- to those that assume Gaussian fluctuations or invoke the central limit theorem. anism of a drug’s adverse effects. This study can be useful for screening target By systematically design experiments to use all of the fluctuation information candidates at the early stages of a drug development pipeline. within modern data, our method enables a key step to improve co-design of ex- periments and models to enhance the efficiency and accuracy of quantitative 1035-Plat systems biology. Dissection of Multiplicity of the GPCR Mediated Signaling Jennifer Chen1, Yue Pan2, Lynn S. Penn1, Jun Xi1. 1032-Plat 1Chemistry, Drexel University, Philadelphia, PA, USA, 2Biology, Drexel Morphology of Embryonic Epidermis: An Empirical Multiscale University, Philadelphia, PA, USA. Biophysics Approach Cells rely on their signaling network to coordinate various downstream effec- Jesse L. Silverberg, Peng Yin. tors in regulation of essential cellular functions, including adhesion, migration, Wyss Institute, Harvard University, Boston, MA, USA. proliferation, and differentiation. Cell signaling network consists of signaling Between nanoscale genotype and mesoscale phenotype, a cascade of physical pathways that often interact in many different ways. Gaining a better under- and biological events take place that translate coded DNA into biological standing of how multiple signaling pathways are coordinated is critical to the

BPJ 8602_8612 206a Monday, February 19, 2018 success of development of medical diagnosis and therapeutic treatment. We time delays to capture reaction intermediates with crystallography and spec- have developed a noninvasive, quantitative approach with the use of the quartz troscopy. We have used the above techniques to study photochemical activation crystal microbalance with dissipation monitoring (QCM-D) for in vitro, real- of the water oxidation reaction of the Photosystem II multi subunit protein com- time examination of the G protein coupled receptor (GPCR)-mediated cell plex, in which the Mn4CaO5 cluster catalyzes the reaction. The current status of signaling. With this approach, we have been able to dissect the multiplicity this research and the mechanistic understanding of this metalloenzyme based of GPCR-mediated cell signaling and obtain mechanistic insight into the dy- on the X-ray techniques is presented. namic coordination and coupling of Gaq,Gas, and Gai pathways. Symposium: Energy Transduction 1039-Symp Efficient Energy Transduction in Respiratory Complexes and Supercom- 1036-Symp plexes Structural Insight into the Role of the Ton Complex in Energy Transduc- Carola Hunte. tion University of Freiburg, Freiburg, Germany. Herve Celia1,2, Nicholas Noinaj3, Stanislov D. Zakarov3, Enrica Bordignon4,5, Istvan Botos1, Monica Santamaria6, Travis J. Barnard1, Symposium: Protein Structure and Dynamics in William A. Cramer3, Roland Lloubes2, Susan K. Buchanan1. the Lipid Bilayer Membrane 1Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD, USA, 2UMR7255 CNRS/Aix-Marseille Universite, Marseille, France, 3Purdue 1040-Symp University, West Lafayette, IN, USA, 4Ruhr-Universitaet Bochum, Bochum, Functional Consequences of Membrane Protein Oligomerization Germany, 5Freie Universit€at Berlin, Berlin, Germany, 6Instituto de Song-I Han, Chungta Han, Matt Idso, Sunyia Hussain. Investigacion Hospital La Paz (IdiPAZ), Madrid, Spain. University of California, Santa Barbara, Santa Barbara, CA, USA. In Gram-negative bacteria, outer membrane (OM) transporters import nutrients It has become increasingly evident that G-protein coupled receptors (GPCRs) by coupling to an inner membrane (IM) protein complex called the Ton com- associate with each other in membranes to form (homo- or hetero-) oligomeric plex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton complexes and that these oligomers broaden the range of cell signaling. A bet- motive force (pmf) at the IM to transduce energy to the OM via TonB. Here, we ter understanding of the factors that drive oligomerization would potentially be structurally characterize the Ton complex from E. coli using X-ray crystallog- key for understanding receptor function. To pursue such ambitious goals of ad- raphy, electron microscopy, DEER spectroscopy, and crosslinking, revealing a dressing structure-function relationship of GPCR oligomers in the future, a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least number of technological and methodological developments are necessary, one TonB. Electrophysiology studies show that the Ton subcomplex forms pH- together with a viable GPCR model to address basic questions regarding the sensitive cation-selective channels, providing insight to the mechanism by functional impact of oligomerization, the lipid membrane environment, and hy- which it may harness the pmf for energy production. dration. We choose the bacterial proteorhodopsin (PR), a 7-helical transmem- brane protein, that serve as an excellent system to test new biophysical tools to 1037-Symp determine their oligomeric state and structure in detergent and lipid mem- Dissociation of the Heterotrimeric G Protein Complex by Nanobodies: Po- branes, as well as to map out its function by multiple methods, including tential uses in the Modulation of Diverse GPCR Signaling pKA of a key Hþ acceptor D97, time-resolved UV/vis absorption spectroscopy Krzysztof Palczewski. of retinal, as well as the light-triggered movement of its E-F loop (also known Case Western University, Cleveland, OH, USA. as ICL3) connecting the 5th and 6th a-helix. I will also present advances we G protein-coupled receptors (GPCRs) comprise the most abundant family of made in developing electron paramagnetic resonance (EPR) methods of cell membrane receptors and share a general mechanism of signal transduction. Gd3þ spin-based labels to sensitively capture multiple distances in the 2-6 GPCRs respond to a wide variety of extracellular signals, including photons, nm regime and Overhauser dynamic nuclear polarization to directly map out ions, lipids, small molecules, peptides, and proteins. Typically, GPCRs activate a membrane and protein surface hydration dynamics. The combination of new heterotrimeric G proteins by mediating GDP to GTP exchange in the subunit. and established tools permits us to pursue important questions: (1) Do lipid This leads to the dissociation of the heterotrimeric into Gaa-GTP and Gbg a bg membrane composition, dynamics and hydration tune oligomerization? (2) Is complexes. While G -GTP facilitates its own signaling events, the -subunits oligomerization a dominant factor in function tuning over that of surfactant can also autonomously exert their effects in cellular signaling. In this study, we bg composition or lipid membrane properties? (3) Do these findings translate used several nanobodies that bind tightly to the -complex causing a receptor- into the study of GPCR oligomerization? I will address these questions in independent dissociation of the G protein. These nanobodies respond to all my presentation. combinations of b- and g-subunits. Further, biochemical, structural, and phys- iological data support a broad application of these genetically encodable nano- bodies to modulate GPCR signaling as well as reveal their potential therapeutic 1041-Symp applications. A (Passive to Active) Chaser: NMR and MD of Membrane Proteins Wonpil Im. 1038-Symp Biological Sciences and Bioengineering, Lehigh University, Bethlehem, PA, Water Oxidation Reaction in Photosystem II Studies with XFELs USA. Junko Yano. I will present and discuss the past and ongoing efforts of my lab on (1) multiple- Lawrence Berkeley National Laboratory, Berkeley, CA, USA. conformer NMR-restrained ensemble dynamics in the context of transmem- The development of XFELs has opened up opportunities for studying the dy- brane helix orientation and dynamics, (2) membrane protein simulations in namics of biological systems. Intense XFEL pulses enable us to apply both various environments (detergent micelles, phospholipid bilayers, and bacterial X-ray diffraction and X-ray spectroscopic techniques to dilute systems or small outer membranes with lipopolysaccharides) with and without NMR restraints, protein crystals. By taking advantage of ultra-bright femtosecond X-ray pulses, and (3) NMR Structure Calculator and other updates in CHARMM-GUI. one can also collect the data under functional conditions of temperature and pressure, in a time-resolved manner, after initiating reactions, and follow the 1042-Symp chemical dynamics during catalytic reactions and electron transfer. Such an Deciphering Transport Mechanisms of Bacterial Efflux Pumps using NMR approach is particularly beneficial for biological materials and aqueous solution Spectroscopy samples that are susceptible to X-ray radiation damage. We have developed Maureen Leninger, Ampon Sae Her, Casey Mueller, James Banigan, spectroscopy and diffraction techniques necessary to fully utilize the capability Nathaniel Traaseth. of the XFEL X-rays for a wide variety of metalloenzymes, and to study their New York University, New York, NY, USA. chemistry under functional conditions. One of such methods is simultaneous Membrane transport proteins provide an important defense mechanism in data collection for X-ray crystallography and X-ray spectroscopy, to look at conferring drug resistance to bacteria. These proteins bind and efflux lethal the overall structural changes of proteins and the chemical changes at metal cat- compounds across the lipid bilayer and in the process reduce drug toxicity in alytic sites. In parallel to the detection techniques, we have also developed an the cytoplasm. Among the smallest known transport proteins are members of efficient sample delivery method that involves deposition of droplets on a the small multidrug resistance (SMR) family composed of four transmembrane conveyor belt. This Droplet on Tape (DOT) method, delivers a single drop domains that assemble into dimers. This family represents an excellent system of the crystal suspension or solution sample onto a tape, which then can be to study ion-coupled transport due to their small size (100-120 residues) and transported to the X ray intersection point with a variable delay in time. In importance in antiseptic resistance and membrane protein evolution. Our the process, the sample is photochemically or chemically activated at various research has focused on EmrE, a member of the SMR family that carries out

BPJ 8602_8612 Monday, February 19, 2018 207a drug efflux as a homodimer by coupling transport with the electrochemical po- helices when both gates are closed simultaneously. This mechanism leads to tential across the inner membrane of E. coli. While it is known that a conserved tight allosteric coupling between gates, preventing them from opening simulta- glutamate residue (Glu-14) within the first transmembrane domain of EmrE neously. Interestingly, the substrate appears to take a ‘‘free ride’’ across the plays a central role in transport, the mechanistic details concerning how this membrane without causing major structural rearrangements in the transporter. anionic residue couples efflux with the pH gradient are not fully understood due to the lack of a high-resolution view of the substrate binding pocket. Our 1045-Plat experiments use a combination of solution and solid-state NMR spectroscopy Neurotransmitter Transporter Conformational Dynamics using HDX-MS to uncover the conformational dynamics and allosteric communication within and Molecular Dynamics Simulation 1 1 2 EmrE that underlie the ion-coupled mechanism. In addition, we offer high- Richard T. Bradshaw , Anu Nagarajan , Suraj Adhikary , 3 3 2 resolution structural insight of EmrE’s binding pocket in lipid bilayers with Daniel J. Deredge , Patrick L. Wintrode , Satinder K. Singh , 1 the goal of defining the multidrug recognition mechanism. Lucy R. Forrest . 1NINDS, National Institutes of Health, Bethesda, MD, USA, 2Department of 1043-Symp Cellular and Molecular Physiology, School of Medicine, Yale University, Unique Insights into the Structural and Functional Biology of Membrane New Haven, CT, USA, 3Department of Pharmaceutical Sciences, School of Proteins from Solid State NMR Spectroscopy Pharmacy, University of Maryland, Baltimore, MD, USA. Timothy Cross1, Joana Paulino1, Huajun Qin2, Yiseul Shin2, Experimental observations of protein dynamics frequently make use of protein Cristian Escobar3, Rongfu Zhang2, Joshua Taylor2, Yimin Miao4, variants with suitably located covalent labels. For mammalian neurotransmitter Riqiang Fu1, Eduard Chekmenev5, Ivan Hung1, Zhehong Gan1, transporters, however, the difficulty in constructing and functionally verifying Petr Gor’kov1. these mutant systems has hampered such investigations. Hydrogen-deuterium 1Natl High Mag Field Lab, Florida State University, Tallahassee, FL, USA, exchange coupled to mass spectrometry (HDX-MS) is an attractive method 2Chemistry and Biochemistry, Florida State University, Tallahassee, FL, for investigating dynamics of fully active transporters, reconstituted into a real- USA, 3Institute of Molecular Biophysics, Florida State University, istic membrane lipid environment, without such modifications. An atomistic Tallahassee, FL, USA, 4City University of New York, New York, NY, USA, rationale for HDX-MS observations can be provided by complementary 5Vanderbilt University, Nashville, TN, USA. computational predictions of deuteration rates. Nevertheless, although success- The Influenza A M2 protein is an extensively studied tetrameric membrane fully applied previously to globular proteins in aqueous solvent, it remains un- protein whose structure in liquid crystalline lipid bilayers from solid state clear whether these computational approaches are transferable to large NMR (ssNMR) has been the basis for detailing the proton conductance mech- transporter proteins in the diverse membrane environment. anism, and has illuminated the role of M2 in the viral budding process, where Here we have used HDX-MS to measure backbone deuteration of the bacterial it takes advantage of cholesterol. Specific proton histidyl proton exchange amino acid transporter LeuT as a model membrane protein system. HDX-MS rates with water have been measured distinguishing futile exchange in which experiments and molecular dynamics (MD) simulations compared LeuT in the proton binds and dissociates from the same site versus protonation of one two distinct conformational ensembles, representing extracellular- or intracel- site and deprotonation of another that could lead to proton conductance. CrgA lular-open states in the transport cycle. Simulations, analyzed with an empirical is a protein associated with the cell division apparatus of Mycobacterium model for estimating deuteration rates from the accessibility of backbone amide tuberculosis. The two helix transmembrane (TM) structure and orientation groups, have shown good agreement with the experimental deuteration data, in liquid crystalline lipid bilayers has been characterized by ssNMR. The and highlight areas of divergence between the outward and inward-facing en- N-terminus is largely disordered except for a short sequence of b-strand close sembles (Adhikary et al., PNAS, 2017, 114, E1786). to the initiation of the first TM helix. CrgA represents the first example of a b- Despite the overall good agreement, some discrepancies were observed be- strand stabilized helical membrane protein dimer. At very high field ssNMR tween predicted & experimental deuteration rates for the TM1a and EL4 pro- of 17O has greatly enhanced sensitivity and resolution. While the high resolu- tein segments. These were investigated in detail to identify deficiencies in tion structure of gramicidin A (gA) was the first peptide or protein character- either the (empirically parameterized) deuteration rate model, or the sampling ized in a liquid crystalline lipid bilayer 25 years ago, the dimer was shown to accessible during our MD timescales. Our results emphasize the potential of ‘perfectly’ symmetric. Not so, now that we have high resolution 17Odataob- HDX-MS complemented with simulations as a strategy for probing conforma- tained 35.2 T (1500 MHz for 1H) that can characterize the sites water and tional dynamics of membrane proteins and highlight improved strategies for cation binding. Single carbonyl 17O labeled gA associated with the cation applying this approach to clinically relevant mammalian neurotransmitter binding site clearly shows two resonances (one from each monomer even in transporters, such as SERT, DAT or NET. the absence of cations suggesting that the water interactions at one binding site influences the water interactions in the other monomer binding site 1046-Plat 20A˚ away. Transport Pathways in Membrane Transporters Sayane Shome1, Edward Yu2, Robert Jernigan3. Platform: Molecular Dynamics I 1Bioinformatics and Computational Biology, Iowa State University, Ames, IA, USA, 2Department of Pharmacology, Case Western Reserve University, 1044-Plat Cleveland, OH, USA, 3Biochemistry, Biophysics, and Molecular Biology, Mechanism of Substrate Translocation in an Alternating Access Trans- Iowa State University, Ames, IA, USA. porter Substrate transport through membrane transporters is critical for many bio- Naomi R. Latorraca, Nathan M. Fastman, Liang Feng, Ron O. Dror. logical processes. One of the most interesting questions is how to understand Stanford University, Stanford, CA, USA. the substrate specificity of transporters. Due to the limitations of experi- Transporters shuttle molecules across cell membranes by alternating among mental methods, computational approaches can be applied advantageously distinct conformational states. Despite a wealth of structural data supporting to screen a large number of possible transported molecules. The experi- the existence of such states, a critical challenge in the field is to capture tran- mental determination of the mechanistic details of transport is difficult. sitions from one conformational state to another, providing insights into how We have employed steered molecular dynamics simulations to determine structural rearrangements regulate substrate translocation. Molecular dynamics the critical factors responsible for the transport and how they interact with simulations represent a powerful means for observing such transitions. Howev- protein components along the pathway. Systems that we have investigated er, many commonly studied transporters, including members of the GLUT and include the transport of: 1) sulfonamide drugs by the AbgT transporter SGLT families, are large (> 50 kDa) and may transport more than one sub- YdaH protein, 2) inorganic carbon (CO2 and bicarbonate ion) by the Low strate, making their spontaneous conformational changes difficult to observe CO2 inducible protein Lci1 and 3) long-chain cyclic lipids (Hopanoid and with unguided simulation. Here, we performed over 175 ms of unguided molec- Steroid) by the RND-like HpnN protein. VMD software has been used. ular dynamics simulations of SemiSWEET, a minimal, model transporter, to Protein-embedded lipid systems were minimized for 250,000 steps, followed capture the substrate translocation process, providing an atomic-level descrip- by equilibration until the system temperature reaches 310 K. Steered Molec- tion of alternating access transport. Simulations of this transporter initiated ular dynamics simulations at constant velocity (cv-SMD) were carried out from an outward-open, glucose-bound structure spontaneously adopt occluded on the equilibrated systems via NAMD, with the direction and magnitude and inward-open conformations (Latorraca, Fastman et al., Cell 2017). Strik- of the pulling being adjusted for the system of interest. Based on the simu- ingly, both of these simulated conformations match existing crystal structures, lation trajectories, we have determined the transport pathway through the including a new inward-open structure. Our results reveal that state transitions protein for the ligand transport in these three cases, as well as the roles of are driven by favorable interactions formed upon closure of extracellular and functionally important residues along the pathway. These pathways have intracellular ‘‘gates’’ and by an unfavorable configuration of transmembrane been confirmed by experimental findings.

BPJ 8602_8612 208a Monday, February 19, 2018

1047-Plat 1049-Plat Virtual Dengue Virus: The INS and OUTS Molecular Mechanism of AB42 Peptide-Fibril Adsorption Jan K. Marzinek1,2, Roland G. Huber1, Daniel Holdbrook1, Mathias M.J. Bellaiche1,2, Tuomas P.J. Knowles2, Robert B. Best1. Priscilla L.S. Boon1,3, Adelene Y.L. Sim1, Ana S. Martins4, Wuan G. Saw5, 1Laboratory of Chemical Physics, National Institutes of Health, Bethesda, Ivo C. Martins4, Ganesh S. Anand2, Gerhard Gruber€ 5,1, Shee-Mei Lok6,7, MD, USA, 2Chemistry, University of Cambridge, Cambridge, United Thorsten Wohland2,7, Yue Wan8, Chandra Verma1,2, Peter J. Bond1,2. Kingdom. 1Bioinformatics Institute (BII) A*STAR, Singapore, Singapore, 2Department Alzheimer’s Disease is quickly becoming the most pressing medical issue fac- of Biological Sciences, National University of Singapore, Singapore, ing a globally ageing population, due in large part to a lack of understanding of Singapore, 3NUS Graduate School for Integrative Sciences and Engineering, the fundamental physics and chemistry responsible for the pathogenesis. The National University of Singapore, Singapore, Singapore, 4Instituto de disease is associated with the aggregation of soluble amyloid-beta (AB) protein Medicina Molecular, Faculdade de Medicina da Universidade de Lisboa, into fibrils and has been found experimentally to demonstrate a nucleation- Lisbon, Portugal, 5School of Biological Sciences, Nanyang Technological elongation polymerisation mechanism, in which the rate-determining associa- University, Singapore, Singapore, 6Program in Emerging Infectious Diseases, tion of few protein monomers into a growth-competent nucleus is followed Duke–NUS Graduate Medical School, Singapore, Singapore, 7Centre for by fast incorporation of additional soluble subunits into insoluble quaternary BioImaging Sciences, National University of Singapore, Singapore, complexes. The reaction cascade is strongly accelerated by secondary nucle- Singapore, 8Stem Cell and Regenerative Biology, Genome Institute of ation, in which the surfaces of existing fibrils drive the formation of further Singapore (GIS) A*STAR, Singapore, Singapore. oligomers and fibrils. This autocatalysis is the positive feedback mechanism Positive single-stranded RNA flaviviruses such as dengue and Zika are by which Alzheimer’s Disease becomes fatal, and as such is critical to under- primary causative agents of infectious disease. The dengue viral core stand in order to be able to design therapeutics. Here we use long all-atom, consists of capsid proteins complexed with the RNA genome, encapsu- explicit-solvent molecular dynamics simulations with a novel enhanced sam- lated by a lipid/protein envelope. A wealth of biophysical data has pro- pling scheme, based on scaling solvation free energy, to probe the interaction vided key insights into the organization of proteins on the virion surface, of a free AB monomer with an AB42 fibril. In doing so we identify possible but the architecture of the lipid membrane and enclosed nucleocapsid binding epitopes on both species, probe the thermodynamics of adsorption core remain unresolved. Furthermore, ongoing hydrogen-deuterium ex- and propose an overall enthalpically driven mechanism of fibril-catalysed change mass spectrometry (HDX-MS) and fluorescence studies indicate peptide collapse that is consistent with experimental findings. Our work is that the virion particle is extremely dynamic, transitioning from important methodologically as we improve on the accuracy and sampling of ‘‘smooth’’ to expanded ‘‘bumpy’’ states in response to environmental peptide interaction simulations, as well as biophysically and biochemically triggers such as changes in temperature or salt, and to the infective as we provide residue-level resolution of a medically salient macromolecular ‘‘spiky’’ form upon a drop in pH within the endosome. In order to fill interaction. existing gaps in the molecular understanding of the viral life cycle, we have integrated structural, biophysical, and genomic experiments 1050-Plat with multiscale modelling, towards the ‘‘virtual dengue virus’’. High- Initial Steps in the PI(4,5)P2 dependent Fibroblast Growth Factor 2 Olig- throughput sequencing (HTS) with chemical probing provided a means omerization 1,2 1 ¨ 3 4 to constrain hierarchical 3D models of the 11kb RNA genome core Fabio Lolicato , Chetan Poojari ,Unal Coskun , Walter Nickel , Ilpo Vattulainen1,2. within the virion. Simulations of the intrinsically disordered capsid pro- 1 2 tein were validated via small-angle X-ray scattering (SAXS), and its University of Helsinki, Helsinki, Finland, Tampere University of Technology, Tampere, Finland, 3Paul Langerhans Institute Dresden, RNA interaction sites mapped via a nuclease digestion assay. Outside 4 of the nucleocapsid complex, reconstruction and simulation-based refine- Dresden, Germany, Heidelberg University Biochemistry Center, ment against cryo-EM maps of the complete 50 nm particle in Heidelberg, Germany. different states of maturation yielded structural insights into envelope The conventional protein secretion process usually starts with the transpor- dynamics, and provided a platform to explore in near-atomic resolution tation of the signaling protein into the inner layer of the endoplasmic retic- the environmentally-triggered processes of virion expansion and endoso- ulum (ER). The secretory protein is then transported to the cell surface via mal membrane fusion. Finally, simulations guided by structural measure- vesicular transport. However, many proteins can be secreted independently ments of virion envelope intermediates inform on how host interactions from the ER-to-Golgi pathway without a signal recognition peptide. Fibro- such as antibodies may facilitate infectivity, with potential consequences blast Growth Factor II (FGF2) is one of those. The unconventional secretory for future therapeutics development. pathway process secreting FGF2 from cells depends on the interactions of FGF2 with PI(4,5)P2 lipids and heparan sulfate proteoglycans at the inner and outer leaflet of the plasma membrane, in respective order. Here, we 1048-Plat used atomistic molecular dynamics simulations to shed light on how Release of Empty Nanodiscs from Charged Droplets in the Electrospray PI(4,5)P2 triggers the oligomerization of FGF2 on the membrane surface. Ionization Process: A Molecular Dynamics Study We demonstrated that the amino acids K127, R128, and K133 that in exper- Beibei Wang1,2, Peter Tieleman1. imental studies have been found to be part of a binding pocket interacting 1Department of Biological Sciences and Centre for Molecular Simulation, with the head group of PI(4,5)P2 are not the only ones able to recruit University of Calgary, Calgary, AB, Canada, 2Center for Informational PI(4,5)P2. Instead, simulations brought up that FGF2 monomers can simul- Biology, University of Electronic Science and Technology of China, taneously interact with several PI(4,5)P2 molecules through additional resi- Chengdu, China. dues (K34, K137, K143) that contribute to the binding process. These Electrospray ionization (ESI) prevents the analyte from being split into interactions gave rise to the high-affinity orientation of FGF2 that fosters fragments and is widely used to analyze biomolecules. Nanodiscs provide its dimerization through the formation of intermolecular disulfide bridges. a native-like environment for membrane proteins, while making them Extensive simulations confirmed dimerization through the C95-C95 bridge, accessible in solution for analysis. We used molecular dynamics simula- in agreement with experimental data. The importance of ion pairs in the sta- tions to provide atomistic insight in the release of intact nanodiscs from bilization of the dimer interface was also evident. The findings highlight that charged droplets. This process proceeds overall according to the charged the FGF2 dimers containing the C95-C95 disulfide bridge represent the residue model, i.e., solvent evaporates leaving a gaseous ion. We observed initial step of PI(4,5)P2 dependent FGF2 oligomerization [1]. two distinct main scenarios, at-center and off-center. In the at-center pro- [1] J.P. Steringer et al., eLife 6, e28985 (2017). cess, the nanodisc stays well in the droplet interior, keeping its original geometry. As solvent evaporates, lipids turn over to protect the hydrophil- 1051-Plat ic surface. In the off-center process, by contrast, the nanodisc migrates to Understanding how Beta-Hairpins Fold using Molecular Dynamics Simu- the water/air interface, leading to lipids escaping from the dissociated lations in Multiple Force Fields dimer. The ESI charge states of the gaseous ions are higher than found Brooke E. Husic, Keri A. McKiernan, Vijay S. Pande. experimentally. By extrapolating our data to lower charge states, our re- Chemistry, Stanford University, Stanford, CA, USA. sults are in excellent agreement with the experimental mass spectra. Understanding how protein substructures fold is a crucial building block for Further simulations without long-range electrostatic interactions or for studying large, complex protein systems. The folding mechanism of beta- neutral droplets confirmed that the electrostatic interactions play an essen- hairpins has been a controversial topic over the past two decades, and many tial role in the behavior of nanodiscs in the ESI process and the shape of contradictory mechanisms have been proposed from both experimental and the resulting gaseous ions. computational studies. Early computational studies of beta-hairpins were

BPJ 8602_8612 Monday, February 19, 2018 209a performed because of computational limits on larger structures; now, increased activates FtsY for downstream steps. Importantly, mutational analyses revealed computational power enables us to completely dissect and understand protein extensive auto-inhibitory mechanisms that prevent free FtsY from engaging substructures on the atomic level with statistically robust methods. Here, we membrane in the Stable mode; an engineered FtsY pre-organized into the Sta- analyze an aggregated CLN025 folding simulation dataset with simulations ble mode led to indiscriminate targeting in vitro and disrupted FtsY function in from several different force fields to study the beta-hairpin formation mecha- vivo. Our results show that the two-step lipid-binding mechanism uncouples nism in a force-field agnostic way. We use Markov state models (MSMs) to the membrane association of FtsY from its conformational activation, thus opti- determine that the extended state first undergoes hydrophobic collapse, and mizing the balance between the efficiency and fidelity of co-translational pro- from this collapsed structure the hairpin turn is formed, which is shown to be tein targeting. the rate-limiting step. These mechanistic conclusions as well as the timescales of the turn formation and hydrophobic collapse events are consistent with 1054-Plat experimental results for the order of events in CLN025 folding, which allows Insight into Coupled Binding and Folding in Insulin Dimer Association us to see a highly detailed process that matches up with coarser experimental from T-Jump Induced Dissociation Experiments data. We are also able to monitor the order of hydrogen bond formation in Xinxing Zhang, Andrei Tokmakoff. atomic detail. This work highlights the possibility of designing MSMs to corre- James Frank Institute, the University of Chicago, Chicago, IL, USA. spond to existing experimental data, to incorporate data from multiple indepen- Molecular recognition in protein-protein interactions is a crucial element of the dent simulations in different force fields, and to coarse-grain models such that biological functions of proteins, involving a number of remarkable conforma- they present interpretable descriptions of beta-hairpin folding that can be veri- tional changes. The association of insulin into a homodimer is a particularly fied by experiment. Finally, this work lends insight into the larger protein interesting example due to the coupled folding and binding processes involved. folding problem by demonstrating a natural means for experiment and modern After initiating insulin dimer dissociation with a nanosecond temperature jump, m computational approaches to come together to better understand biophysical transient infrared spectra show a fast and a slow response that occurs on sub- s phenomena: namely by the coupling of advanced theory and modern force and sub-ms time scales. Using a rate distribution map analysis reflecting both fields with experimental validation. spectral and time evolution, we assign these processes to the partial melting of a helical structure followed by the interfacial b sheet unfolding. Rate distri- Platform: Protein Dynamics and Allostery I butions variations are observed by varying the initial temperature, pH, and co- solvent. To extract thermodynamic parameters and kinetic rate constants, the T- 1052-Plat jump data is analyzed self-consistently with FTIR, CD, and isothermal titration The Conformational Landscape of SMC: A FRET Study calorimetry. By analyzing kinetic models, we conclude that insulin dimer Gemma L.M. Fisher1, Benji C. Bateman2, Timothy D. Craggs3, dissociation requires significant conformational changes within the monomer Mark S. Dillingham1. in concert with the folding of the interfacial b sheet in order to dimerize. 1School of Biochemistry, University of Bristol, Bristol, United Kingdom, 2Central Laser Facility, Rutherford Appleton Laboratory, Didcot, United 1055-Plat 3 Dynamics of Human Telomerase Kingdom, Department of Chemistry, University of Sheffield, Sheffield, 1 2 3 1 United Kingdom. Linnea Jansson-Fritzberg , Joseph Parks , Rhiju Das , Michael Stone . 1UC Santa Cruz, Santa Cruz, CA, USA, 2University of Colorado Boulder, In concert with its loading partner ParB, the Bacillus subtilis SMC complex is 3 important for nucleoid compaction and efficient chromosome segregation. Like Boulder, CO, USA, Stanford University, Santa Cruz, CA, USA. all SMC proteins, it displays an extraordinary architecture comprising ‘‘hinge’’ Telomerase is a specialized reverse transcriptase that maintains the ends of and ATP-hydrolysing ‘‘head’’ domains, connected by 50 nm long coiled coils. linear chromosomes in order to counteract the end replication problem. Over SMC monomers associate to form dimers which adopt a range of conforma- 90% of cancers over-express telomerase, leading it to be an attractive drug tions, as observed for example in AFM and EM studies. Dimerization occurs target for cancer therapeutics. While the general mechanism of telomerase at the head and hinge domains, with the former controlled by binding of the reg- catalysis is understood, the structure of human telomerase and the mechanism of the various steps in the telomerase catalytic cycle remain unknown. Human ulatory subunits ScpAB2 and ATP, thereby forming a huge proteinaceous ring. Models for SMC function often invoke the idea that this ring is topologically telomerase has remained a difficult enzyme to structurally characterize due to engaged with the chromosome. This could occur by breaking and resealing its naturally low abundance and requirement for multiple cofactors and chaper- the SMC ring (V-shaped conformations) or by threading an O-shaped ring ones to fold properly. Because of this, telomerase is not amenable to large scale onto a DNA loop, possibly followed by the closing of the coils to form a clamp expression and reconstitution. Single molecule techniques are ideally-suited for (I-shaped conformation). It has been widely presumed that such conformational the study of telomerase because of the need for only small amounts of material transitions in SMC are driven by the ATP hydrolysis cycle. and the capacity to analyze dynamics of individual enzymes. We have devel- We present single-molecule and stopped-flow FRET analysis of full-length oped a single molecule FRET structure probing assay that permits direct anal- SMC, specifically labelled at the heads, coiled coils or hinges. Our data ysis of telomerase in multiple functional states. Recently, we have used this show that for SMC alone: (1) the hinges are closed for SMC dimerization, approach paired with automated Rosetta modeling to map conformational (2) the heads are in close proximity prior to full engagement upon ATP binding changes in the essential RNA pseudoknot domain within the active telomerase and (3) the coiled-coil arms occupy a range of conformations, in which they complex. We are currently extending this approach using site-specific protein may be juxtaposed (I-shaped) or somewhat further apart (O-shaped). Upon labeling to monitor conformational changes in evolutionarily conserved do- mains within the telomerase protein component. addition of ATP and ScpAB2 the heads undergo small local structural rear- rangements, which propagate to a larger opening of the coiled-coil arms, whilst 1056-Plat the hinges remain closed. Our data support the idea that ATP binding and hy- Visualization of Asymmetric Structure of Ca2D/Calmodulin-Dependent drolysis promotes transition to an O-shaped conformation, and are inconsistent Protein Kinase II Oligomers by High-Speed Atomic Force Microscopy with functional models involving the opening of the homodimer at the hinge Mikihiro Shibata1,2, Hideji Murakoshi3,4. domain. 1Institute for Frontier Science Initiative, Kanazawa Univiersity, Kanazawa, Japan, 2Bio-AFM Frontier Research Center, Kanazawa University, 1053-Plat Kanazawa, Japan, 3Supportive Center for Brain Research, National Institute Two-Step Membrane Binding by the Bacterial SRP Receptor Enables Effi- for Physiological Sciences, Okazaki, Japan, 4Prest, JST, Kawaguchi, Japan. cient and Accurate Co-Translational Protein Targeting Ca2þ/calmodulin-dependent protein kinase II (CaMKII) is especially en- Yu-Hsien Hwang Fu1, William Y.C. Huang2, Kuang Shen1,3, Jay T. Groves2, riched in the nervous system. Pharmacological analysis or knock-out Thomas Miller1, Shu-ou Shan1. mice of CaMKII revealed that CaMKII plays an important role for the 1Chemistry, California Institute of Technology, Pasadena, CA, USA, memory formation. Because CaMKII forms dodecameric holoenzyme 2Chemistry, University of California at Berkeley, Berkeley, CA, USA, that responds to the frequency of the activating signal of Ca2þ,ithas 3Whitehead Institute, Cambridge, MA, USA. been believed that oligomeric structures of CaMKII and its structural The signal recognition particle (SRP) delivers 30% of the proteome to the eu- changes are key elements of a memory mechanism. However, because karyotic endoplasmic reticulum, or the bacterial plasma membrane. The precise of a lack of experimental techniques, there is no direct evidence that dy- mechanism by which the bacterial SRP receptor, FtsY, interacts with and is namics of CaMKII oligomers relates to a memory mechanism. Here we regulated at the target membrane remain unclear. Here, quantitative analysis used high-speed atomic force microscopy (HS-AFM) to directly visualize of FtsY-lipid interactions at single-molecule resolution revealed a two-step the dynamics of CaMKII protomers in oligomeric structures. HS-AFM is mechanism in which FtsY initially contacts membrane via a Dynamic mode, a unique technique to visualize the conformational changes of proteins at followed by an SRP-induced conformational transition to a Stable mode that the single molecule in the near physiological conditions [1-3]. HS-AFM

BPJ 8602_8612 210a Monday, February 19, 2018 images of full-length CaMKIIa showed two typical structures. One is and thus, complement the DENV structural studies. Both, viral structure centered solid structures, which were 15 nm in diameter and 10 nm and its related dynamics, are important determinants in viral activity and in height. Another is surrounding globular structures, which fluctuated their knowledge can open new path in vaccine design. dramatically. Using truncated CaMKIIa expressed the hub domain, centered and surrounding structures were assigned to the hub and the ki- 1059-Plat nase domains which are connected by linkers, respectively. Interestingly, High-Resolution Neutron Scattering Data Reveal the Decoupling of Pro- symmetrical dodecamer or tetradecamer ring structures were observed teins and Water at the Dynamical Transition a Antonio Benedetto1,2. only in truncated CaMKII (mainly tetradecamer), while asymmetrical do- 1 2 decamer ring structures were observed in full-length CaMKIIa.Inthepre- School of Physics, University College Dublin, Dublin, Ireland, Laboratory sentation, we would like to discuss the relationship between the function for Neutron Scattering, Paul Scherrer Institut, Villigen, Switzerland. and asymmetrical structures of CaMKII oligomers. [1] M. Shibata et al. The dynamics of a model protein (Lysozyme) and its hydration water have been Nat. Nanotech. 5, 208-212 (2010). [2] T. Ando et al. Chem. Rev. 114, investigated by neutron scattering at very high-energy resolutions, up to 3 times 3120-3188 (2014). [3] M. Shibata et al. Nat. Commun. in press. better than the best ones used until now (0.3 ueV versus 1 ueV). Because of this improvement, I was able to measure the decoupling between water and protein 1057-Plat dynamics in proximity of the protein dynamical transition [1]. This decoupling, Molecular Switch-Like Regulation Enables Global Coordination in a Viral never observed before, is a clear sign that the transition in the dynamic of hy- Ring ATPase dration water does not directly drive a corresponding change in the dynamic of Sara Tafoya1, Shixin Liu2, Juan P. Castillo3, Rockney Atz4, Marc Morais5, proteins. As a result, the scenario generally accepted during the last 30 years Grimes Shelley4, Paul Jardine4, Carlos Bustamante6. has eventually been challenged, and a new theory on the connection between 1Biophysics PhD program, University of Californa, Berkeley, Berkeley, CA, these two dynamics and protein function has to be formulated. USA, 2Rockefeller University, New York City, NY, USA, 3University of [1] A. Benedetto, Low-Temperature Decoupling of Water and Protein Dy- Californa, Berkeley, Berkeley, CA, USA, 4Department of Diagnostic and namics Measured by Neutron Scattering, J. Phys. Chem. Lett. (2017), 8, Biological Sciences and Institute for Molecular Virology, University of 4883-4886, DOI: 10.1021/acs.jpclett.7b02273. Minnesota, Minneapolis, MN, USA, 5Department of Biochemistry and Molecular Biology, University of Texas, Galveston, TX, USA, 6Department Platform: Muscle and Motors Biophysics of Molecular and Cell Biology, Chemistry and Physics, University of California, Berkeley, Berkeley, CA, USA. 1060-Plat Subunits in multimeric ring-shaped motors must coordinate their activities ATPase Cycle Analysis Predicts that Mutations Linked to Dilated Cardio- to ensure correct and efficient performance of their mechanical tasks. myopathy in Human Beta Myosin Will Impair Force Generation Here, we study wild-type and arginine finger mutants of the pentameric Michael Geeves1, Zoltan Ujfalusi2, Carlos Vera3, Srbolujub Mijailovich4, bacteriophage 429 DNA packaging motor. Our results reveal the molec- Marina Svicevic5, Leslie Leinwand3. 1 2 ular interactions necessary for the coordination of ADP-ATP exchange Biosciences, University of Kent, Canterbury, United Kingdom, Biophysics, 3 during the dwell phase and ATP hydrolysis during the burst phase of University of Kent, Pecs, Hungary, Molecular & Developmental Biology, 4 the motor’s mechano-chemical cycle. We show that two distinct regulato- University of Colorado, Boulder, CO, USA, Biology, Illinois institute of 5 ry mechanisms determine this coordination. In the first, an arginine finger Technology, Chicago, IL, USA, Sciences, University of Kragujvac, in each subunit promotes ADP-ATP exchange inand stimulates ATP hy- Kragujevac, Serbia. drolysis ofits neighbor. Accordingly, we suggest that the subunits We expressed and studied the motor domain of human b-cardiac myosin car- perform the roles described for GDP exchange factors and GTPase- rying one of 5 Dilated CardioMyopathy (DCM) mutations in mouse C2C12 activating proteins observed in small GTPases. In the second mechanism, cells. Using the purified protein we completed a biochemical kinetic analysis the DNA up-regulates a single subunit, transforming it into a global regu- of the individual events in the ATPase cycle. This revealedl that each mutation lator that initiates the dwell and burst phases. We propose that these alters different steps in the cycle depending upon its location in the motor mechanisms are fundamental to inter-subunit coordination and are likely domain. For example different mutation have enhanced or reduced ATP, present in other ring ATPases. ADP or actin affinity, enhanced or reduced rate constants of ATP binding, ATP hydrolysis or ADP release. Thus local effects dominate with no common pattern for the changes observed that could account for the similar clinical 1058-Plat phenotype resulting from each mutation. Nor is there a distinct set of changes Dengue Virus Strain 2 Conformations and its Structural Dynamics - Roles that distinguish DCM mutations from the Hypertrophic CardioMyopathy of Divalent Ions and Temperature (HCM) mutations we have previously analysed. However when using the 1 1 1 Kamal Kant Sharma , Xin-Xiang LIM , Sarala N. Tntirimudalige , data for each step in the cycle to model the complete ATPase contraction cycle 1 1,2 1,3 1,3 Anjali Gupta , Jan K. Marzinek , Xin Yin Elisa LIM , Shee-Mei Lok , some themes emerge. All DCM linked mutations result in a lower duty ratio 1,2 1 1,4 Peter J. Bond , Ganesh S. Anand , Thorsten Wohland . due to reduced occupancy of the force holding, AM.ADP complex in the 1Biological Sciences, National University of Singapore, Singapore, 2 steady-state. Under load the AM.ADP complex is predicted to increase due Singapore, Bioinformatics Institute (A*STAR), National University of to a reduced rate constant for ADP release, and this effect is blunted for each Singapore, Singapore, Singapore, 3Graduate Medical School, Duke-National 4 DCM mutation. These results predict that sarcomeres expressing myosin University of Singapore, Singapore, Singapore, Chemistry, National with DCM mutations would have impaired force generation & force holding University of Singapore, Singapore, Singapore. capacity and a lower efficiency of ATP usage compared to wild type protein. Dengue is a mosquito-borne virus with dire health and economic impacts. This result is the opposite of that we reported recently for a HCM mutation, Among its five serotypes, Dengue 2 (DENV2) is the most virulent strain R453C (Mijailovich et al 2017 Biophys J 112: 984-996) and the same is shoiwn responsible for an estimated 390 million infections per year. Interest- here for the R403Q HCM linked mutation. Grant: NIH-R01 GM29090 ingly, it is also for strains of this serotype that large scale conformational changes, called ‘‘breathing’’ have been observed. Although, the structure of 1061-Plat these conformations has been solved to 3.5 A˚ resolution the dynamics of The Enzymatic Activity and Cellular Localization of Drosophila Myosin 7a viral envelope are unknown. Here, we combine fluorescence and mass is Regulated by a Novel Binding Protein spectroscopy and computational studies to provide insights into DENV2 Rong Liu1, Verl Siththanandan1, Yi Yang2, Amy Hong1, Fang Zhang1, structural dynamics in comparison to DENV1. Using amide hydrogen/ Xufeng Wu1, Neil Billington1, Yasuharu Takagi1, James R. Sellers1. deuterium exchange mass spectrometry (HDXMS), we captured DENV2 1National Institutes of Health, Bethesda, MD, USA, 2Human Agricultural Envelope-protein motifs that undergo either temperature or divalent-ions University, Changsha, China. dependent conformational reorganization. While using time resolved Fo¨r- All myosin 7 isoforms have a conserved motor domain, 5 IQ motifs, a putative ster Resonance Energy Transfer (trFRET), we measured the extent of coiled-coil motif, and two MyTH4-FERM domains separated by an SH3 such reorganization. Furthermore, using FRET fluctuation spectroscopy domain. Myosin 7a was found in regions of high actin density such as the ster- (FRETfs), we delineated structural fluctuation dynamics of virus in 0.9 eocilia of the hair cells and bristles in flies. We have previously shown that the ms and 2.3 ms range and correlated virus dynamics to the virus infec- MgATPase of myosin 7a from Drosophila requires large amounts of actin to tivity. We observe hitherto unseen conformational changes and structural obtain Vmax (KATPase = 30 uM). Electron microscopy (EM) reveals that dynamics of DENV2 that are influenced by both, temperature and divalent baculovirus-expressed myosin 7a is single-headed and the tail bends back ions. Our results show, at least for DENV2 and DENV1, that the structural upon the head. EM and truncation experiments suggest that an interaction be- dynamics but not the viral conformations are correlated to viral infectivity tween the third subdomain (termed MyTH7) of the second MyTH4-FERM

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domain and the myosin head are responsible for this regulation. A yeast-two- load-dependence of the power and its maximum (6aWatF 0.3 F0, V 1 hybrid screening uncovered a myosin 7a binding partner, termed M7BP by us- mm/s). A kinetic model fit to mechanical and energetic outputs of fast mamma- ing the last FERM domain as bait. M7BP has a C-terminal myosin binding lian muscle explains the power and the efficiency of the machine. The results domain and a putative Rab binding domain at the N-terminus. M7BP binds show how the degree of coordination between motors varies depending on to the MyTH4-FERM domain of myosin 7a and activates myosin’s MgATPase the mechanical conditions and demonstrate the power of this novel tool for activity at low actin concentration (KATPase =2 mM), suggesting that M7BP quantitative assays on mutant or engineered motor proteins. Supported by may unfold myosin 7a. Co-expression of GFP-myosin 7a and M7BP- IIT-SEED, Genova (Italy) and ECRF, 2015 (Italy). mCherry in Drosophila S2 cells resulted in a marked shape change with large areas of ruffling membrane, extensive polymerization of actin within the cell 1064-Plat body, and the extension of numerous filopodia. The two proteins are exten- Myosin Va Vesicular Transport is Modulated by Actin Filament Density, In Vitro sively co-localized and are found predominantly in actin-rich regions, Orientation, and Polarity in an 3D Actin Network 1 1 1 including filopodia. There is a tendency for the two proteins to be localized Andrew T. Lombardo , Shane R. Nelson , Guy G. Kennedy , 1 2 1 near the tips of filopodia and large punctuates of fluorescence can be observed Kathleen M. Trybus , Sam Walcott , David M. Warshaw . 1Molecular Physiology and Biophysics, University Of Vermont, Burlington, moving in both directions in these structures. Present findings support that the 2 novel drosophila myosin 7a binding-partner M7BP activates its ATPase enzy- VT, USA, Mathematics, University Of California, Davis, Davis, CA, USA. matic activities and motor functions. The cell’s dense three dimensional (3D) actin filament network presents an obstacle course that physically and directionally challenges vesicular transport 1062-Plat via teams of Myosin Va (MyoVa) motors. Complicating matters is that actin Challenges in TIRF-Microscopy Based Single Molecule ATPase and Bind- filament density, orientation and polarity within such networks can vary within ing Assays for Myosin and Actin a cell. To define how a MyoVa team maneuvers its cargo through such complex Alf Mansson, Marko Usaj. networks, we created actin filament networks in vitro that were suspended from Linnaeus University, Kalmar, Sweden. silica structures and then characterized the 3D trajectories of fluorescent, Total internal reflection fluorescence (TIRF) microscopy has permitted 350nm fluid-like, lipid vesicles transported by a MyoVa team (10 motors). visualization of myosin and actin mechanochemistry on a single- Every filament’s 3D spatial orientation was defined using STORM microscopy molecule level. It has, for instance, been possible to study complex phe- as was its polarity (plus ends), as reported by the movement of single MyoVa. nomena such as temporal relationship between ATP turnover and the force Model networks of randomly oriented actin filaments were assembled as well producing transition. However, despite quite large number of studies using as branched actin networks using the Arp2/3 complex to create an inherent fluorescent ATP analogs, several challenges remain in the interpretation of plus end polarity bias. For every vesicle, its modes of motion (i.e., stationary, TIRF data in terms of ATP binding to the active site of myosin. We here diffusive-like, directed) and the spatial orientation/polarity for each filament present improvements in assay conditions for more reliable detection of the vesicle engaged during its trajectory were used to constrain an in silico ATP on-time (i.e. dwell time) at the active site(s) of myosin using ap- model (Lombardo et al., 2017). The model predicts that the modes of vesicular proaches to suppress fluorophore blinking events related to the photophy- motion result from tug-of-wars between motors on the vesicle surface that are sics of the fluorescent probe. We also suggest assay designs for analysis engaged with different filaments. For example, directed vesicular transport is of myosin-binding to actin filaments on a surface in the presence of nucle- enhanced when filament polarity is biased, as in Arp2/3-based networks, where otide. The experiments were performed using a custom built TIRF micro- force vectors are predicted to be aligned and cooperative for engaged motors on scopy platform and fluorescent Alexa Fluor 647 ATP (Alexa-ATP) whose separate filaments. As filament density in the network increases, a bias in fila- turnover to Alexa Fluor 647 ADP (Alexa-ADP) and inorganic phosphate ment polarity is even more critical for directed transport. This model transport (Pi) by myosin and actomyosin occurs by a similar mechanism as for un- system provides a broader platform to understand how cellular regulation of the labeled ATP. Using this system we present solutions for improved single cytoskeleton’s architecture can be employed to fine-tune intracellular cargo molecule assays for actin and myosin with emphasis on the use of reducing transport. and oxidizing agents (trolox/trolox-quinone; TTQ) in assay buffers to sup- press photo-blinking while maintaining long-lasting fluorescence signals. 1065-Plat The cumulative frequency distribution of dwell-time events exhibited A Minimal Model for the Effects of pH and Phosphate on Muscle Provides more than one exponential phase both in the presence and absence of a Molecular Basis for Cellular Measurements Katelyn Jarvis1, Edward P. Debold2, Sam Walcott1, Mike Woodward2. TTQ. The relative amplitude of the slowest phase increased appreciably 1 2 in the presence of TTQ as well as the overall half-time. Mathematics, UC Davis, Davis, CA, USA, Kinesiology, U. Mass, Amherst, Amherst, MA, USA. 1063-Plat Fatigued skeletal muscle has elevated phosphate (Pi) and acidity (decreased The Power of a Synthetic Machine Based on the Fast Myosin Isoform of pH). How pH and Pi affect muscle at the molecular level remains unclear, Skeletal Muscle partly because although pH and Pi cause observable changes in muscle fibers, Irene Pertici1, Lorenzo Bongini1, Luca Melli1, Giulia Falorsi1, Pi has little effect on single molecule measurements in the laser trap. We there- Danut-Adrian Cojoc2, Tama´s Bozo´3, Miklo´s S.Z. Kellermayer3, fore measured force production of mini myosin ensembles (10 molecules) in- Vincenzo Lombardi1, Pasquale Bianco1. teracting with a single actin filament, the minimal system affected by both pH 1Department of Biology, University of Florence, Sesto Fiorentino, Italy, and Pi. We analyzed these data with a mathematical model wherein myosin in- 2IOM-CNR, Trieste, Italy, 3Department of Biophysics and Radiation teracts with actin via four states: 1. weakly-bound (pre-powerstroke, ADP and Biology, Semmelweis University, Budapest, Hungary. Pi in the active site); 2. strongly-bound (post-powerstroke, strongly bound to The emergent properties as a collective motor of the muscle myosin are studied actin, ADP in the active site); 3. rigor (post-powerstroke, strongly-bound, with a synthetic machine, where HMM fragments (100 mg/ml) of myosin II pu- empty active site); and 4. unbound (post-powerstroke, ATP in the active rified from fast skeletal muscle (rabbit psoas) are randomly dispersed on the site). This model describes the effects of Pi, given a Pi-dependent branch at lateral surface of a chemically etched optical fibre (diameter 4 mm) and interact the strongly bound state (Debold et al. 2013). Simulating the model with with a single actin filament attached to a bead trapped in the focus of a Dual Monte-Carlo methods and introducing pH-dependent changes to ADP release Laser Optical Tweezers (DLOT, Bianco et al. Biophys J. 101:866, 2011). (state 2-3) and attachment (state 1-2), we fit our mini ensemble measurements The mechanical output of the machine is measured by means of the DLOT, at variable pH and Pi, in addition to previous measurements in the single mole- which acts as a force transducer (range 0.5-200 pN, compliance 3.7 nm/pN), cule laser trap and motility assay. The model’s prediction of a pH-dependent and a piezoelectric nano-positioner carrying the support for the motors, which decrease in attachment rate is consistent with decreases in myosin’s ATP turn- acts as a length transducer. The number of HMM available for actin interaction, over we measured in solution. Further, the model predicts Pi- and pH- estimated from the rupture events in rigor, is less than ten. Isometric and dependent changes in peak power, isometric force and unloaded shortening isotonic contractions are reproduced by the motor ensemble in 2 mM ATP that have been measured in muscle fibers. In the model, isometric force is and 23C switching the control from position to force feedback. Following a largely pH-independent due to a balance of decreased myosin attachment (in- drop in force from the maximum isometric value (F0) to a lower value (F), hibiting force) and decreased ADP release (enhancing force). This mechanism the actin filament slides at a constant velocity (V). Up to five F-V points may explain conflicting experimental results and provides molecular insight for each interaction can be determined, allowing the definition of the into the process of fatigue.

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1066-Plat Platform: Calcium Channels and Signaling Ovarian Hormone Affects the Regulation of Super-Relaxation in Skeletal Muscle 1068-Plat 1 2 3 1 Lien A. Phung , Sira Karvinen , Brett A. Colson , Karl J. Petersen , Role of NPC1 in Regulating Store-Operated Calcium Entry: Lessons from Dawn A. Lowe2, David D. Thomas1. 1 2 Niemann Pick Type C Disease BMBB, University of Minnesota, Minneapolis, MN, USA, Department of Scott A. Tiscione, Oscar Vivas, Eamonn J. Dickson. Rehabilitation Medicine, University of Minnesota, Minneapolis, MN, USA, 3 University of California Davis, Davis, CA, USA. Department of Cellular and Molecular Medicine, University of Arizona, Niemann-Pick Type C is an autosomal recessive neurodegenerative disease Tucson, AZ, USA. characterized by mutations in the gene encoding NPC1, a protein critical for We have used refined quantitative epifluorescence microscopy of fluores- the transport of cholesterol to the endoplasmic reticulum (ER) and other cent MANT-ATP to measure single-nucleotide turnover of myosin in cellular membranes. Mutations in NPC1 result in accumulation of cholesterol skinned skeletal muscle fibers from aging mouse models of both sexes. Pre- and other lipids in endo-lysosomes of cells. It has previously been established vious work on female muscle showed that estradiol (E2)-mediated that non-physiological depletion of cholesterol can alter the activity of the mo- signaling reversibly regulates slow ATP turnover by myosin super- lecular components comprising store-operated calcium entry (SOCE). This relaxed state (SRX). This result suggested that reduction of ovarian hor- event is choreographed by interaction of ER membrane protein STIM1 and mone production with menopause contributes to the age-related decline plasma membrane Orai channels to facilitate the movement of calcium ions in muscle strength and function. In muscle, myosins use the energy from across the plasma membrane in response to depletion of ER calcium. Despite ATP hydrolysis to perform mechanical work during muscle contraction, this relationship, little is known regarding the mechanism by which mammalian and help maintain basal metabolic rate at rest. SRX is emerging as an cells can regulate cholesterol to control SOCE and its subsequent downstream important player in muscle mechanics and regulation, yet its physiological signaling pathways. Thus, we sought to examine SOCE in the context of this role in skeletal muscle remains elusive. We hypothesized that SRX myosin neurodegenerative disorder whose principal characteristic is an alteration of serves as a reservoir of motors that can be activated in response to myosin cholesterol homeostasis. Using a combination of dynamic confocal and super function attenuation over time. We examined the change in myosin SRX resolution microscopies, and proteomics we have discovered that SOCE- state in response to the natural aging process by measuring the overall turn- mediated calcium flux is enhanced in Niemann Pick Type C disease. Moreover, over rate of MANT-ATP on fibers isolated from young and aged female there are larger conducting STIM1/Orai1 puncta at steady state and enhanced mice. We found that the nucleotide turnover by the SRX population was formation of these puncta following depletion of ER calcium. We also deter- faster in aged fibers compared to the young ones (p=0.03). This result mine that a downstream consequence of these dynamics is enhanced transloca- agrees with previous study that examined the effect of E2 on the SRX tion of the nuclear transcription factor, NFAT. Pharmacological inhibition of state. However, we did not find any difference in turnover rate (p=0.80) NPC1 recapitulates this SOCE phenotype in mouse hippocampal neurons. or population of SRX species (p=0.92) between young and aged male These neurons also exhibit increased basal calcium signaling and enhanced mice. Together, this suggests that ovarian hormone level is a driving force excitability, a consequence of NPC1 inhibition. Thus, a protein of the lyso- behind the age-related decline in skeletal muscle myosin function. From some, NPC1, has the ability to tune SOCE through its ability to transfer choles- this work, we are making significant progress toward understanding the terol at ER-lysosome membrane contact sites, facilitating gene and protein biochemical mechanism of aging skeletal muscle regulation. This work expressional changes which may contribute to the disease phenotype. was supported by NIH R01AR032961-33 and R37AG26160 to DDT. LAP was supported by NIH T32GM008244 and T32AG29796-10. 1069-Plat Molecular Insights into the Pathophysiology of the Ca2D Sensing Protein 1067-Plat STIM1 Mechano-Chemical Coupling in Sarcomere Lattice Modulated by Romana Schober1, Irene Frischauf1, Victoria Lunz1, Christoph Romanin1, Nonlinear Crossbridge Elasticity Rainer Schindl2. 1 1 2 Djordje Nedic , Boban Stojanovic , Michael A. Geeves , 1JKU, Linz, Austria, 2Medical University, Graz, Austria. 3 Srboljub M. Mijailovich . The store-operated Ca2þ channel components STIM1 and Orai1 are among 1Faculty of Science, University of Kragujevac, Kragujevac, Serbia, 2School 3 others important for T cell activation and consequently lead to severe diseases of Biosciences, University of Kent, Canterbury, United Kingdom, Biology, upon malfunction. The pathophysiological role of STIM1/Orai1 Ca2þ signaling Illinois Institute of Technology, Chicago, IL, USA. has also been shown to contribute to critical steps in malignant cancer cell devel- Coupling between the ATPase cycle and sarcomere mechanics is defined opment. Here, we analyzed several STIM1 mutants potentially associated either by the strain dependence of the crossbridge cycle, which is dependent with different cancer types, with tubular aggregate myopathy, or are a natural upon crossbridge compliance. Recent measurements of nonlinear cross- variation of the protein. We examined the mutants for constitutively active bridge compliance (Kaya et al., Science 329:686-688) and our theoretical Ca2þ-influx, store-independent interaction with Orai1, altered cytosolic calcium investigation demonstrated that energy landscapes are asymmetric because levels and therefore uncontrolled activation of calcium dependent transcription the crossbridges are much more compliant in compression than in tension factors. Transcription factor activation was studied in HEK 293 cells upon due to a combination of buckling and bending of S2. The strain-dependent over-expression of STIM1 mutants at resting state. We identified 4 cancer derived transition rates between actin-myosin states are strongly modulated by mutants that drastically enhanced the activation of NFAT, without store- nonlinear crossbridge compliance and geometrical factors reflecting three- depletion. Additionally, most of the tubular aggregate myopathy mutants showed dimensional nature of myosin binding to actin in the sarcomere lattice. uncontrolled NFAT activation, but not all of them. In order to study the patho- We implemented the modified strain dependent rates in the computational physiological role of the STIM1 mutants in more detail, we also examined the platform MUSICO. Comparison of model predictions between linear and transcription factor EB (TFEB) and the microphthalmia-associated transcription nonlinear crossbridge compliances for classical experiments in muscle re- factor (MITF). Both transcription factors, as well as NFAT, are dependent on the vealed new insights into force development, isotonic shortening or length- Ca2þ dependent phosphatase calcineurin. The STIM1 cancer database mutants ening and T1-T2 transitions. In order to fit the observations the simulations that stimulated NFAT also lead to an enhanced amount of MITF and TFEB nu- including the nonlinear crossbridge compliance required significant changes clear translocation. Moreover, these STIM1 mutants showed constitutive in the crossbridge cycle including two step binding process i.e. weakly and coupling to Orai1, independent of ER store depletion, as determined by FRET strongly bound states, the detachment rates of compressed crossbridges measurements. Further experiments will focus on the pathophysiological rele- during shortening, distorted profile power stroke transition rates and Pi vance regarding enhanced autophagy and more mechanistic insights will be release. The key strength of the approach is that all experiments are fitted achieved by MD simulations of the mutated STIM1 protein. (This work was sup- with the same set of model parameters typically obtained from independent ported by FWF project P2606700 (to Ra.S.) and P27263 to C.R.)). measurements or extracted from specific features of the experiments. For example, during To-T1 transition T1 force is strongly nonlinear at values 1070-Plat of T1 0 and at higher values of applied length step shows small compres- Mixed Signals: Interaction between RyR and IP3R Mediated Calcium sive force consistent with observations. Also recovery tension T2 reached Release Shapes the Calcium Transient for Hypertrophic Signalling in Car- the observed values. These findings invite further re-examination of models diomyocytes of muscle contraction and revision of basic understanding of actomyosin Hilary Hunt1, Gregory Bass1, Llewelyn Roderick2, Christian Soeller3, cycle in the 3-D sarcomere lattice. Vijay Rajagopal1, Edmund Crampin1. Supported by: NIH R01-AR048776, P41-GM103622, and Serbian Ministry of 1The University of Melbourne, Melbourne, Australia, 2University of Leuven, Science grants III41007 and OI174028. Leuven, Belgium, 3University of Exeter, Exeter, United Kingdom.

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Calcium plays a central role in mediating the contractile function of heart bound to RyR1 and RyR2 in isolated sarcoplasmic reticulum membranes at cells. However, calcium is also the second messenger in a wide variety of 0.03 and 30 mMCa2þ, we used TR-FRET between fluorophores targeted to other intracellular signalling pathways, including hypertrophic signalling in the cytoplasmic assembly of RyR1 or RyR2 via donor-FKBP12.6 labeled at cardiomyocytes. How intracellular calcium can encode several different, one of eight sites and acceptor-CaM labeled at one of four sites. Donor- specific signals at once is not well understood. In heart cells, calcium acceptor distances were used to trilaterate the acceptor locations within release from RyRs triggers contraction. Under hypertrophic stimulation, RyR. On both RyR1 and RyR2, we detect a Ca2þ-driven decrease in the 2þ calcium released from IP3R channels triggers dephosphorylation and nu- separation between CaM lobes, suggesting a Ca -CaM structure that is clear import of the transcription factor NFAT, with resulting gene expres- more compact than apo-CaM. On RyR1 (but not on RyR2), we found sion linked to cell growth. Yet this must occur on a background of rising that the position of probes attached to N-lobe of Ca-insensitive CaM and falling cytosolic calcium with each heart beat. We show experimen- (CaM1234) overlapped with probes bound to WT-CaM in nM Ca2þ, sug- 2þ tally that, after addition of IP3, there is an increase in the full width at gesting an apo-CaM/RyR1 complex at nM Ca . On RyR2, the half maximum (FWHM) of the cytosolic calcium transient in adult ventric- CaM1234 N-lobe is shifted ‘‘upward’’, suggesting that WT-CaM is at least ular myocytes. Computational modelling indicates that this can be attrib- partly Ca2þ-loaded when bound to RyR2 in nM Ca2þ. By integrating our uted solely to activation of IP3R channels within the cytosol. This work, TR-FRET system in a stopped-flow experiment, we resolved millisecond together with a recent study which shows that dephosphorylation and nu- Ca2þ-driven structural transitions of CaM-bound to RyR. These fast time clear import of NFAT responds to the cumulative calcium load, suggest constants highlight the importance of understanding CaM’s action as a 2þ a plausible mechanism for the IP3-dependent hypertrophic signalling Ca sensor in modulating RyR channel function during muscle contrac- pathway in cardiomyocytes. tion. This work was supported by NIH grants R01HL092097 (RLC/ DMB) and R37AG26160 (DDT), and by American Heart Association Post- doctoral Fellowship 16POST31010019 (RTR). 1071-Plat 2D IP3-Induced SR-Ca Release Functions as an Anti-arrhythmogenic Mechanism in Ventricular Myocytes 1073-Plat Joaquim Blanch Salvador, Marcel Egger. Structural Dynamics of Calmodulin in Regulation of Calcium Release in Department of Physiology, University of Bern, Bern, Switzerland. Health and Disease 2þ 2þ In cardiac muscle, besides Ca -induced Ca release (CICR), a second Megan R. McCarthy, Robyn T. Rebbeck, Razvan L. Cornea, 2þ Ca release mechanism activated by hormone binding to G-protein David D. Thomas. coupled receptors is present. This prompt intracellular production of the Biochemistry, Molecular Biology and Biophysics, University of Minnesota, signaling molecule Inositol-1,4,5-trisphosphate (IP3)whichthensubse- Minneapolis, MN, USA. 2þ quently triggers sarcoplasmic reticulum (SR)-Ca release through open- We have used time-resolved fluorescence resonance energy transfer (TR- 2þ ings of IP3 receptors type 2 (IP3R2s). IP3-induced SR-Ca release FRET) spectroscopy to study the structural changes in calmodulin (CaM) (IP3ICR) may modulate Ryanodine receptor (RyR2s) function via locally that are relevant to regulation of the skeletal muscle calcium release channel, regulated interactions and fine-tune excitation-contraction coupling in ven- the ryanodine receptor (RyR1 isoform). Regulation of RyR1 by CaM is dis- tricular myocytes. A functional local interplay between IP3R2s and RyR2s rupted by oxidation and disease-causing mutations. Several studies suggest may be significantly pronounced under several cardiac pathologies where that the modulatory role of CaM is closely tied to its conformation when IP3R2 has been found to be overexpressed. We examined IP3ICR and bound to RyR, but the correlation between structure and function in physio- CICR in ventricular myocytes on a local scale in a cardiac specific logically relevant conditions is largely unknown. To test the hypothesis that IP3R2-overexpressing mouse model (IP3/tTA) with a phenotype of cardiac the modulatory action of CaM on RyR1 is caused by structural changes in hypertrophy. Protein analysis confirmed a 12-fold increase in IP3R2 expres- the CaM-RyR1 complex, we used TR-FRET between two fluorescent probes 2þ sion together with a reduction in RyR2 protein levels by 61.7%. IP3- covalently bound to each lobe (N and C) of CaM to measure Ca -dependent pathway stimulation in wild-type permeabilized cells increased spontaneous changes in the distance between lobes when CaM is bound to RyR1 in sarco- 2þ Ca events by 24.2%. Surprisingly, activation of IP3ICR in IP3/tTA myo- plasmic reticulum membranes, isolated RyR1, and a peptide corresponding to 2þ cytes induced a decrease in Ca spark frequency by 25.2% together with a a CaM-binding domain of RyR1 (RyRp; RyR residues 3614-3643). [3H]-rya- 2þ reduction of the SR-Ca content by 14.4% that cannot be explained by nodine binding measurements were performed to ensure functional integrity 2þ RyR2 Ca spark occurrence alone. We examined this phenomenon in of labeled CaM constructs, and to directly correlate the FRET structural 2þ more detail in intact myocytes by applying specific SR-Ca leak/load pro- readout with the CaM-RyR functional interaction. At low Ca2þ,wefind tocols. We found that in IP3/tTA mice IP3ICR functions below the threshold that CaM adopts a different structure when bound to full-length RyR1 2þ 2þ for Ca sparks, via efficient modulation of the SR-Ca leak. Activation of compared with binding to RyRp. However, this structural difference, as re- 2þ this pathway acts as a protective mechanism against arrhythmogenic Ca flected by the distance between CaM’s lobes, is only apparent at low Ca2þ. wave occurrence. We conclude that the overexpression of IP3R2 in ventric- This suggests that CaM-RyRp binding may only be representative of CaM ular myocytes may represent a new and so far not recognized anti- binding to full-length RyR1 at high, not low Ca2þ. Ongoing studies utilize 2þ arrhythmogenic mechanism that can prevent SR-Ca overload and reduce this CaM biosensor to determine the structural changes that contribute to 2þ the propensity of aberrant SR- Ca release preconditioned for arrhythmo- regulation of the cardiac RyR isoform (RyR2). This work was supported by genicity. This mechanism could be a potential target for precision medicine NIH grants AG26160 (DDT), HL092097 (RLC) and 1F31AG052329-01A1 therapies treating ventricular tachycardia. (MRM) and an American Heart Association Predoctoral Fellowship 15PRE25700131 (MRM). 1072-Plat FRET-Based Mapping and Millisecond Structural Kinetics of Calmodulin 1074-Plat Bound to Ryanodine Receptor Channels Microstructural and Functional Imaging of the Intact Sinoatrial Node De- Robyn T. Rebbeck1, Bengt Svensson1, John A. Rohde1, Montserrat Samso2, tects Heterogenous Ca2D-Driven Intra and Intercellular Communications Donald M. Bers3, David D. Thomas1, Razvan L. Cornea1. that Lead to Pacing Perfection 1University of Minnesota, Minneapolis, MN, USA, 2Department of Rostislav Bychkov, Kenta Tsutsui, Magdalena Juhaszova, Steven Sollott, Physiology and Biophysics, Virginia Commonwealth University, Michael D. Stern, Victor A. Maltsev, Edward G. Lakatta. Minneapolis, VA, USA, 3Department of Pharmacology, University of Laboratory of Cardiovascular Science, National Institute on Aging, NIH, California at Davis, Davis, CA, USA. Baltimore, MD, USA. Using time-resolved fluorescence resonance energy transfer (TR-FRET), Cells within the sinoatrial node (SAN), the primary pacemaker of the heart, we resolved distinct structural states underlying CaM regulation of skeletal are extremely heterogeneous in size, morphology and electrophysiological and cardiac ryanodine receptor isoforms (RyR1 and RyR2) at relaxed and characteristics. A coupled-clock theory suggests that rhythmic action poten- þ þ contracting [Ca2 ], and determined the kinetics of Ca2 -driven transitions tial (AP) firing is generated by tight interactions of rhythmic local Ca re- between these structural states. Cryo-EM and FRET-based mapping place leases with cell surface membrane ion channels and transporters. This þ Ca2 -CaM in similar locations on RyR1 and RyR2. However, the position mechanism was reported in single cells, but has not been systematically of apo-CaM remains controversial. To resolve the structural states of CaM explored within cells residing in intact SAN tissue. We developed novel

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Ca imaging techniques to visualize the spread of the impulse across the 1077-Plat entire network of SAN cells and local Ca release dynamics within individual Calculation of Ion-Dependent RNA Folding Free Energy using Coarse- cells. Immunolabeling of Connexin-43 and HCN4 was employed to correlate Grained Simulation cytoarchitecture with Ca activity. Rhythmic AP-induced Ca transients Hung T. Nguyen, Dave Thirumalai. (APCTs), which correlated with APs recorded simultaneously using sharp Chemistry, University of Texas at Austin, Austin, TX, USA. microelectrode, were employed to monitor the impulse conduction across Magnesium ions (Mg2þ) are fundamentally important in RNA biology, play- the SAN. The site at which the earliest APCTs occurred, identified by phase ing indispensable roles in RNA structure, folding and function. In order to shift analysis, was between crista terminalis and superior vena cava. The develop a model that treats monovalent ions implicitly while retaining explicit APCT travels from this area across the entire SAN along curvilinear bundles divalent cations we developed a theory based on liquid state integral equation of striated SAN cells rich in Connexion-43 and weak in HCN4 immunolab- theory that captures both inner shell and outer shell bindings of Mg to phos- eling. LCRs were observed in all areas of SAN, including cells within the phate groups. Coupling this approach with our thermodynamically consistent conduction bundles. Cells close to the APCT initiation site generated only RNA model reproduces Mg2þ-RNA free energies for several RNA molecules, rhythmic LCRs (without APCTs) and these LCRs preceded APCTs in the ranging from a small pseudoknot to the aptamer domain of adenine riboswitch. cells of the APCT-initiation area. Cells close to APCT initiation site were The model not only works for the RNA folded state, but also can be used to give rich in HCN4, but essentially devoid of Connexin-43 and striations. Our re- accurate Mg2þ-RNA thermodynamics for intermediate and unfolded states. In sults are consistent with the hypothesis that a coupled-clock system operates addition, we provide here one of the first attempts to elucidate the full three both within and among cells comprising the SAN network: some specialized dimensional distribution of Mg2þ ions around RNA. Comparing Mg2þ with cells generate only rhythmic LCRs that ignite adjacent cells to initiate the Ca2þ , it is revealed that magnesium binds using inner shell and outer shell impulse that propagates throughout the SAN cell network. equally well while calcium prefers to bind directly with phosphate groups due to the ease of dehydration of the lower charge density cation. Our model 1075-Plat provides a comprehensive treatment of Mg2þ-RNA interaction in terms of Modulation of Ca2D Influx at Hyperpolarized Membrane Potentials Alters both energetic and structural features, allowing us to study ion-dependent Depolarization-Triggered Exocytosis in Bovine Chromaffin Cells RNA folding. Alla F. Fomina, Lukun Yang. University of California, Davis, Davis, CA, USA. 1078-Plat Perturbations in Ca2þ entry via neuronal transient receptor potential (TRP) and Alternative SRP RNA Folded States Accessible Co-transcriptionally can ORAI family proteins have been shown to alter synaptic signaling and architec- Modulate SRP Protein-Targeting Activity ture, and suggested to play a role in a spectrum of neuropathological conditions. Shingo Fukuda, Shannon Yan, Mingxuan Sun, Carlos J. Bustamante. Specific mechanisms by which of TRP/ORAI channels modulate synaptic HHMI/Biophysics/MCB/QB3, University of California, Berkeley, Berkeley, transmission are not completely understood. Here we employed a combination CA, USA. of imaging and electrophysiological techniques to monitor changes in intracel- Signal recognition particle, SRP—a ribonucleoprotein complex conserved 2þ 2þ across all kingdoms—is an essential protein biogenesis machinery that medi- lular Ca concentration ([Ca ]i), membrane currents at hyperpolarized mem- brane potentials, voltage-gated Ca2þ currents, and changes in membrane ates co-translational targeting of nascent signal peptides displayed on ribo- capacitance to investigate effects of modulation of Ca2þ entry at hyperpolar- somes to their proper membrane localization. Throughout this critical ized membrane potentials on exocytosis in bovine chromaffin cells. A small cellular pathway, a series of elaborate conformational rearrangements within SRP, and the associated SRP receptor (SR), provides fidelity checkpoints for inwardly directed membrane current (I hold), whose amplitude was dependent on the concentration of extracellular Ca2þ and Naþ, was present at resting accurate delivery of ribosome complexes. In E. coli, SRP comprises SRP54 protein (FfH) and 4.5S SRP RNA, whose functionality hinges on its correct membrane potential. Augmentation of I hold either by hyperpolarization (from 50 mV to 90 mV) or stimulation with bradykinin (1 mM), a catechol- folding into a native hairpin. SRP RNA first recruits FfH with pico-molar affin- amine secretagogue, elevated [Ca2þ] at hyperpolarized potentials and facili- ity via the tetra-loop at its hairpin tip. The indispensible distal stem region i tated exocytotic responses triggered by short depolarizations (<500 ms in ( 16-bp) of SRP RNA hairpin then configures the GTPase domains from duration). Exocytotic responses triggered by long depolarizations (>500 ms both FfH and SR, which are then activated to trigger the loading of ribosome in duration) were smaller at 90 mV holding potential compared with those complexes to the translocons. at 50 mV holding potential. Reversal of Naþ/Ca2þ exchanger by removal Using high-resolution optical tweezers, we have resolved in real-time the co- of Naþ from bath solution did not cause depolarization-independent exocytosis transcriptional folding pathways of SRP RNA on the E. coli RNA polymerase at 50 mV holding potential. Membrane hyperpolarization from 50 mV to surface. We identified critical conformational transitions between an on- 90 mV in Naþ -free bath solution evoked depolarization-independent exocy- pathway—yet inactive—intermediate fold that lacks the crucial distal stem, tosis with the maximal release rate of about 6 fF/s. Our findings provide first and the final functional hairpin fold of SRP RNA. We showed that specific direct evidence that 1) Ca2þ influx via native Ca2þ channels operating at nega- nucleotide mutations tune the population ratio and the switching rate between tive voltages is functionally coupled with Naþ/Ca2þ exchanger and that 2) the two structures. The adoption of alternative structures by SRP RNA can then perturbation in Ca2þ influx an negative voltages with physiologically relevant modulate SRP protein-targeting activity, with the RNA component acting like a stimuli significantly modulate exocytotic responses to depolarization-triggered molecular switch. Hence, the relative population of SRP RNA folded states— Ca2þ influx in neuroendocrine cells. accessed via co-transcriptional folding pathways—likely underpins the molec- ular origin of various SRP phenotypes. To test this idea, we designed RNA mu- tants that predominately adopt ‘‘switch-off’’ intermediate folds, to sequester Platform: RNA Structure and Dynamics SRP proteins from binding to the ‘‘switch-on’’ native fold. We aim to exploit regulated in vivo expression of ‘‘switch-off’’ SRP RNA mutants to interfere 1076-Plat with protein targeting, thereby manipulating bacterial viability. Different Cations Change the Rates of an RNA Folding Pathway Robb Welty, Kathleen B. Hall. 1079-Plat Biochem & Molecular Biophys, Washington University, St. Louis, MO, Simulations of Optical Tweezers Experiments Reveal Details of RNA USA. Structure Unfolding RNA tertiary structure often depends on divalent ions for electrostatic shielding Wojciech K. Kasprzak1, Taejin Kim2, My-Tra Le3, Feng Gao3, or binding that stabilize the folded structure. We employed machine learning to Megan Y.L. Young3, Xuefeng Yuan3, Joonil Seog4, Anne E. Simon3, analyze 2-aminopurine stopped-flow fluorescence data to measure how Bruce A. Shapiro2. different cations modify folding kinetics of the 60 nucleotide E. coli rRNA 1Basic Science Program, Leidos Biomedical Research, Inc., Frederick, MD, GTPase Center. The folding pathway of the GAC RNA in the presence of USA, 2RNA Biology Laboratory, National Cancer Institute, Frederick, MD, Mg2þ,Ca2þ, and Sr2þ, appears identical. Rate constants for the five folding USA, 3Department of Cell Biology and Molecular Genetics, University of transitions span timescales from sub-microsecond to tens of seconds, and are Maryland, College Park, MD, USA, 4Department of Materials Science and ion-dependent for transition times longer than >10 ms. Divalent ions appear Engineering, University of Maryland, College Park, MD, USA. to modulate intermediate populations in conformational ensembles along the Genomic RNA can fold into alternative structures with distinct biological folding pathway and at equilibrium. The sensitivity of RNA tertiary structure functions, such as replication and translation. We have previously identified to cation identity impacts all but the fastest events in RNA folding. and characterized a structure of a 3’ cap-independent translation enhancer

BPJ 8602_8612 Monday, February 19, 2018 215a within the 30 UTR of the positive strand RNA turnip crinkle virus (TCV). directly to the telomerase catalytic protein subunit, and is absolutely This T-shaped Structure (TSS) is similar in shape and size to a tRNA, required for telomerase function in vitro and in vivo. Generally, 3WJs despite a distinct secondary structure involving three hairpins and two co- have been shown to be highly dynamic and capable of adopting multiple axially stacked helix-pseudoknot motifs at the 5’-end and the 3’-end of tertiary conformations. In the case of the CR4/5 domain, two dramatically the structure. TSS binds ribosomes and enhances translation, but upon bind- different structures have recently been reported for the 3WJ in the absence ing to the RNA-dependent RNA-polymerase (RdRp), it can change its or presence of its cognate protein. These structures highlight the ability of conformation and foster viral replication. We present a study of the unfold- a particular RNA sequence to fold into exceedingly distinct structures. Pre- ing pathway of the extended TCV TSS, combining steered molecular dy- cisely how the intrinsic folding properties of the CR4/5 domain contribute namics simulations (SMD) and optical tweezers (OT) experiments. to functional RNP assembly remains to be elucidated. In the present work, Developing an SMD protocol for a structure of this size and complexity we are characterizing the 3WJ RNA folds from several model telomerase was an important and novel part of the study. Simulations and experiments systems using high-throughput chemical probing and the recently estab- indicate that the TSS unfolding starts with the 3’-side structures, followed by lished mutate-and-map strategy. In this way, we will investigate the folding the long central hairpin, and ends with the 5’-side pseudoknot-hairpin motif. heterogeneity of telomerase 3WJs and will generate experimentally vali- This order could not be deduced from the ranking of the free energies of the dated structural models for distinct RNA conformations present in the equi- TSS motifs. The SMD simulations explained this sequence of unfolding librium ensemble. To complement these bulk biochemical experiments, we based on the 3D organization of the TSS and formation of transient tertiary use single-molecule FRET, a technique that is uniquely suited for studying interactions that maintain the 5’-end pseudoknot topology and delay its full dynamic and heterogeneous RNAs. Taken together, these studies will shed unfolding until all other structures have opened. The SMD results also ex- light on mechanisms of telomerase RNA folding and RNP assembly, as plained the differences between the estimated and measured contour lengths well as more fundamental principles of RNA structural dynamics and for the TCV TSS domains, while mutational studies pinpointed the RdRp function. binding site. Thus, combined biochemical analysis, single molecule force spectroscopy and SMD simulations yielding complementary information at 1082-Plat multiple levels of resolution provide a means for studying complex struc- A Nanoimaging Approach for Identification of the Secondary Structural tures. Funded in part by HHSN261200800001E. Domains in Long ssRNA Molecules Jamie L. Gilmore1,2, Aiko Yoshida2, Hideki Aizaki3, Masahiro Nakano1, Takaji Wakita3, Shige Yoshimura2, Kunio Takeyasu2, Takeshi Noda1. 1080-Plat 1 Relationship between Folding and Catalysis in the GLMS Ribozyme Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan, 2Graduate School of Biostudies, Kyoto University, Kyoto, Japan, Riboswitch 3 Andrew Savinov1, Steven M. Block2. Department of Virology II, National Institute of Infectious Diseases, Tokyo, 1Biophysics Program, Stanford University, Stanford, CA, USA, Japan. 2Departments of Applied Physics and Biology, Stanford University, Stanford, Many biologically important single-stranded RNA (ssRNA) molecules are CA, USA. thousands of bases in length, and often contain structural domains which In many Gram-positive bacteria, expression of glucosamine-6-phosphate syn- have a variety of functions and activities. However, due to technological thase (GlmS) is regulated by the glmS riboswitch. This riboswitch, a regulatory limitations, discovery of these structures is generally a tedious, time- element found in the 50 UTR of the glmS mRNA, controls gene expression by consuming process, and many important RNA structural domains remain an unusual mechanism: the element folds to form a self-cleaving ribozyme. The undiscovered. We have endeavored to create a high-throughput approach self-cleavage reaction, in turn, is activated by the GlmS enzymatic product and to identify RNA structural domains to overcome these limitations. Towards cell wall precursor, glucosamine 6-phosphate (GlcN6P), which serves as a this goal, we have developed sample preparation strategies for observing cofactor for the ribozyme. Self-cleavage of the riboswitch targets the entire the secondary structural arrangement of domains in ssRNA molecules glmS mRNA for degradation by intracellular RNAses, thereby downregulating with Atomic Force Microscopy (AFM) by depositing RNA molecules in GlmS expression. We used optical tweezers to investigate folding and catalysis low salt conditions on a spermidine-modified mica surface. In addition, of the ribozyme core domain, a minimal version that retains GlcN6P-dependent we have developed automated MATLAB-based algorithms for identifica- self-cleavage activity. Measurements of the folding dynamics revealed a series tion of domains in these molecules and prediction of the nucleotides of distinct intermediate states. By applying controlled optical loads and scoring involved in each domain. Since ribosomal RNA molecules (rRNA) are the transition probabilities among these states as a function of force, we deter- the only ssRNA molecules longer than a kilobase with existing 3D struc- mined the free energy landscape of ribozyme formation, from the fully tures, we applied these procedures to the 5.0 kb human 28S rRNA as a unfolded state to the fully folded state. Single-molecule measurements of proof-of-concept. We found large domains which have sizes and morphol- self-cleavage in optically trapped ribozymes revealed that the fully folded state ogies which corresponds well to 5 out of the 6 well-established domains was catalytically active in a GlcN6P-dependent manner. The self-cleavage rate known to exist in this molecule, with the 6th domain divided into two was also load-dependent, decaying in sigmoidal fashion with increasing force. smaller domains. In addition, we have further applied this algorithm to To understand the structure-function relationship responsible for this behavior, characterize the domain structure of viral RNA molecules, including struc- we investigated the folding dynamics of the ribozyme further, assessing the ef- tures present at the splice sites of influenza mRNA molecules, and struc- fects of the non-cleaving cofactor analog glucose 6-phosphate. Combined, our tures present in the 9.6 kb Hepatitis C Virus (HCV) genome. The single measurements of folding and catalysis indicate that glmS ribozyme core self- molecule nature of this method further allows visualization of the confor- cleavage activity is mainly controlled by the folding of the P2.2 element, which mational variability present in both the molecule as a whole and in individ- is labile and not further stabilized by cofactor interactions. ual domains. Following identification of the nucleotides and the morphology of RNA structural domains, models of the secondary structure in these molecules have been generated. 1081-Plat Investigating the Function of Conformational Heterogeneity in Telome- 1083-Plat rase RNA using Multi-dimensional Chemical Mapping and Single- Cellular Imaging of Small RNAs using Fluorescent RNA-Mango Molecule Spectroscopy Aptamers Christina Palka1, Rhiju Das2, Yehuda Tzfati3, Michael Stone1. Adam Cawte1, Sunny Jeng2, Alexis Autour3, Michae¨l Ryckelynck3, 1University of California Santa Cruz, Santa Cruz, CA, USA, 2Stanford Peter Unrau2, David Rueda1,4. University, Stanford, CA, USA, 3The Hebrew University of Jerusalem, 1MRC London Institute of Medical Sciences, Imperial College London, Jerusalem, Israel. London, United Kingdom, 2Department of Molecular Biology and Cells preserve genomic integrity and prevent the DNA damage response at Biochemistry, Simon Fraser University, Burnaby, BC, Canada, 3Architecture the ends of chromosomes by maintaining specialized chromatin structures et Reactivite de l’ARN, CNRS, Universite de Strasbourg, Strasbourg, France, called telomeres. Telomere DNA is synthesized by the reverse transcriptase 4Section of Virology, Imperial College London, London, United Kingdom. telomerase, a multi-subunit ribonucleoprotein (RNP) enzyme. Telomerase In recent years, there has been an explosion of fluorescent RNA aptamers that RNA contains several structurally conserved domains that contribute to have been isolated using SELEX. Since their discovery, fluorogenic RNA ap- RNP biogenesis and function. One of these domains is the conserved re- tamers, such as Spinach and Mango have held great potential to enable the vis- gion 4 and 5 (CR4/5), which folds into a three-way junction (3WJ), binds ualisation of RNA molecules in cells. However, resolving complex RNA

BPJ 8602_8612 216a Monday, February 19, 2018 structures has been limited primarily to bacterial cells due to inefficiency in spray ionization. We have a new way of generating ions, and it promises to maintaining adequate aptamer stability and fluorescence. Evolving new RNA expand the scope of MS to studies of small numbers of (even single) biomol- aptamers with improved physicochemical properties (i.e. thermal stability, ecules in a wide variety of contexts. A single molecule approach could drasti- fluorescence brightness and ligand affinity) should better their use in cellular cally increase the speed and reduce the cost of protein sequencing, which imaging. remains slow and expensive, especially in comparison with DNA sequencing. Three new Mango-like aptamers have recently been evolved using microflui- My research is focused on establishing sets of conditions that will allow us to dic-assisted in vitro compartmentalization, mutagenesis and fluorescent selec- identify all 20 AA. AA are challenging to identify due to their wide range of tion, which improved fluorescence brightness, ligand binding affinity and iso-electric points. In order for AA to be identified by mass spectrometry, thermal stability. We show that these aptamers are readily useable to image they must be charged. My results demonstrate that we can detect single AA. small non-coding RNAs (such as 5S rRNA and U6 snRNA) in both live and They can be transferred directly from the liquid meniscus from the nanopore fixed human cells with improved sensitivity and resolution. The Mango tagged into the mass spectrometer. A specificity of our MS is, it electrosprays directly RNAs sub-cellular localisation pattern is conserved, as validated using immu- into high vacuum, no external gas so no nebulization of the compound before nofluoresence. Our data show that these new aptamers are significantly reaching the detector. Not having a gas should leave the analyte surrounded by improved for cellular imaging over previous fluorogenic aptamers and can in a lot of water molecule, it is not the case, we observe our analytes with one or principle be incorporated into a wide range of coding and non-coding RNAs. no water molecule. Another advantage, I was able to detect hydrophobic ana- We anticipate that these new aptamers will drastically improve RNA imaging lytes in water (eg: valine, /) in cells. 1086-Plat Active Transport by a Membrane Embedded Biomotor Nanopore Platform: Micro- and Nanotechnology Ke Sun, Yuejia Chen, Changjian Zhao, Xialin Zhang, Xiaojun Zeng, Xin Jiang, Jia Geng. 1084-Plat State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China. Detection of Methylation on dsDNA at Single-Molecule Level using Solid- Helicase plays a vital role in all living organisms to unpackage genes. Its struc- State Nanopores ture and function have been studied by various methodology. Molecular trans- 1 2 3 4 Julian Bello , younghoon Kim , Shouvik Banerjee , Kirby Smithe , port through nanoscale channels including membrane channels, biological and 5 2 6 4 2 David Estrada , SuA Myong , Ann Nardulli , Eric Pop , Rashid Bashir , artificial nanopores are of great importance in several key biological processes. 1 Jiwook Shim . Both biological and artificial nanopores have been constructed and reconsti- 1Biomedical Engineering, Rowan University, Glassboro, NJ, USA, 2 tuted into planar lipid bilayer for biosensing and DNA sequencing. However, Bioengineering, University of Illinois at Urbana - Champaign, Urbana, IL, most of the DNA and molecule translocation through those pores are passive USA, 3Material Science and Engineering, University of Illinois at Urbana - 4 transport aided by external electrical field force. Here we report that a helicase Champaign, Urbana, IL, USA, Electrical Engineering, Stanford University, from single-stranded DNA bacteriophage with ring shape can be reengineered Stanford, CA, USA, 5Material Science and Engineering, Boise State 6 and inserted into planar lipid bilayers. We designed this novel biological nano- University, Boise, ID, USA, Molecular and Integrative Physiology, pore with the capability of active DNA transport fueled by the chemical energy University of Illinois at Urbana - Champaign, Ur, IL, USA. from ATP hydrolysis. The membrane-embedded helicase nanopore acts as a Methylation on DNA is an epigenetic modification of DNA in which methyl conductive channel to allow the translocation of single-stranded DNA, while groups are added at the 5-carbon position of cytosine. Aberrant DNA still retain the helicase activity to unwind double-stranded DNA on membrane. methylation, which has been associated with carcinogenesis, can be assessed The helicase activity could be inhibited by removal of Mg2þ or ATP in vitro. in various human biological fluids and potentially be used as biomarkers for DNA with abasic furans can be distinguished during transmembrane- detection of cancer at early-stage. Analytically sensitive and specific assays unwinding process. This 1.3 nm-diameter channel can also form pores in live for methylation targeting low-abundance and fragmented DNA are needed cell membranes. This helicase nanopore is expected to provide an interesting for optimal clinical diagnosis and prognosis. We present a solid-state nano- tool to study motor activity at single-molecule level, and have potential appli- pore-based direct methylation detection assay that circumvents bisulfite con- cations in nanotechnology and nanomedicine. version and PCR amplification. We used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay 1087-Plat selectively detects methylated DNA/MBP complexes through a 19 nm nano- Label-Free Detection of Single-Molecule Melting Kinetics with Laser pore with significantly deeper and prolonged nanopore ionic current block- Heated Nanopore ing, while unmethylated DNA molecules were not detectable due to their Hirohito Yamazaki1, Rui Hu2,1, Robert Henley1, Justin Halman3, smaller diameter. Discrimination of hypermethylated and unmethylated Kirill Afonin3, Dapeng Yu2, Qing Zhao2, Meni Wanunu1. DNA on 90 bp, 60 bp, and 30 bp DNA fragments was demonstrated using 1Northeastern University, Boston, MA, USA, 2Peking University, Beijing, sub 10 nm nanopores. Hypermethylated DNA fragments fully bound with China, 3University of North Carolina at Charlotte, Charlotte, NC, USA. MBP are differentiated from unmethylated DNA at 2.1-fold to 6.5-fold cur- Here, we present a photothermal effect in which a visible laser irradiant on a rent blockades and 4.5-fold to 23.3-fold transport durations. These nanopore silicon nitride nanopore results in non-radiative heating, which creates a highly assays can also detect CpG dyads in DNA fragments and could someday localized thermal gradient around the pore. Controlled temperatures can there- profile the position of methylated CpG sites on DNA fragments. Further- fore be achieved instantly by tuning the laser power, affording nanopore tem- more, we present a novel nanopore-based assay using a nanopore in a peratures from 20-100C, which can be used to study rapid thermal effects on MoS2 membrane. We show that the dsDNA translocation was effectively biomolecular stability. In addition to the ion-current enhancement profile, the slowed down using an asymmetric concentration of buffer and explore the highly localized thermal gradient near the pore induces thermophoresis near possibility of profiling the position of methylcytosines on the DNA strands the pore, which is seen to affect DNA capture and the ion current signal. We as they translocate through the 2D membrane. Our findings advance us one have developed an analytical model that combines temperature-dependent so- step closer towards the possible use of nanopore sensing technology in med- lution conductivity and thermophoresis, which quantitatively matches our ical applications such as cancer detection. experimentally obtained current enhancement vs. laser power. Using this analytical model, the current enhancement factor can be used as a pore ther- 1085-Plat mometer, reporting on the nanopore temperature. We use this principle to The Nanopore Mass Spectrometer demonstrate single-molecule melting of three different biomolecules, two con- Mathilde Lepoitevin, William Maulbetsch, Benjamin Wiener, Derek Stein. trol DNA molecules and one transfer RNA (tRNA) molecule. We induce Department of Physics, Brown University, Providence, RI, USA. melting by controlling the nanopore temperature, which is observed as a sharp We are developing a technique to sequence proteins at the single molecule transition in dwell times as a function of temperature. This transition occurs at level. Our approach merges the ability of nanopores to force polymers to trans- different temperatures for the different molecules, and further changes with locate in a linear fashion with the ability of mass spectrometry (MS) to identify applied voltage. To resolve the equilibrium melting temperature at zero- individual amino acids (AA). The nanopore in our setup takes the form of a force, we have developed true single-molecule thermoscopy, in which temper- capillary that has been pulled into a needle-like tip with an opening on the ature is ramped for each molecule after its capture. From the voltage- 10 - 100 nm scale. AA are drawn out of solution and into the mass spectrometer dependence of the thermoscopy-induced melting of tRNA, we arrive at a by the application of strong electric fields through a process known as electro- zero-force meling temperature that matches the bulk melting point of the

BPJ 8602_8612 Monday, February 19, 2018 217a tRNA. These findings confirm that current enhancement is a result of increased 1Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA, nanopore temperature, and further allow applications of rapid heating of single 2Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA, molecules in future studies. 3Department of Medicine and Center for Immunotherapy, Roswell Park Cancer Institute, Buffalo, NY, USA, 4Department of Immunology, University 1088-Plat of Pittsburgh, Pittsburgh, PA, USA, 5Bioengineering, University of Nanoparticle-Guided Biomolecule Delivery for Transgene Expression and Pittsburgh, Pittsburgh, PA, USA. Gene Silencing in Mature Plants Gradients of soluble molecules regulate many biophysical processes by guiding Gozde S. Demirer, Roger Chang, Huan Zhang, Linda Chio, complex biological events such as tissue regeneration, embryogenesis, angio- Markita P. Landry. genesis, cancer invasion and formation of metastases or immune responses. UC Berkeley, Berkeley, CA, USA. One common aspect of these biological responses is the chemotactic motility Genetic engineering of plants is at the core of environmental sustainability ef- of the cells. Generation of physiologically-relevant concentration gradients is forts, natural product synthesis of pharmaceuticals, and agricultural crop engi- particularly important to understand how chemokines affect cell migration in neering to meet the needs of a growing population and changing global climate. physiology and many pathologic conditions, including inflammatory diseases The physical barrier presented by the cell wall has limited the ease and and cancer progression. Developing chemotaxis assays that examine cell throughput with which exogenous biomolecules can be delivered to plants. response to precisely tailored complex microenvironments that can better Current techniques suffer from host range limitations, low transformation effi- mimic physiological conditions are crucial to study and characterize directed ciencies, toxicity, and unavoidable DNA integration into the host genome. The cell motion. Here, we describe a novel microfluidic device, which is able to central aim of our research is to bring plant science up to speed with modern generate complex chemokine patterns in flow-free gradient chambers. The de- genome editing biology. Herein, we demonstrate efficient diffusion-based vice has multiple gradient chambers in which combinatorial gradients of plasmid DNA and small interfering RNA (siRNA) delivery into two species different chemokines can be generated simultaneously while being imaged of mature plants with a suite of pristine and chemically-functionalized high with high-magnification objectives for direct tracking of chemotactic cells. aspect ratio nanomaterials. Efficient DNA delivery and strong transient protein We simulated and experimentally validated the generation of various concen- expression is accomplished in mature Eruca sativa (arugula) leaves with tration gradients and presented design parameters for proper gradient genera- covalently-functionalized or pristine single-walled and multi-walled carbon tion. Using this microfluidic device, we studied Jurkat cell chemotaxis in nanotubes, with efficiencies comparable to agrobacterium. We also demon- different CXCL12 gradients and fibronectin surface concentrations. Jurkat strate nanotube-based transient protein expression in cell wall-free arugula pro- cell motility ratio and average migration speed have been found to increase toplasts with 85% transformation efficiency. Lastly, we demonstrate a second with increasing fibronectin surface concentration in the absence of CXCL12 nanoparticle-based strategy in which siRNA is delivered and activated in the gradients while remaining constant in different CXCL12 gradients. On the Nicotiana benthamiana plant cell cytosol, effectively silencing a gene with other hand, increasing fibronectin concentration is shown to reduce the Chemo- 95% efficiency. Our work provides a promising tool for species-independent, tactic Index of Jurkat cells in CXCL12 gradients. Lastly, increasing mean targeted, and passive delivery of genetic material, without transgene integra- CXCL12 concentration with the same gradient steepness have been observed tion, into plant cells for rapid and parallelizable testing of plant genotype- to cause saturation of Jurkat cells and lead to random motion. In summary, phenotype relationships. we describe an enhanced microfluidic system for cell migration studies and pro- vide novel data on the migratory response of Jurkat cells to CXCL12, relevant 1089-Plat to diverse areas, ranging from organogenesis to cancer research. Gold Nanowire Fabrication with Surface-Attached Lipid Nanotube Templates 1091-Plat Kristina Jajcevic, Kaori Sugihara. High Speed Motors Driven by a Molecular Tension Gradient University of Geneva, Geneva, Switzerland. Aaron T. Blanchard, Khalid Salaita. The fabrication of conductive nanostructures is the key technology in semicon- Biomedical Engineering, Emory University, Atlanta, GA, USA. ductor industry and has gained importance in biology for applications such as Synthetic molecular motors have important applications in drug delivery, biosensors and drug delivery. There is a growing interest in the use of lipid chemical sensing, nanoscale cargo transport, and molecular computing. nanotubes (LNTs) as templates in the fabrication of one-dimensional nano- Our group previously developed the first DNA-based rolling motors which structures such as gold nanowires. displayed a maximum speed and processivity that is 1000 times faster The lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is known to than conventional ‘‘bipedal’’ DNA walkers or ‘‘DNA spiders’’. These mo- self-assemble into single-wall synthetic LNTs on polyelectrolyte- tors are made of DNA-coated microparticles that bind to complementary functionalized surfaces. DOPE is the main component of bacterial cell mem- RNA on a surface and degrade the RNA (Yehl, Salaita et. al. Nat. Nanotech. branes and its self-assembly into tubes is similar to the tube formation in bac- 2016). For reference, these rolling motors translocate at velocities approach- teria. LNT structures have been shown to have biological applications such as ing that of biological motors. Here we will describe our efforts to elucidate in the transport of intercellular organelles and in transmitting electrical signals the molecular basis through which DNA-mediated RNA hydrolysis drives between cells. We have demonstrated a high-throughput approach to fabricate particle translocation. Using biophysical models for the force-distance rela- gold nanowires on surfaces with a LNT template. First, biotin-tagged DOPE tionships of oligonucleotide duplexes, we propose that DNA micromotor LNTs are formed from lipid blocks in inverted hexagonal phase adsorbed on motion is best described by a mechanical tension gradient (MTG) model polymer-coated surfaces upon application of shear force. Streptavidin-coated which differs from Brownian ratchet models observed in other synthetic gold nanoparticles were then attached to the biotin-tagged LNTs and gold and natural molecular motors. We start by presenting simulations which nanoparticle-encapsulated LNTs were cross-linked by chemical fixation. Sam- illustrate how the distance-dependent association and dissociation kinetics ples were dried and treated with oxygen plasma to remove the organic template of oligonucleotide duplexes give rise to the MTG model and, by extension, and connect the particles. The created nanowires were characterized by cryo- the emergent behavior of our micromotors. We next provide experimental transmission electron microscopy, atomic force microscopy and electrical mea- data supporting the MTG model. For example, our experiments and simula- surements. The method is advantageous because the small size of LNTs enables tions show that micromotor velocity increases when the density of oligonu- the fabrication of solid nanostructures with a higher throughput without using cleotides is increased, while Brownian ratchet models would suggest the expensive electron beam lithography. The approach can further be combined opposite trend. Finally, we demonstrate how the MTG model predicts chem- with single LNT patterning with a micromanipulator to create distinct patterns ical sensing capabilities. Conventional molecular detection techniques rely instead of random networks. on equilibrium binding between capture and target molecules, but molecular sensing in biology is typically performed in an out-of-equilibrium manner 1090-Plat and can achieve much higher sensitivity. By engineering our motors to stall Chemotaxis of Immune Cells in Microfluidic Flow-Free Concentration in the presence of target biomolecules via a ‘‘molecular lock’’ mechanism, Gradient Generator we demonstrate that small numbers of target molecules (one or two per mo- Utku M. Sonmez1, Philip R. LeDuc2, Pawel Kalinski3,4, Lance A. Davidson5. tor) can slow motion in an out-of-equilibrium manner.

BPJ 8602_8612 218a Monday, February 19, 2018

Posters cell. One such partner is RecQ, an ATP-dependent DNA helicase that performs multiple roles in genome maintenance. We elucidated the mechanistic role of the RecQ-SSB interaction by using a combination of single-molecule magnetic twee- Posters: Protein Structure and Conformation I zers and rapid kinetic experiments. We show that, via binding to SSB’s C-termi- nal segment, RecQ triggers a change in the ssDNA binding mode of SSB. This 1092-Pos Board B1 allows RecQ to gain access to previously SSB-bound ssDNA and to initiate Conserved Domain Architecture of Human BLM Helicase Maintains Bal- double-stranded (ds) DNA unwinding. We also show that the interaction en- ance between D-Loop Disruption and Extension hances recruitment of RecQ to ssDNA-dsDNA junctions. Moreover, the interac- Ga´bor M. Harami1, Yeonee Seol2,Ja´nos Pa´linka´s1,Ma´te Gyimesi1, tion of RecQ with the SSB C-terminal segment slightly enhances the DNA Zolta´n J. Kova´cs1,Ma´te Martina1, Anna Budai1, Julianna B. Nemeth1, unwinding rate of RecQ. Based on our results, we propose a mechanism that Keir C. Neuman2, Miha´ly Kova´cs1. can explain how SSB can efficiently assist RecQ in initiating dsDNA unwinding 1Biochemistry, Eo¨tvo¨s Lora´nd University, Budapest, Hungary, 2Laboratory at ssDNA-dsDNA junctions instead of blocking its action. Taken together, these of Single Molecule Biophysics, National Heart, Lung and Blood Institute, results suggest a reciprocal interaction in which each protein modulates the National Institutes of Health, Bethesda, MD, USA. behavior of the other. As SSB binds to a set of diverse proteins, it is likely that Cells must protect their genome from harmful alterations, caused directly by some aspects of the proposed mechanisms are universal. DNA damage or indirectly by inappropriate repair, to avoid cell death or 1095-Pos Board B4 cancerous transformation. DNA double-strand (ds) breaks, the most severe Potential Disruption of Ebola Virus Matrix by Graphene Nano-Sheets type of DNA damage, are repaired in a potentially error-free manner by homol- Rudramani Pokhrel1, Jeevan GC2, Nisha Bhattarai1, Prem Chapagain1, ogous recombination (HR). During HR, broken dsDNA ends are transformed into Bernard Gerstman1. 3’ single-stranded (ss) overhangs that form nucleoprotein filaments with recom- 1FIU, Miami, FL, USA, 2WSU, Pullman, WA, USA. binases. These filaments can search for intact, homologous dsDNA regions that Ebola virus infections cause hemorrhagic fever that often results in very high serve as template for repair and can invade into the homologous DNA molecule fatality rates. In addition to exploring vaccines, development of drugs and dis- to form a displacement loop (D-loop). D-loops are thus key HR DNA- infectants is also essential for treating the disease and preventing the spread of intermediate structures. D-loops are thought to be processed by multiple path- the infection. The Ebola virus matrix protein VP40 is a multifunctional protein ways with different outcomes and can serve as early decision points in HR regu- that can assume various conformational and oligomeric forms and is a potential lation. Members of the RecQ helicase family are implicated both in D-loop pharmacological target for disrupting the virus life-cycle. Here we explored stabilization and disruption. The human Bloom’s syndrome RecQ helicase VP40-graphene interactions using molecular dynamics simulations and gra- (BLM) is thought to channel HR into error-free pathways partially by processing phene pelleting assays. Results show that graphene sheets can associate D-loops. However, the exact mechanism of this action is poorly understood. Via a strongly with VP40 at various interfaces. The graphene is able to disrupt the novel kinetic assay, here we show that the conserved domain architecture of BLM CTD-CTD interface that is formed due to VP40 hexamer-hexamer association. maintains an almost 1:1 balance in D-loop disruption and stabilization. Interest- This interface is crucial in forming the Ebola viral matrix and disruption of this ingly, the activity profile of BLM markedly differs from that of the very effective interface may provide a method to use graphene or similar nanoparticle based D-loop disruptor Escherichia coli RecQ. However, our single-molecule magnetic therapy to inhibit viral budding or as a disinfectant that can significantly reduce tweezers experiments reveal that the similar domain architecture of BLM and the spread of the disease and prevent an Ebola epidemic. RecQ, while having different roles in D-loop processing, allows both proteins to sense DNA substrate geometry and unwind dsDNA in a repetitive fashion. 1096-Pos Board B5 Our results highlight how functions of the conserved architecture of RecQ heli- Toward High Resolution Structures of the HIV-1 In/LEDGF/DNA cases evolved to specialize these enzymes to different cellular requirements. Complex Julien Batisse, Eduardo Bruch, Nicolas Levy, Benoit Maillot, Sylvia Eiler, 1093-Pos Board B2 Oyindamola Oladosu, Marc Ruff. Secret From the ABYSS: Structures of the D-Family DNA Polymerase IGBMC, Illkirch, France. (POLD) Reveal that DNA Replication and DNA Transcription Share a After retroviral infection of a target cell, during the early phase of replication, Joint Evolutionary History in Archaea the HIV-1 genomic RNA is reverse transcribed by the viral reverse transcrip- Pierre Raia1, Pierre Beguin2, Ghislaine Henneke3, Marc Delarue1, tase to generate the double-stranded viral DNA that interact with viral and Ludovic Sauguet1. cellular proteins to form the pre-integration complex (PIC). Viral integrase 1DBSC, Institut Pasteur, Paris, France, 2Department of Microbiology, Institut 3 (IN) is the key component of the PIC and is involved in several steps of repli- Pasteur, Paris, France, LMEE, Ifremer, Plouzane, France. cation notably in reverse transcription, nuclear import, chromatin targeting and Archaeal replicative DNA polymerases D (PolD) constitute an atypical class of integration. Viral components such as IN cannot perform these functions on DNA polymerases made of a proof-reading exonuclease subunit (DP1) and a their own and need to recruit host cell proteins to efficiently carry out the larger polymerase catalytic subunit (DP2), both with unknown structures. We different processes. IN is a flexible protein, property allowing its interaction have determined crystal structures of Pyrococcus abyssi PolD, revealing a cata- with multiple partners and enabling its multiple functions in viral replication. lytic core strikingly different from all other known DNA polymerases (DNAPs). To study the molecular mechanisms of viral integration we use a bottom - up Rather, the PolD DP2 catalytic core has the same ‘‘double-psi b-barrel’’ architec- strategy by assembling in vitro and/or in cellulo multiprotein complexes around ture seen in the RNA polymerase (RNAP) superfamily, which includes multi- the integrase protein (core protein of the PIC) and DNA. This strategy enabled subunit transcriptases of all domains of life, homodimeric RNA silencing us to solve cryo-EM structures of the IN/LEDGF and IN/LEDGF/INI1 com- pathway RNAPs and atypical viral RNAPs. This finding bridges together, for plexes at low resolution [1, 2]. With the recent progress of the cryo-EM tech- the first time in cellular life, DNA transcription and DNA replication within niques and our improvement in the complexes preparations [3], new cryo-EM the same protein superfamily. This study documents further the complex evolu- datasets are collected which will enable us to increase the structure quality to tionary history of the DNA replication apparatus in different kingdoms of life and near atomic resolution for the IN/LEDGF/DNA complex. The latest results proposes a novel paradigm on the classification of all extant DNAPs. will be presented at the meeting. [1] Michel et al. (2009) EMBO J., 28, 980-991 1094-Pos Board B3 [2] Maillot et al. (2013) PLoS ONE 8(4): e60734 RECQ Helicase Triggers a Binding Mode Change in the SSB-DNA Com- [3] Levy et al. (2016) Nature comm. 7 : 10932 plex Efficiently Initiate DNA Unwinding Ga´bor M. Harami1, Maria Mills2, Yeonee Seol2,Ma´te Gyimesi1, 1097-Pos Board B6 Ma´te Martina1, Zolta´n J. Kova´cs1, Mihaly Kovacs1, Keir C. Neuman2. Can SAXS Tell Us Whether Antibody Shape Evolves? 1Dept. of Biochemistry, Eotvos University, Budapest, Hungary, 2Laboratory Rosaleen A. Calvert, Katy A. Dore, Brian J. Sutton, Andrew J. Beavil. of Single Molecule Biophysics, National Heart, Lung and Blood Institute, Randall Centre, King’s College London, London, United Kingdom. National Institutes of Health, Bethesda, MD, USA. IgM, found in jawed vertebrates, which diverged from the jawless fish 500 The single-stranded (ss) DNA binding protein (SSB) of Escherichia coli plays million years ago (mya), is still important in mammals. It can be pentameric essential roles in DNA metabolism. Besides sequestering ssDNA, SSB has also or hexameric: each ‘‘monomer’’ has a Fab region which binds pathogens and been shown to interact with numerous DNA-processing enzymes via its highly an Fc region mediating biological activity. The common ancestor of birds conserved, unstructured C-terminal segment. The interaction has a general stim- and mammals diverged 325 mya and chicken IgY has the function of ulatory effect on the DNA-processing activities of binding partners and appears to mammalian IgG. IgY, like each IgM ‘‘monomer’’, has an Fc of six immuno- be essential for organization of DNA-processing enzyme complexes within the globulin domains, two of which have been lost from IgG (but not from IgE).

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The oldest extant IgE is that of the platypus (a monotreme, which diverged that bind at the HBGA pocket and block attachment to HBGAs are believed to from other mammals 166 mya). Antibody Fc binding to complement (for neutralize the virus. However, additional neutralization epitopes elsewhere on IgM and IgG), or to Fc receptors on cells (IgM, IgG and IgE) can lead to the capsid likely exist and impeding the intrinsic structural dynamics of the both protective activity or uncontrolled effects such as chronic inflammation, capsid could be equally important. In the current study, we investigated a panel allergy and autoimmunity. IgM and complement co-exist in serum and so a of Nanobodies in order to probe functional epitopes that could trigger capsid mechanism is needed to control initiation of the destructive complement rearrangement and/ or interfere with HBGA binding interactions. The precise cascade. There is evidence that IgM-Fc may bend on antigen binding to reveal binding sites of six Nanobodies (Nano-4, Nano-14, Nano-26, Nano-27, complement-binding sites. Conflicting evidence suggests that IgM-Fc is consti- Nano-32, and Nano-42) were identified using X-ray crystallography. We tutively bent, and activity is controlled by the position of the Fab fragments. showed that these Nanobodies bound on the top, side, and bottom of the noro- There are crystal structures of both bent and extended human IgE-Fc, but it virus protruding domain. The impact of Nanobody binding on norovirus capsid is not known whether the extended conformation exists in solution. The degree morphology was analyzed using electron microscopy and dynamic light scat- of bending is known to affect receptor binding. The conformation of IgY-Fc tering. We discovered that distinct Nanobody epitopes were associated with and of platypus IgE-Fc is unknown. varied changes in particle structural integrity and assembly. Interestingly, We have investigated the solution conformation of ‘‘monomeric’’ IgM-Fc, certain Nanobody-induced capsid morphological changes lead to the capsid chicken IgY-Fc, platypus IgE-Fc and human IgE-Fc by SEC-SAXS to explore protein degradation and viral RNA exposure. Moreover, Nanobodies employed whether each is bent, extended or can adopt different states. Thus we can multiple inhibition mechanisms to prevent norovirus attachment to HBGAs, observe whether there is an evolutionary progression for these antibodies in which included steric obstruction (Nano-14), allosteric interference (Nano- terms of their structure and function. 32), and violation of normal capsid morphology (Nano-26 and Nano-85). Finally, we showed that two Nanobodies (Nano-26 and Nano-85) not only 1098-Pos Board B7 compromised capsid integrity and inhibited VLPs attachment to HBGAs, but Accounting for Specificity and Cross-Reactivity in T Cell Receptor Molec- also recognized a broad panel of norovirus genotypes with high affinities. ular Recognition Consequently, Nano-26 and Nano-85 have a great potential to function as novel Brian M. Baker. therapeutic agents against human noroviruses. Chemistry & Biochemistry, University of Notre Dame, Notre Dame, IN, USA. 1101-Pos Board B10 In orchestrating cellular immunity, T cell receptors (TCRs) bind short peptide Mercury and Alzheimers Disease: HG(II) Ions Display Specific Binding to antigens presented by major histocompatibility complex (MHC) proteins. As the Amyloid-Beta Peptide and Modulate its Aggregation the number of possible peptide antigens far exceeds the number of different Sebastian Warmlander1, Cecilia Wallin1, Sabrina Sholts2, Per Roos3, T cell receptors in an organism, T cell receptors must be cross-reactive, with Jyri Jarvet1, Astrid Graslund1. some studies concluding that a single TCR must be capable of productively 1Division of Biophysics, Stockholm University, Stockholm, Sweden, engaging at least 1 million different peptides. Yet, antigen specificity is a hall- 2Smithsonian Institution, Washington, D.C., DC, USA, 3Institute of mark of cellular immunity. Here we deconstruct this ‘‘dichotomy’’ or Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. ‘‘duality’’ of specificity/cross-reactivity and demonstrate how structural and Increasing evidence connects Alzheimer’s disease (AD) to metal exposure. biophysical principles can account for both high specificity and high cross- Aberrant metal concentrations are observed in AD brains, but the role of metals reactivity in TCR binding reactions. The principles and conclusions discussed in AD pathology remains unresolved. Animal models exposed to mercury have far-reaching implications for the utilization and optimization of TCR- display the typical pathological signs of AD, and many studies (but not all) based molecular and cellular therapies for cancer and infectious disease. indicate elevated mercury levels in brain tissue of AD patients. At a molecular level, however, no mechanisms have been described that would link mercury to 1099-Pos Board B8 AD pathogenesis. The pathological hallmark of AD brains is deposition of am- Flavivirus Capsid Protein Binding to Host Lipid Systems yloid plaques, consisting mainly of amyloid-b (Ab) peptides aggregated into Ana S. Martins, Andre Nascimento, Andre F. Faustino, amyloid fibrils. Previous research has shown that Ab peptide aggregation Filomena A. Carvalho, Nuno C. Santos, Ivo C. Martins. and fibrillation can be modulated by specific binding between Ab and metal Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de ions such as Cu(II) and Zn(II). Here, we study in vitro the molecular interac- Lisboa, Lisbon, Portugal. tions between Ab(1-40) and Hg(II) ions. NMR spectroscopy results show Flaviviruses, such as Dengue (DENV), West Nile (WNV) and Zika (ZIKV) vi- that Hg(II) ions bind the N-terminal part of the Ab(1-40) peptide, likely via ruses are transmitted to humans mainly by the bite of mosquitos and are a the same ligands that bind Cu(II) and Zn(II) ions, i.e. histidines H6, H13, serious public health treat. Despite the global spread and disease severity, there and H14. Fluorescence quenching of Ab residue Y10 by Hg(II) ions indicates is no specific and effective treatment, in part due to a poor understanding of the the apparent Hg(II)/Ab dissociation constant to be around 1-3 mM. Fluores- flavivirus life cycle. The capsid (C) protein, conserved in terms of sequence and cence spectroscopy and solid state atomic force microscopy (AFM) show a structure among flaviviruses, is a major drug target. For example, DENV C me- concentration-dependent effect of Hg(II) ions on Ab fibrillation, which is diates viral life cycle, namely by binding to host lipid droplets (LD), which is completely inhibited at a 1:1 Ab:Hg(II) ratio. Thus, together with e.g. Cu(II), essential for viral replication. Here, we investigated WNV and ZIKV C protein Fe(II), Mn(II), Pb(IV) and Zn(II), the Hg(II) ion belongs to a family of metal binding to host lipid systems. Zeta potential shows that WNV C interacts with ions that modulate Ab aggregation by binding to specific Ab ligands. This in- þ LD surface proteins, requiring K ions, as previously shown for DENV C by dicates that AD metal chemistry is more complex than previously considered, þ þ us. ZIKV C also binds to LD, although in this case Na and K ions are inter- and may have implications for AD pathology and pathogenesis, especially as changeable. Dynamic light scattering shows that WNV C binds very low- in most countries mercury continues to be an important environmental density lipoproteins (VLDL) but not low-density lipoproteins (LDL). ZIKV contaminant. C also binds to VLDL, similarly to DENV C. Following, WNV C (un)binding forces upon interaction with LD and VLDL were determined by atomic force microscopy (AFM)-based force spectroscopy. AFM confirmed that WNV C 1102-Pos Board B11 binds specifically to LD and VLDL (but not LDL), in a process requiring Spectroscopic Studies of Buffer and Metal Ion Effects on Amyloid-Beta Kþ ions. Furthermore, ZIKV, WNV and DENV C protein sequences display Peptide Structure and Aggregation similar predicted hydrophobicity, a-helical propensity and tertiary structure Keyon Carter. that can thus be targeted via similar approaches. Combining all this with our James Madison University, Stafford, VA, USA. b background on DENV C protein and pep14-23 peptide (an inhibitor of Amyloid- peptides are found in brains obtained from Alzheimer’s patients. b DENV C binding to host lipid systems, designed and patented by us), we These peptides aggregate forming insoluble Amyloid- plaques. It is uncertain will now use this information for Flavivirus drug development strategies. how the presence of these peptide plaques correlate with the onset of the disease, however environmental conditions are known to alter disease progression. Previ- 1100-Pos Board B9 ous studies have proven that metal ions such as zinc and copper are co-localized Nanobodies Targeting Norovirus Capsid Reveal Functional Epitopes and within Ab plaques. Recent studies suggest that these metal ions may serve a role þ þ Potential Mechanisms of Neutralization in inducing Ab peptide misfolding and aggregation. The Cu2 and Zn2 ions are Anna D. Koromyslova, Grant S. Hansman. believed to bind to the Ab peptide structure and increase aggregation. The metal Tumor Virology, German Cancer Research Institute, Heidelberg, Germany. ion concentrations play a factor as higher metal ion concentrations correlate with Norovirus is the leading cause of gastroenteritis worldwide. Little is known on a higher aggregation rate. Understanding the environmental conditions that affect how norovirus infects the host cells, except that histo-blood group antigens Ab peptide structure and aggregation will provide a greater understanding about (HBGAs) are important binding factors for infection and cell entry. Antibodies the role of metals in disease. Current studies are aimed at understanding how

BPJ 8613_8616 220a Monday, February 19, 2018 metal ions influence peptide structure, solvation, and aggregation. Infrared spec- domain of DnaG among the eubacteria, we determined the crystal structure troscopy was used to monitor aggregation and structural changes of control Ab of the Se-Met-labeled helicase binding domain of DnaG from M. tuberculosis peptide and Ab peptide in the presence of Cu2þ and Zn2þ over time. Infrared (MtDnaG-CTD) at 1.58 A˚ . The overall structure of MtDnaG-CTD displayed spectra show that peptide length, buffer and metal concentration influence Ab two subdomains, the N-terminal glob region (GR) and the C-terminal helix peptide structure, solvation, and aggregation. hairpin region (HHR) connected with a small loop. Further, to study the AcknowledgementsNSF-REU #1461175JMU Department of Chemistry and helicase-primase interaction in M. tuberculosis, a complex was modeled using Biochemistry the MtDnaG-CTD and MtDnaB-NTD crystal structures. By using this model, a nonconserved hydrophobic residue Ile605 on helicase binding interface of 1103-Pos Board B12 DnaG-CTD was identified as a potential key residue. Mutation guided by mo- Combining DNP NMR with Segmental and Specific Labeling to Study the lecular dynamics and biophysical studies validated our model. Biosensor bind- Quaternary Structures of Yeast Prion Protein Strains ing studies show ten-fold higher binding affinity of MtDnaB-NTD with native Yiling Xiao, Whitney Costello, Carla Madrid, Kendra Frederick. MtDnaG-CTD (Kd-250 nM) to mutant Ile605Ala MtDnaG-CTD (Kd-2.5 mM). Biophysics, UT Southwestern Medical Center, Dallas, TX, USA. Both in silico calculations and in vitro binding sensor experiments suggest the Yeast prions are self-templating protein-based mechanisms of inheritance crucial role of the Ile605 (MtDnaG) in the stabilization of the helicase-primase whose conformational changes lead to the acquisition of diverse new pheno- complex in M. tuberculosis. Investigation of specificity in the interaction of types. The best studied of these is the prion domain (NM) of Sup35, which DnaB with non-cognate DnaGs was studied by the in silico complex model forms an amyloid that can adopt several distinct conformations (strains) that and the biophysical interaction of MtDnaB-NTD with other DnaG-CTDs produce distinct phenotypes. Despite intense study, there is no consensus on from E. coli, H. pylori, and V. cholerae. The complex model, together with the organization of monomers within Sup35NM fibrils. Some studies point to mutagenesis and binding analysis, explain the role of loop region of HHR in a a -helical arrangement, whereas others suggest a parallel in-register organiza- the stability of DnaG-DnaB complex by aligning in the proper orientation for tion. Intermolecular contacts are often determined by experiments that probe maximizing the binding affinity with species-specific helicases. Apart from hy- long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 15 drophobic interaction, electrostatic surface potentials also influence the C- and N-labeled monomers. However, for Sup35NM, like many large pro- species-specific DnaB-DnaG complex interactions. teins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degener- 1106-Pos Board B15 acy and increase experimental sensitivity, we combined specific and segmental Evolution of Antibody Structure and Function through Studies of IgE and isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Us- IgM ing this combination, we examined two of the amyloid forms of Sup35NM that Rosemary Nyamboya. do not have a parallel in-register structure. The combination of a small number Randal, King’s College London, London, United Kingdom. of specific labels with DNP NMR enables determination of architectural infor- Crystallographic and solution studies of IgE-Fc have shown that the molecule is ε ε mation about polymeric protein systems. acutely bent between the C 2 and C 3 domains, a bend that is further enhanced in IgE/sFcεRI complex. Structural studies of IgE have proved crucial in map- 1104-Pos Board B13 ping receptor interactions to inform mutagenesis for functional studies and en- Understanding the Structural Basis of Recognition between Plasmodium gineering effector functionality, as well as drug discovery. Despite its crucial Falciparum and Human Sumoylation Machinery role in primary immune response, the structure of IgM remains elusive. A struc- Jai Shankar Singh1, Vaibhav Kumar Shukla2, Mansi Gujarati3, Ram Kumar tural framework is urgently required to underpin the current research activity in Mishra3, Ashutosh Kumar4. the functions of IgM and interplay between its Fc receptors. Based on the struc- 1Bioscience & Bioengineering, IIT Bombay, Mumbai, India, 2Centre for tural similarity of IgE and IgM in the Fc domain, the hypothesis of this study is Excellence in Basic Sciences, Mumbai, India, Mumbai University, Mumbai, that IgM and IgM-receptor interactions also involve conformational changes. India, 3Department of Biological Sciences, Indian Institute of Science The key question is whether IgM displays the acutely bent Fc structure that Education & Research, Bhopal, India, Bhopal, India, 4Bioscience & was discovered in IgE-Fc, and whether conformational changes play a role in Bioengineering, IIT Bombay, Mumbai, India. its functions. Fluorescence resonance energy transfer (FRET) analysis of an Malaria is one of the deadliest infectious diseases, affecting millions of lives IgE-Fc biosensor, Alexa 488-IgE-Fc-TAMRA, designed using the sortase annually and is caused by parasitic protozoans of the genus Plasmodium. P. mediated ligation technique confirms the structure and conformational changes falciparum (Pf) adopts various survival strategies including post-translational that accompany IgE interaction with its receptors as earlier reported in crystal modifications (PTMs) to stabilize and potentiate its crucial proteins for suc- and solution studies. Higher FRET efficiencies were obtained with this new cessful infection cycle in the mosquito and human host. One of the important biosensor in comparison with the previously used mRFP-IgE-Fc-eGFP PTMs is called sumoylation, which is essential for normal cellular functions. biosensor, an indication of higher sensitivity. Using similar technique, an It is known that SUMO interacts with both E1-activating (hetero-dimeric IgM-Fc biosensor will be designed to assess the disposition of the Cm2, Cm3 Aos1/Uba2), and E2-conjugating (Ubc9) individually. Can the interaction and Cm4 domains and its implications for receptor interaction. This study of Pf-SUMO with E1and/or E2 have the cross-species interaction element also aims to investigate the binding sites of IgM Fc Receptors, FcmR and that can be targeted? Moreover, the structure of Pf-SUMO is not known, Fca/mR. we present the first structure of Pf-SUMO solved using solution state NMR. The residue specific interactions of Pf-SUMO with Pf-Ubc9 and the 1107-Pos Board B16 possibilities of cross-interaction of host and parasite sumoylation machin- Spectroscopic Study of Cu(II) Binding to the Light Chain 6aJL2 and its eries were also checked by NMR titration studies. We have identified the Effect on Amyloid Fiber Formation 1 2 1 important residues of Pf-SUMO proteins involved in sumoylation that gov- Angel Pelaez-Aguilar , Carlos Amero , Lina Rivillas-Acevedo . 1Centro de Investigacio´n en Dina´mica Celular, Universidad Auto´noma del erns the specificity of interaction in Plasmodium as well as human host. 2 The residues at the interacting interface that displayed prominent interactions Estado de Morelos, Cuernavaca, Mexico, Centro de Investigaciones have been mutated to probe their specificity. Overall, all these results empha- Quı´micas, Universidad Auto´noma del Estado de Morelos, Cuernavaca, size that during disease condition the parasite may use the host sumoylation Mexico. machinery to maintain its survival. This information can be used Light chain amyloidosis or primary amyloidosis is one of the most common for designing drugs that specifically block the interface. The dynamics of systemic amyloidosis, characterized by the deposition of immunoglobulin light Pf-SUMO protein will also help in ascertaining role of protein plasticity in variable domain as insoluble amyloid fibers in vital organs and tissues, leading substrate recognition and specificity. to death of patients in a few months. Germline VI are closely related with this disease and has been reported that 25 % of proteins encoded by this germline 1105-Pos Board B14 have a mutation at position 24 where Arg is replaced by a Gly (R24G). In vitro Structural Basis of an Essential Interaction between DnaG and DnaB in studies have shown that this mutation reduce the stability and increase the pro- Mycobacterial tuberculosis pensity to form amyloid fibrils. In different systems, like Alzheimer, Parkinson Dhakaram P. Sharma, Ramachandran Vijayan, Arif Abdul Rehman, and Prion, the role of metal ions have proved to be relevant. For this reason, we Samudrala Gourinath. studied the role of Cu(II) in the amyloid fibrillation of the recombinant protein School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. 6aJL2-R24G by different spectroscopic techniques. It was observed that Cu(II) The helicase-primase interaction is a critical event in DNA replication and is accelerate the 6aJL2-R24G fibrillation by binding, with micro molar affinities, mediated by the helicase (DnaB) interaction domain within the primase in the His99 and His8. These results provide important information in the mo- (DnaG). To better understand the poor conservation of the DnaB binding lecular characterization of amyloidogenic diseases.

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1108-Pos Board B17 quasi-RNA Recognition Motifs (H/FqRRM12) and a third C-terminal qRRM3 Electrostatic Interactions at the Dimer Interface Stabilize the E. coli b embedded within glycine-rich repeats. The tandem qRRMs are connected sliding Clamp through a 10-residue linker with most of the amino acids strictly conserved be- Anirban Purohit. tween hnRNP H and F. A significant difference occurs at position 99 of the Arizona State University, Tempe, AZ, USA. linker where hnRNP H contains a proline and hnRNP F an alanine. To inves- E.coli processivity clamp is a translocating homodimeric protein which encir- tigate the influence of P99 on the conformational properties of hnRNP H, we cles DNA during replication to help DNA polymerase bind and move along the probed the structural dynamics of its HqRRM12 domain with x-ray crystallog- DNA template strand.An oligomeric protein called clamp loader opens the raphy, NMR spectroscopy, and Small Angle X-ray Scattering (SAXS). We clamp through one interface to load it on DNA strand.This opening doesn’t observed in the HqRRM12 contains multiple structures in solution by SAXS. require ATP hydrolysis, thus the dynamic behavior at the interface becomes These exchangeable conformations that located on the linker region and crucial to understand whether clamp loader is taking advantage of that.This RNA recognition sites reach certain equilibrium in different temperature by study focuses on understanding the contribution of charged amino acids present NMR relaxation dispersion. The collective results best describe that HqRRM12 at the dimer interface to the stability of the interface by studying the fluores- exists in a conformational equilibrium between compact and extended struc- cence properties of TMR dye tagged at the interface.Fluorescent correlation tures. The compact structure displays an electropositive surface formed at the spectroscopy (FCS) was used to investigate if any conformational dynamics qRRM1-qRRM2 interface; the surface is abrogated within the extended struc- is present at the interface.TMR dye was tagged on the Cysteine residues, which ture. Comparison of NMR relaxation parameters between HqRRM12 and were introduced in the place of Arg103(charged)S109(hydrophilic)I305(hydro- FqRRM12 indicate that FqRRM12 primarily adopts an extended conformation. phobic) at the interface to understand which interaction plays significant role in Introducing the P99A mutation into HqRRM12 alters its conformational dy- stability of the clamp. NaCl was used to screen electrostatic charges at the inter- namics to favor the more extended structure. Thus, our work demonstrates face which destabilizes the interface as it’s present in the middle of the hof- that the linker compositions confer different structural properties between meister series, ensuring no change in the ionic atmosphere of the protein.A hnRNP H/F family members that might contribute to their functional diversity. subunit exchange experiment was performed with labeled and unlabeled pro- tein samples and found that Arg103 weakens the protein significantly compare 1111-Pos Board B20 to other two mutations which indicates that electrostatic interactions are the Amyloid BETA Peptide Aggregation Process in the Presence of Sugar- major contributing factor to the dimer stability.FCS experiment was performed Based Surfactants- Conformational and Structural Studies 1 1 2 in different salt concentrations to monitor monomer formation by measuring Michalina Wilkowska , Weronika Andrzejewska , Ryszard Zielinski , Maciej Kozak1,3. diffusion coefficients.The results confirmed significant monomer formation 1 with increasing salt concentration.Although, it is not conclusive if microsecond Department of Macromolecular Physics, Adam Mickiewicz University, 2 fluctuations come from conformational dynamics at the interface lead to forma- Poznan, Poland, Department of Technology and Instrumental Analysis, 3 tion of open states or it is only an artifact of dye dimer.In conclusion, this study Poznan University of Economics, Poznan, Poland, Joint SAXS Laboratory, helped us to understand effect of electrostatic interactions in protein Adam Mickiewicz University, Poznan, Poland. stabilization. The progression of Alzheimer’s Disease (AD) is caused by aggregation process of amyloid b peptide (Ab) [1]. The aggregation mechanism of Ab peptides is b 1109-Pos Board B18 still under discussion, because the process of A oligomerization is relatively b Dissociation of Factor XIII Homodimer during Activation Process: Solu- fast and it can be affected by many variables [2]. The control of A aggregation tion Evidence Weighs in on Decades-Long Debate is very hard to achieve, but use of selected specific surfactants may contribute Boris Anokhin1, Vilius Stribinskis2, William Dean2, Muriel Maurer1. to a better understanding of this process. The aim of this study was character- b 1Chemistry, University of Louisville, Louisville, KY, USA, 2Brown Cancer ization of the structure and conformational changes in -amyloid peptides Center, University of Louisville, Louisville, KY, USA. induced by the presence of zwitterionic, sugar-based surfactants comprising b Oligomeric assemblies have been increasingly realized to regulate protein func- a lactose moiety. Several different variants of -amyloid peptides were selected tion. As the scope of this research area expands, it has been estimated that 60% for the study. The effect of surfactant concentration in solution was tested on b b of enzymes form oligomers. While many of these enzymes require self- A 1-42 peptide and its shorter variants. Selected solutions of tested -amyloid association to fulfill their catalytic function, the spotlight of the current project peptides with surfactants were subjected to a series of solution scattering exper- is on a protein that is inactive in its oligomeric form. Factor XIIIA (FXIIIA) is a iments using the synchrotron radiation (SR-SAXS). The SAXS data for these transglutaminase that cross-links an array of intra- and extracellular protein surfactants were collected on P12 beam line of EMBL (DESY). SR-SAXS substrates in a calcium-dependent manner. FXIIIA is the only member of the studies exhibited the structure of various mixed (peptide-surfactant) assem- blies. In our study we analyzed also the secondary structure of Ab peptide in transglutaminase family found as a homodimer (A2) in zymogen form. It can be activated by thrombin-mediated cleavage of the activation peptides (AP) solutions with different concentrations of surfactant, using FTIR spectroscopy or non-proteolytically by Ca2þ ions. The oligomeric composition of activated and circular dichroism (CD) methods. Atomic force microscopy (AFM) was b FXIIIA is controversial. Although the enzyme has long been considered a used to characterize the morphology of A assemblies. Particular attention dimer, recent indirect evidence suggests the possibility of a monomeric state. was paid to analysis of small soluble oligomers that are currently believed to In the present study, size exclusion chromatography and analytical ultracentrifu- initiate development of brain disfunction in AD. [1] J. Hardy and D. J. Selkoe, Science, 2002. gation were used to study global rearrangements in FXIIIA2 accompanying its activation. For the first time, a quantitative assessment of FXIIIA intersubunit in- [2] J. A. Loureiro, S. Rocha, and M. do C. Pereira, J. Pept. Sci. Off. Publ. Eur. teractions was performed. Those interactions were tight (Kd 8 nM) in the dimeric Pept. Soc., 2013. Acknowledgments zymogen form. Both non-proteolytic and thrombin-mediated FXIIIA activation : The study was supported by research grant, Grant Dia- resulted in monomeric species (with Kd values ranging from 90 to 220 mM). Inter- mentowy’’ from Ministry of Science and Higher Education (Poland) - DEC: 0011/DIA2015/44. estingly, cleavage of a single AP on the FXIII A2-homodimer resulted in dissoci- ation and in expression of full enzymatic activity for both cleaved and noncleaved protomers. By contrast, non-proteolytic activation was much less efficient. 1112-Pos Board B21 Thus, direct experimental evidence was obtained for the monomeric state of Mapping the Regions in PCNA that Mediate Nucleosome Assembly activated FXIIIA in solution. Dimerization of the zymogen is proposed to sta- Lynne Dieckman, Molly Carrig, Claire Embree, Kurt Shaffer, bilize FXIIIA in a physiological setting and to prevent premature protein cross- Hunter VanDolah. linking. Consequently, dissociation of the homodimer is crucial for full expres- Chemistry, Creighon University, Omaha, NE, USA. sion of FXIIIA function. The eukaryotic genome must be accurately organized into nucleosomes, the basic units of chromatin, immediately following DNA replication to maintain 1110-Pos Board B19 genomic and epigenetic integrity. This process, called replication-coupled Structure and Conformational Dynamics of the Splicing Factor hnRNP H nucleosome assembly, is mediated by two key factors: CAF-1, the protein com- Liang-Yuan Chiu, Blanton S. Tolbert, Srinivasa Rao Penumutuchu. plex that deposits histones onto the newly synthesized DNA, and PCNA, the Chemistry, Case Western Reserve University, Cleveland, OH, USA. ring-shaped sliding clamp that recruits and regulates the proteins that replicate Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H/F family DNA. Although the mechanism by which nucleosome assembly and DNA are multi purpose RNA binding proteins that participate in most stages of RNA replication are coupled remains poorly understood, studies show that the inter- metabolism. Despite having similar RNA sequence preferences, hnRNP H/F action between CAF-1 and PCNA is essential for this process. Here, we deter- proteins function in overlapping but distinct cellular processes. The domain or- mined the X-ray crystal structure of three mutant forms of PCNA that cause ganization of hnRNP H/F proteins is modular consisting of N-terminal tandem decreased CAF-1 association with replicating DNA and exhibit gene silencing

BPJ 8613_8616 222a Monday, February 19, 2018 defects. Although these amino acid substitutions are located in three drastically stem-loop DNA structure. Using ADAAD I created F507A and F507W site different locations within the PCNA protein structure, all three caused large directed mutant of motif IV and S558A and T560A site directed mutant of perturbations of a surface cavity on the front surface on the PCNA ring. Results motif V of ADAAD to investigate the function of these motifs in the ADAAD. from these structural studies suggest that this surface cavity on PCNA repre- Results showed that F507A was inactive whereas S558A and T560A had the sents a novel, secondary binding site for CAF-1. In addition, preliminary same activity as compare to ADAAD. Kinetic studies of S558A and T560A protein-protein binding studies suggest that disruption of this region does not showed that the kinetic parameters of S558A and T560A were lowered as interfere with the canonical CAF-1-PCNA interaction or the binding site for compared to ADAAD. So these residues are important for the turnover number other PCNA-interacting proteins. Overall, we propose that the integrity of and catalytic effieciency of the ADAAD. Binding studies showed that F507A this cavity plays an essential role in facilitating replication-coupled nucleosome binding was lowered with either ligand in presence of another ligand. Circular assembly and that it may be indispensible for other PCNA-mediated processes. dichroism studies showed that the conformation of these mutants were altered as compare to ADAAD. Thermal denaturation data by circular dichroism 1113-Pos Board B22 showed that Tm was almost same of all these mutants as compare to ADAAD. TAU Peptide Interactions with Lipid Membranes: Secondary Structure In conclusion, motif IV and V are required for the conformational integrity of Analysis the protein. Sam Ealy. Chemistry, Hamline Universtity, Saint Paul, MN, USA. 1116-Pos Board B25 For nearly two decades the rate of mortality in the most common form of dementia, Human Norovirus Inhibition through Combination Drug Treatment Alzheimer’s disease, has been rising. Physiological changes seen in AD are caused in Alessa Ringel1, Turgay Kilic1, Jessica Devant1, Kerstin Ruoff1, part by neurofibrillary tangles or the formation of plaques by b-amyloid protein. Tan- Anna Koromyslova1, Alexander Hempelmann1, Michelle Haas2, gles are formed by tau protein whose normal function is aiding in stabilization of the Celina Geiß2, Imme Roggenbach2, Juliane Graf2, Grant Hansman1. cytoskeleton through the microtubule binding domain (MBD). The MBD contains 1German Cancer Research Center, Heidelberg, Germany, 2Ruprecht-Karls four repeat segments in the domain: R1, R2, R3, and R4; and this is the location University, Heidelberg, Germany. of misfolding. Studies have shown that tau aggregates localize to the Golgi mem- Human noroviruses are the leading cause of acute gastroenteritis outbreaks brane, which has an anionic surface. In this study, we used membranes composed worldwide. Norovirus recognize histo-blood group antigens (HBGAs) and of either DOPC (zwitterionic) or DOPC/DOPG (80:20, anionic) to explore structural the binding interaction is known to be important for infection. Blocking the changes of the isolated repeat segments R1, R2, R3, and R4. Fourier transform HBGA pocket might represent one possible approach for norovirus antiviral infrared spectroscopy (FTIR) was used to determine the secondary structure of the development. Human milk oligosaccharides (HMOs) structurally mimic peptides in the presence of these membranes, and monitor any time dependent HBGAs and may function as receptor decoys. In this study, we identified changes that occur. The Amide I peak was peak fitted using random coil, a-helix, two nanobodies, Nano-62 and Nano-94, against human norovirus GI.1. b-sheet, and b-turn conformations to determine the overall percent composition of Nano-62 and Nano-94 were both found to recognize the GI.1 capsid protruding each fold. We found that anionic membranes induce structural changes in each repeat (P) domain, but only Nano-94 detected intact virus-like particles (VLPs). X-ray segment and that these segments change their structural composition over the period structural analysis revealed that Nano-62 bound to an epitope on the P domain, of days, with an enhanced population b-sheet . Such data is important in consider- which was hidden on intact VLPs. Interestingly, analogous Nanobodies against ation of mechanisms that explain misfolding events that are led by mislocalization GII.4 noroviruses could bind VLPs, despite recognizing a similarly occluded to anionic membranes or infection from pathogenic bacteria. epitope. Our data indicated that prototype GI.1 norovirus capsid was more rigid compared to the prevalent GII.4 norovirus. Nano-94 bound to an exposed site 1114-Pos Board B23 on the top of the P domain nearby the HBGA pocket with a nanomolar affinity. Deciphering the Interactions between an Anticancer Bacteriocin and the Moreover, Nano-94 blocked norovirus VLPs from binding to HBGAs. Interest- P53 DNA Binding Domain ingly, we discovered that Nano-94 treatment of VLPs lead to particle aggrega- Yongqi Huang, Jingjing Zhou, Xiyao Cheng, Zhengding Su. tion, which likely reduced the attachment to HBGAs. Intriguingly, we found Hubei University of Technology, Wuhan, China. that a combination of the HMO 2’FL and Nano-94 lead to a significantly Azurin is an anticancer bacteriocin secreted by Pseudomonas aeruginosa. The enhanced HBGA blocking. Structural analysis showed different binding sites intact protein of azurin or its derived peptide p28 preferentially can enter a va- of HMO 2’FL and Nano-94 on the GI.1 P domain, corresponding to the Bliss riety of human cancer cells and induce apoptosis or growth inhibition. criterion of ‘‘drug independence’’. In consequence, Bliss Independence could Although extensive studies have confirmed that azurin and p28 exert their anti- be used to characterize the interaction of HMO 2’FL and Nano-94 as synergis- cancer activity mainly by interacting with the p53 DNA binding domain (DBD) tic. In conclusion, this work identified a combination of Nanobody and HMO and interfering in the p53 degradation signaling pathway, the detailed interact- treatment against norovirus and demonstrated for the first time a synergistic ing mechanisms and the complex structures remain unknown. In this work, we effect. extensively characterized the interactions between azurin and p53 DBD and the interactions between p28 and p53 DBD by various biophysical techniques. P28 is a helical segment isolated from azurin, but it is disordered in the free state. 1117-Pos Board B26 CD spectrum indicated that p28 folds into helical structures upon binding with Monitoring of Alzheimer’s Amyloid-b Peptide Aggregation via Fluores- p53. Site-directed mutagenesis and protein NMR characterization showed that cence Correlation Spectroscopy and Total Internal Reflection Microscopy the p28 binding site on p53 DBD is different from the azurin binding site on p53 Juri€ Jarvet1,2, Astrid Gr€aslund1, Ann Tiiman3, Vladana Vukoevic3. 1 DBD although they overlap with each other. Binding of p28 or azurin did not Department of Biochemistry and Biophysics, Stockholm University, 2 affect the interactions between p53 DBD and DNA, allowing us to obtain a p53/ SE-10691 Stockholm, Sweden, National Institute of Chemical Physics and 3 DNA/p28 ternary complex. Determination of the complex structure in the Biophysics, Tallinn, Estonia, Center for Molecular Medicine, Department of future will allow us to rationally design p28 derived peptides that will exhibit Clinical Neuroscience, Karolinska Institutet, SE-17176 Stockholm, Sweden. higher affinity with p53 DBD and stronger anticancer activity. The aggregation mechanisms of amyloid-b peptide are complex. They have been investigated using a variety of different analytical techniques, including fluorescence spectroscopy and fluorescence correlation spectroscopy 1115-Pos Board B24 (A. Tiiman, J. Jarvet, A. Gr€aslund, and V. Vukojevic, Biochemistry 2015 54, Motif IV and V of Active DNA Dependent ATPase a Domain, a SWI2/SNF2 7203-7211 and references therein). The benzothiazole salt Thioflavin T Protein, Are Required for Both Ligand Binding and Conformational Integrity (ThT) fluorescence assay is a standard method for amyloid detection by fluores- Vijendra Arya, Rohini Muthuswami. cence spectroscopy and ThT fluorescence could also be used for total internal School of Life Science, Jawaharlal Nehru University, New Delhi, India. reflection microscopy. It is known that amyloidogenic aggregates enriched with ATP dependent chromatin remodeling proteins use the energy released by ATP pleated b-sheet secondary structure readily bind ThT and significantly alter its hydrolysis to alter the chromatin structure to make it accessible for fundamental spectral properties, shifting the absorption spectrum towards longer wave- processes e.g. replication, transcription and repair etc. SMARCAL1 is a distant lengths and significantly increasing the fluorescence quantum yield. ThT acts member of SNF2 family of chromatin remodelers and has been reported to as a smart dye becoming fluorescent only when bound to amyloid thereby interact with RPA at the DNA damage site and exhibits reannealing helicase strongly reducing the fluorescence background. Fluorescence correlation spec- activity and maintain the genome integrity. Mutations in hSMARCAL cause troscopy (FCS) is a time-resolved spectroscopic technique that can measure the a rare autosomal recessive disorder known as Schimke Immunoosseous concentration and size of fluorescently labeled particles. The method will Dysplasia (SIOD). ADAAD is the N-terminal proteolytic fragment of SMAR- benefit from the fact that many ThT molecules bind to a single amyloid aggre- CAL1 that possesses all the characteristic helicase motifs (Q, I, Ia, II, III, IV, V gate. Total internal reflection microscopy (TIRF) combined with ThT fluores- and VI) of SMARCAL. It can hydrolyse the ATP only in presence of a cence was used to follow the elongation of fibrils in time. We identified

BPJ 8613_8616 Monday, February 19, 2018 223a aggregates of different sizes with molecular weight from 260 kDa to more than following rehydration. Addition of plasmin at a concentration of 10 nM re- 1 106 kDa and revealed the hitherto unobserved kinetic turnover of interme- sulted in complete fibrinolysis within 15 minutes. By following the topology diates during amyloid-b aggregation. of the matrix and individual filaments during lysis, we were able to uncover the molecular mechanisms of the process. Fibrils became flexible but retained 1118-Pos Board B27 axial continuity for an extended time period, indicating that lateral interac- Biochemical and Biophysical Characterisation of Influenza a Virus tions between protofibrils are disrupted first, but the axial interactions remain Proteins stable. Sometimes small fibrils even fused into bundles, suggesting that a Muhd Faiz-Hafiz Mohd Kipli, Jolyon Claridge, Jason Schnell. cohesive force persists between them. Axial fragmentation rapidly took place Oxford University, Oxford, United Kingdom. in the final stage of the lysis. Conceivably, the persisting axial integrity and Influenza A viruses are important pathogens that cause major global health cohesion of the fibrils assist to maintain global clot structure, to prevent problems. The influenza A virus core is made of 8 viral ribonucleoprotein microembolism, and to generate a high local plasmin concentration for the (vRNP) complexes. Each vRNP is made of a segment of the viral RNA pack- rapid, final axial fibril fragmentation. The nanoscale fibrin matrix developed aged around the nucleoprotein and viral RNA polymerase. The influenza A vi- and tested here provides a unique insight into the molecular mechanisms rus Matrix protein 1 (M1) is the most abundant protein in the virus particles. behind the structural and mechanical features of fibrin and its proteolytic Aside from its role in stabilising the 3D structure of the virus, M1 has other degradation. essential functions that include regulating the transport of vRNPs and recruiting other viral proteins in the budding process during virus assembly. vRNPs are 1121-Pos Board B30 bound by M1 and studies have shown that the nuclear export protein (NEP) as- Characterization of the Molecular Mechanism for Maturation Inhibitors sociates with M1, mediating the export of vRNPs from the nucleus. However, against the HIV-1 Capsid-SP1 Domain there is still no structural evidence for this. In the formation of virus particles at Carly A. Sciandra1, Pengfei Ding1, Eric O. Freed2, Michael F. Summers1. the plasma membrane, M1 interacts with the M2 protein, as well as other viral 1HHMI at University of Maryland, Baltimore County (UMBC), Baltimore, constituents. There is evidence that the direct interaction of M1 with the cyto- MD, USA, 2HIV Dynamics and Replication Program, NCI/CCR, Frederick, plasmic tail of M2 is important for virus assembly but the exact residues MD, USA. involved has not been mapped. The aim of this project is to first optimise the HIV/AIDS is a global pandemic and infects roughly 40 million people world- purification of M1, NEP and M2 proteins and eventually characterise the mo- wide. Current therapies target fusion, reverse transcription, and integration lecular basis of interaction of M1/M2 and M1/NEP using nuclear magnetic processes, but serve only to minimize the effects of the disease. Only the resonance (NMR) and other biophysical techniques. In this report, the purifica- maturation process is targeted in the late phase of the HIV replication cycle, tion of M1 (N-term and C-term domain), M2 C-term and NEP are discussed. which involves proteolytic cleavage of the Gag polyprotein. During viral as- sembly, thousands of Gag molecules form a hexagonal lattice, while the CA- 1119-Pos Board B28 SP1 junction region transitions from a random coil to a helical structure in Loop Substitution with BETA Turns Disrupts Fibril Formation in Amy- order to assemble into a 6-helix bundle. This CA-SP1 junction is cleaved loid Model Sequences last during viral maturation, and previous work has suggested that maturation Heng Chi. inhibitors prevent the cleavage of the CA-SP1junctionbytargetingandsta- Pharmacy, Jiangsu Food and Pharmaceutical Science College, Huai’an, bilizing the 6-helix bundle structure. The 6-helix bundle is only formed in China. assembled Gag molecules, hindering the solution study of its interaction b b D-Pro-Gly (pG) segment was proposed to promote a type II’ -turn in model - with maturation inhibitors. Meanwhile, efforts to crystallize the assembled hairpin peptides. Combined thermodynamic and kinetic studies have shown CA-SP1/inhibitor complex have not been successful. Thus, the molecular that with a compact dual hairpin structure locked with pG turns, 19mer peptide mechanism for the inhibitory function of these compounds still remains B3pGD formed an oligomeric aggregate before morphing into fibrils under elusive. This work aims to provide insight to the mechanism of maturation b thermal perturbation. Here, -turn inducing moieties such as pG, NG and BG inhibition, by utilizing a hexameric scaffolding protein to construct an iso- b (Aib-Gly) were incorporated into Amyloid 16-37 sequence and the mutated lated CA-SP1 junction hexamer. This protein will facilitate probing into b peptides (Ab16pG/Ab16BG/Ab16NG) would have both core -strands origi- the binding stoichiometry and binding affinity of maturation inhibitors, b nally from the A 1-42 sequence connected with an artificial 2-residue turn exemplified by Bevirimat, to the hexameric protein by isothermal titration instead of a 10mer loop. UV-CD spectra of all the mutants have features of calorimetry (ITC) and solution nuclear magnetic resonance (NMR). Once b -sheet secondary structure. IR and VCD spectra of these peptides confirmed the binding ratio is better understood, NMR structural studies can be per- b their structures as typical -sheet based aggregates with poor thermal revers- formed to probe the binding mechanism of maturation inhibitors to the ibility. None of the three mutants demonstrated any lag phase on a ThT assay, CA-SP1 junction helix. neither did they grow into fibril like aggregates which could enhance the quan- tum yield of ThT dramatically as their wild type Ab16-37 did. These mutant pep- tides can convert into oligomer-fibril hybrids detectable by TEM, whereas the Posters: Protein-Small Molecule Interactions II morphology of the aggregates formed by Ab16-37 shows fibril like structures. We proposed that the insertion of b-turn promoting segments into the amyloid 1122-Pos Board B31 beta core sequences disrupts the elongation process, resulting in an oligomer/ Engineering Targeted Lectins by Computer-Guided Directed Evolution proto fiber complex. This study provides a method of isolating non-fibril aggre- Ismail C. Kazan1, Prerna Sharma1. 1 gates for further mechanistic investigation on formation and toxicity of oligo- Arizona State University, Tempe, AZ, USA. meric species in disease related amyloids. (NSFC 21503087) Glycan-binding proteins also known as lectins are important in regulation of viral infections, inflammation, and cancer progression by connecting with 1120-Pos Board B29 highly glycosylated regions. Thus, characterization of a complete map of Topology of Plasmin-Driven Fibrinolysis in a Nanoscale Fibrin Matrix cell glycosylation and correlating glycan composition to physiological and Tı´mea Feller1, Jola´n Ha´rsfalvi1, Csilla Csa´nyi1, Balazs Kiss2, pathologic cell functions represents a major goal in glycoscience. However, Miklo´s Kellermayer1. designing specific lectins for target glycans with high specificities and bind- 1Biophysics and Rad. Biol., Semmelweis University, Budapest, Hungary, ing affinities is a current challenge. Here we develop a computational 2Department of Cellular and Molecular Medicine, The University of Arizona, approach to guide the design panel of stable, robust lectins specific for gly- Tucson, AZ, USA. cans of choice. We use Cyanovirin-N (CVN), a 101aa lectin that exhibits Fibrin plays a fundamentally important, mechanical role during hemostasis. In high-affinity binding to the mannose-rich glycans that cover the surface of order to withstand the shear forces of blood flow and prevent embolization, gp120 as our model system. Since our earlier work has shown that positions fibrin monomers form a three-dimensional network that serves as an elastic distal from binding sites involve with binding site conformational dynamics, scaffold for the blood clot. The complex spatial hierarchy of the fibrin mesh- thus impact binding affinities in CVN; we utilize our recent method of dy- work severely complicates the exploration of structural features, mechanical namic coupling index (DCI) to identify these sites. After identifying these properties and molecular changes in the clot. Here we developed a quasi- distal positions important for the binding event of CVN to di-mannose, we two-dimensional nanoscale fibrin matrix that enables the investigation of deploy multiple sequence alignment (MSA) to statistically identify distal po- fibrin properties by topographical analysis using atomic force microscopy sitions co-evolved with binding site positions along with their amino-acid fre- (AFM). The matrix was prepared and stabilized by clotting fibrin on mica sur- quencies. We then screen the change in binding energies upon mutations on face for 2 hours followed by drying with nitrogen gas. The average thickness these sites through our Adaptive-BP dock method. Our approach provides the of the matrix was 50 nm, and component fibrils could be easily identified mutational landscape to increase the binding affinity of our model lectin to- with AFM. The matrix retained functionality as it could be lysed with plasmin ward Di-Mannose.

BPJ 8613_8616 224a Monday, February 19, 2018

1123-Pos Board B32 binding studies of NADPþ to the apoprotein or a ternary complex with NADPþ Probing the Interaction of ABETA42 Amyloid Species with an Aggrega- and DHF showed characteristic spectra for each complex. The apoprotein gave tion Suppressor Molecule by Infrared Nanospectroscopy rise to a sharp and a broad peak. Upon addition of NADPþ, the sharp peak for Francesco Simone Ruggeri, Johnny Habchi, Sean Chia, W38 shows line broadening while the broad peak for W45 remains unchanged. Michele Vendruscolo, Tuomas P.J. Knowles. In the ternary complex, the sharp peak in the apo and binary complex splits into Department of Chemistry, University of Cambridge, Cambridge, United three peaks while the broad peak remains unchanged. The appearance of three Kingdom. new resonances can be explained by how NADPH and DHF bind in the active A wide class of neurodegenerative disorders is due to the failure of a peptide or site pore. Two symmetry related lysine 32s at the edge of one side of the pore protein to keep its native functional conformational state and undergo a confor- constrain the position of NADPH by forming ionic interactions with the phos- mational change into a misfolded state, triggering the formation of fibrillar phate groups. However, on the other side of the pore, the glutamate tail of DHF cross-b sheet amyloid aggregates. Tremendous efforts have been devoted in is disordered and switches between ion pairs with the K32s on that side of the the last twenty years with the aim to understand the molecular basis of Alz- pore. This results in two different environments for the nearby W38 residue. heimer’s disease and to develop small molecules that could interfere with the Our NMR results are consistent with computational simulations of the aggregation pathway of Ab peptides. Several disease-modifying small mole- glutamate-tail interacting with symmetry related lysine 32 residues at the cules inhibiting Ab aggregation have been proposed. However, despite the edge of the pore. This approach will be valuable for determining the binding great effort, no compound has entered the clinical use to date. One of the affinity of DHF towards R67 DHFR by in-cell NMR. main reasons leading to this failure is the incomplete knowledge of the molec- ular mechanisms underlying the process by which small molecules interfere 1126-Pos Board B35 with the aggregation pathway. Indeed, inhibiting Ab aggregation per se could The Regulation of Surface Charge by Biological Osmolytes have unexpected consequences on the toxicity. Roy Govrin. Here, we apply infrared nanospectroscopy to unravel the effect of a small mole- Department of Physics and the Russell Berrie Nanotechnology Institute, cule, bexarotene, on Ab aggregated species from the morphological and struc- Technion, Haifa, Israel. tural points of view. We provide for the first time a direct measurement at the Osmolytes, small molecules synthesized by all organisms, play a crucial role in nanoscale of the interaction between a small molecule and Ab42 aggregated tuning protein stability and function under variable external conditions. Despite species. Our results prove that fibrils incubated with bexarotene show the their electrical neutrality, osmolytes action is entwined with that of cellular typical signature of the molecule related to the carbonyl group C=O at 1690 salts and protons in a mechanism only partially understood. To elucidate this cm1. This specific adsorption corresponds to the IR vibration of monomeric mechanism, we utilize an ultra-high resolution FM- AFM for measuring the ef- bexarotene, while free molecules in solution form a dimer with shifted absorp- fect of two biological osmolytes, urea and glycerol, on the surface charge of tion of the C=O at 1725 cm1. Together with bulk approaches, we demonstrate silica, an archetype protic surface with pK value similar to that of acidic amino that hydrophilic moiety of bexarotene binds to Ab42 mainly through a acids. We find that addition of urea, a known protein destabilizer, enhances sil- hydrogen bond to the proteins aggregates. These results constitute a key finding ica deprotonation and hence surface charge by more than 50%, an effect equiv- for the development of therapeutics molecule against AD. alent to a 4 units increase of pH. Conversely, addition of glycerol, a protein stabilizer, practically neutralizes the silica surface, an effect equivalent to 2 units reduction of pH. Simultaneous measurements of the interfacial liquid vis- 1124-Pos Board B33 cosity indicate that urea accumulates extensively near the silica surface while Interactions of Quinazoline Derivatives with Beta-Amyloid glycerol depletes there. Comparison between the measured surface charge Praveen Nekkar Rao, Tarek Mohamed, Arash Shakeri. and Gouy-Chapman-Stern model for the silica surface teaches that the modifi- School of Pharmacy, University of Waterloo, Waterloo, ON, Canada. cation of surface charge is 4 times too large to be explained by the change in Protein misfolding and aggregation is involved in the pathophysiology of a dielectric constant upon addition of urea or glycerol. The model hence leads number of diseases. In this context, beta-amyloid fragment 1-42 (Aß42) is to the conclusion that surface charge is chiefly governed by the effect of osmo- known to form neurotoxic aggregates which include dimers, trimers, tetramers, lytes on the surface reaction constants, namely, on silanol deprotonation and on oligomers and fibrils. These Aß42-aggregates are known to exhibit varying de- cation binding. These findings highlight the unexpectedly large effect that grees of neurotoxicity which is associated with Alzheimer’s disease (AD). The neutral osmolytes may have on surface charging and Coulomb interactions. steps involved in the self-assembly and fibrillogenesis is complex. In this re- gard, small molecules can be used as chemical tools to understand the mecha- 1127-Pos Board B36 nisms of Aß42 misfolding and their self-assembly into higher order structures. Mechanism of Allosteric Modulator Binding to the Adenosine A1 Receptor We synthesized a series of quinazoline derivatives and investigated their inter- Yinglong Miao. actions with the recently solved NMR solution structure of Aß42. A combina- Center for Computational Biology and Department of Molecular Biosciences, tion of fluorescence aggregation kinetics, transmission electron microscopy University of Kansas, Lawrence, KS, USA. (TEM) and molecular modeling (Discovery Studio - Structure-Based-Design, The adenosine A1 receptor (A1AR) is a key G-protein-coupled receptor BIOVIA Inc) studies were used to determine the binding of quinazoline deriv- (GPCR) that has served as a therapeutic target for treating cardiovascular atives using the Aß42-monomer, dimer and oligomer models. These studies and neurological diseases. Activation of the A1AR is able to protect the indicate that quinazolines with N2 (alkylamino), C4 (benzylamino) and C8 myocardium against ischemia-reperfusion injury and reduce neuropathic (halogen) substituents were capable of preventing Aß42-aggregation. Compu- pain. In contrast to traditional agonists that often cause off-target side effects, tational studies show that they can bind either at the Ser8-Glu22 loop (consist- positive allosteric modulators (PAMs) that increase the receptor responsiveness ing of the N-terminal region) or at the solvent exposed region consisting of to endogenous adenosine in local regions are promising selective drug candi- His14, Gln15, Leu34 and Met35 in the monomer and dimer models of Aß42. dates of the A1AR. However, the binding modes of PAMs in the A1AR and Comparison of protein-ligand complex energies favors the His14, Gln15, mechanism of the receptor allosteric modulation remain unclear. Here, all- Leu34 and Met35 region as the most likely site to be exploited to prevent atom computer simulations using the robust Gaussian accelerated molecular Aß42 aggregation into higher order structures. Detailed structure activity dynamics (GaMD) method are performed to capture spontaneous binding of studies provide insights into the applications of molecular modeling studies prototypical PAMs to the A1AR. The GaMD simulations allow free energy cal- to understand the mechanisms of Aß42-aggregation. culations to identify low-energy binding modes of the PAMs in the receptor. The predicted binding modes are consistent with previous mutation and binding 1125-Pos Board B34 assay experiments. These results provide important insights into the mechanism Ligand Binding Studies of a Plasmid Encoded Dihydrofolate Reductase by of A1AR allostery at an atomistic level. The work will be also useful for future 19 F NMR computer-aided drug design efforts targeting the A1AR and other GPCRs. Gabriel J. Fuente Gomez. Biochemistry and Cellular and Molecular Biology, University of Tennessee, 1128-Pos Board B37 Knoxville, TN, USA. Effects of Trimethylamine-N-Oxide on the Conformation of Peptides and Plasmid encoded-R67 dihydrofolate reductase (DHFR) catalyzes the NADPH- Proteins dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. R67 DHFR is Zhaoqian Su, Farbod Mahmoudinobar, Cristiano Dias. a homotetramer with a single active site pore and two tryptophans per monomer New Jersey Institute of Technology, Newark, NJ, USA. (W38 and W45). W38 and its symmetry-related partners occur at the dimer- To provide insights into the stabilizing mechanisms of trimethylamine-N-oxide dimer interfaces while the W45 residues occur at the monomer-monomer inter- (TMAO) on protein structures, we perform all-atom molecular dynamics sim- faces. We have labeled these tryptophans using 19F-labeled indole with the 19F ulations of peptides and the Trp-cage miniprotein. Effects of TMAO on the atoms at different positions (4-, 5-, 6- or 7-) of the indole ring. In vitro ligand charged residues of peptides are found to stabilize compact conformations,

BPJ 8613_8616 Monday, February 19, 2018 225a whereas effects of TMAO on nonpolar residues lead to peptide swelling. This mutations, however, are unfortunately unknown. Here, we investigated the ho- suggests competing mechanisms of TMAO on proteins which would accounts mophilic binding of wildtype Dsg2 and two mutations (p.D105E (p.D154E) for swelling of hydrophobic cores and stabilization of charge-charge interac- and p.V343I (p.V392I)), which are associated with ARVC. Using single mole- tions. These mechanisms are studied from replica exchange molecular dy- cule force spectroscopy and applying Jarzynski’s equality we determined the namics simulations of the Trp cage miniprotein. kinetics und thermodynamics of Dsg2 dimerization. Moreover, we modelled the Gibbs free energy landscape of Dsg2 homophilic binding which strengthens 1129-Pos Board B38 the picture of highly specific but low affinity interaction. Interestingly, even Inducible Gene Expression and Protein Localization using CRISPR/ though the strand-swap binding motif is not directly affected by the mutations DCAS9 and Antiviral Protease Inhibitors the binding kinetics differs significantly from the wildtype. Furthermore, we Elliot P. Tague, John Ngo. tested cell network stability against tensile stress by means of a dispase based Biomedical Engineering, Boston University, Boston, MA, USA. cell dissociation assay using HT1080 cell lines overexpressing Dsg2 wildtype In many cases, dissecting biophysical processes in cells requires tools to elicit and mutants, respectively. We found that wildtype cell clusters are apparently time-dependent gene expression and/or protein localization. However, many of more fragile than cell networks bearing specific mutations. Merging our molec- the drugs used in presently available inducible systems possess endogenous ular and cellular data we demonstrate that Dsg2 mutations can heavily affect cellular targets, which can cause undesirable side-effects that make them homophilic Dsg2 interactions. Furthermore, our results allow drafting a consis- incompatible for use in therapeutic applications. With the increasing prospect tent picture of the Dsg2 binding mechanism within the desmosome. of the use of gene modulation in human therapies (i.e., gene therapy, cell- based therapies, etc.), orthogonal drug-inducible systems that use safe ligand 1132-Pos Board B41 molecules will be needed. Here we present a novel method for drug- Discerning Mercury Methylation: Interdisciplinary Approaches to Solve a inducible gene expression control using existing (FDA-approved) anti-viral Complex Puzzle drug compounds that are able to bind and inhibit the cis-proteolytic activity Swapneeta Date1, Katherine Rush2, Xiangping Yin1, Judy Wall3, of the Hepatitis C virus (HCV) protease NS3/4a. Stephen Ragsdale2, Jerry Parks1, Dwayne Elias1, Baohua Gu1, We show that the protease can be used to preserve an artificial transcription factor Alexander Johs1. based on dCas9 subject to drug control via insertion of the viral enzyme between 1Oak Ridge National Laboratory, Oak Ridge, TN, USA, 2University of the dCas9 scaffold and a C-terminal transactivation domain. In the absence of Michigan, Ann Arbor, MI, USA, 3University of Missouri, Columbia, MO, drug, the protease serves as a self-immolating linker that leads to dismemberment USA. of the chimera. Upon exposure to drug, the protease is inhibited and intact Bioaccumulation of highly toxic methylmercury (MeHg) in the food web is a chimera are preserved. Intact copies of the protein are able to translocate to the significant public health concern. The transformation of inorganic mercury nucleus to activate the expression of sgRNA-specified target genes. Use of deposited in soils and sediments to MeHg has been linked to a two-gene clus- NS3/4a and its anti-viral inhibitors as a preserveable linker have proven to be ter (hgcAB), which has been identified in the genomes of more than 180 bac- highly modular and can be inserted into a host of other chimera, including alter- teria and archaea to date. The two genes are predicted to encode a corrinoid- nate RNA hairpin binding transcription factors. Overall, these results demonstrate dependent membrane protein, HgcA, and a 2[4Fe-4S] ferredoxin, HgcB, the versatility of using the HCV NS3/4a domain as a drug-sensitive module for acting as methyl carrier and electron shuttle, respectively. However, the mo- regulating the activity and localization of engineered transcription factors. lecular structures of HgcA and HgcB, and the specific mechanism of mercury 1130-Pos Board B39 methylation have not been determined. Furthermore, there is only limited Systematic Development of Small Molecules to Inhibit Specific Micro- knowledge about metabolic pathways linked to mercury methylation. Our scopic Steps of Amyloid-Beta42 Aggregation in Alzheimer’s Disease experimental efforts are focused on examining enzyme kinetics and identi- Sean Chia1, Johnny Habchi1, Ryan Limbocker1, Benedetta Mannini1, fying cellular factors affecting mercury methylation activity in whole-cell ex- Minkoo Ahn1, Michele Perni1, Oskar Hansson2,3, Paolo Arosio1, tracts of the model methylator Desulfovibrio desulfuricans ND132. Janet R. Kumita1, Pavan Kumar Challa1, Samuel I.A. Cohen1, Sara Linse4, Furthermore, tandem affinity-tagged constructs of HgcA and HgcB in Christopher M. Dobson1, Tuomas P.J. Knowles1, Michele Vendruscolo1. ND132 cells are used for pull-down and cross-linking analysis of cellular 1University of Cambridge, Cambridge, United Kingdom, 2Memory Clinic, components involved in MeHg formation. Bioinformatic analyses of interac- Ska˚ne University Hospital, Malmo, Sweden, 3Department of Clinical tion and co-occurrence networks are used to complement the experimental Sciences, Lund University, Lund, Sweden, 4Department of Biochemistry & data in delineating the biomolecular machinery involved in the enzymatic Structural Biology, Center for Molecular Protein Science, Lund University, MeHg formation and the associated cellular import and export of mercury Lund, Sweden. species. In a broader context, the molecular level studies are complemented The aggregation of the 42-residue form of the amyloid-beta peptide (amyloid- by quantum chemistry calculations to reveal reaction mechanisms, pure cul- beta42) is a pivotal event in Alzheimer’s Disease (AD). Here, we describe a ture and microbial community studies, field observations and integration into chemical kinetics-based approach that allows us to quantify precisely the ef- reactive transport models to understand mercury cycling across many spatial fects of small molecules on the aggregation of amyloid-beta42. We thus report and temporal scales. A comprehensive understanding of the various the identification of a library of compounds that can target specific microscopic geochemical and biochemical factors culminating in the production of steps in amyloid-beta42 aggregation. By validating the effects of these com- MeHg will facilitate the development of effective strategies to reduce expo- pounds in human cerebrospinal fluid, and in a Caenorhabditis elegans model sure to this pervasive neurotoxin. of AD, we show that this approach provides us a diverse pool of small mole- cules that can be taken forward in drug discovery programs for AD. This sys- 1133-Pos Board B42 tematic concept also offers an exciting opportunity in enabling us to rationally Understanding the Toxicity and Repurposing Potential of Kinase design candidate molecules that can both inhibit specific microscopic steps of Inhibitors the protein aggregation process, and at the same time, possess good drug phar- Hammad Naveed. mocokinetic characteristics. Computer Science, National University of Computer & Emerging Sciences, Islamabad, Pakistan. 1131-Pos Board B40 The promiscuity of drugs towards protein binding sites and vice versa hinders Arrhythmogenic Cardiomyopathy Related DSG2 Mutations Affect Deso- our understanding of the metabolism of healthy and diseased states. The iden- mosmal Binding Kinetics tification of all protein targets is therefore important to understand a drug’s Mareike Dieding1, Jana D. Debus2, Raimund Kerkhoff1, Anna Gaertner- (side) effects, and to reuse existing drugs. Current FDA approved kinase inhib- Rommel2, Volker Walhorn1, Hendrik Milting2, Dario Anselmetti1. itors have significant side-effects, many of which might be due to the off-targets 1Biophysics Department, University of Bielefeld, Bielefeld, Germany, 2Erich of these drugs. We have developed a novel computational approach for drug & Hanna Klessmann Institute for Cardiovascular Research and Development, target prediction for large-scale discovery of new targets for existing drugs. Heart and Diabetes Center NRW, Bad Oeynhausen, Germany, Bad For a given drug, we construct a probabilistic pocket ensemble (PPE) that cap- Oeynhausen, Germany. tures the promiscuous structural features of drug binding sites. We were able to Cadherins are calcium dependent adhesion proteins that establish the mechanic predict the interaction of six FDA approved kinase inhibitors (Sorafenib, Ima- cell-cell contact by bridging the gap between adjacent cells. Desmoglein-2 tinib, Gefitinib, Dasatinib, Sunitinib, Pazopanib) with its known targets with a (Dsg2) is a specific cadherin of the intercellular contact in cardiac desmosomes. sensitivity of 50% and specificity of 56%. 4 out of the 5 top predicted targets for Mutations in the DSG2-gene are known to cause arrhythmogenic (right) ven- Sorafenib were shown to interact with it using in vitro experiments. Our method tricular cardiomyopathy (ARVC) which is a rare but severe heart muscle dis- is broadly applicable for the prediction of protein-small molecule interactions ease. The molecular pathomechanisms of the vast majority of DSG2 with several novel applications to biological research and drug development.

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1134-Pos Board B43 allows label-free measurements at physiological concentrations for applica- Binding Pathways of Phenylalanine to the Dimeric Regulatory Domain of tions in drug discovery. In this work, we demonstrate dual-gated FET struc- Human PAH Reveal a LID Gating Mechanism tures that improve measurement sensitivity and stability in a test-tube or in Yunhui Ge1, Eileen K. Jaffe2, Vincent A. Voelz1. microfluidic channels. Monte Carlo and molecular dynamics simulations 1Chemistry Department, Temple University, Philadelphia, PA, USA, are used to gain molecular insight into the mechanism of inhibition, and 2Temple University Health System, Fox Chase Cancer Center, Philadelphia, form a powerful complement to experiments. These measurements are PA, USA. possible, in part, due to the recent dramatic improvements in genomics and Phenylalanine hydroxylase (PAH) is an enzyme that catalyzes the conver- proteomics that have allowed the synthesis of libraries of thousands of sion of phenylalanine to tyrosine, and functions in humans to control free drug candidates that can cross the blood-brain barrier. Rapid screening of phenylalanine (Phe), an essential amino acid that is neurotoxic at elevated therapeutic molecules is critical to the development of new treatments, for levels. Mutations in PAH can result in phenylketonuria, which is the most example to target Alzheimer’s disease, and are a crucial challenge in the common inborn error of amino acid metabolism. The transition from drug discovery pipeline. resting-state PAH to activated PAH requires formation of a new intersubunit interface that can be stabilized by Phe binding in an allosteric manner; for- 1137-Pos Board B46 mation of the new protein:protein interface is coupled to exposure of the The pH Dependence of Ketamine Binding to G-Protein Coupled Receptors active site, thus activating the enzyme. A recent crystal structure of this Thomas T. Joseph1, Roderic G. Eckenhoff1, Grace Brannigan2. 1Department of Anesthesiology and Critical Care, University of interface bound to Phe enables new insight into phenylalanine binding 2 mechanism from molecular dynamics (MD) simulations. We performed Pennsylvania, Philadelphia, PA, USA, Department of Physics, Rutgers massively parallel explicit-solvent simulations on the Folding@home University, Camden, NJ, USA. distributed computing platform to elucidate pathways and rates of binding Ketamine has various desirable clinical effects, acting as an anesthetic, of Phe to the interface, which is comprised of a dimer of ACT domains. analgesic, and antidepressant, and it is useful as an opioid adjunct. These Time-lagged independent component analysis (tICA) of binding trajectories effects are thought to be primarily mediated through N-methyl-D-aspartate suggest a conformational selection mechanism. Markov state models (NMDA) receptor antagonism. However, experiments suggest that keta- (MSMs) constructed from the trajectory data reveal a key loop motion mine also binds, albeit relatively weakly, to G protein coupled receptors which acts as a ‘‘gatekeeper’’ of allosteric ligand binding. Binding rates (GPCRs), but the structural basis for this is not currently known. Using mo- estimated by different methods (MSMs, Transition Path Theory and lecular dynamics (MD), we studied the interaction of S-ketamine - for Bayesian inference) agree well with each other. These results warrant which we carefully derived molecular mechanics parameters - with three further MD studies of the conformational dynamics involved in regulation GPCRs: mu opioid receptor, kappa opioid receptor, and 5HT2B (serotonin) of PAH activity, the effect of disease-associated mutations, and suggest receptor. We hypothesized that S-ketamine would bind to GPCRs in their future directions in simulation-based drug discovery. orthosteric binding sites, and found that the protonated form of S-ketamine (pKa 7.5) did so in each receptor. Using free energy perturbation (FEP) MD, we found that the binding affinity of neutral S-ketamine for each 1135-Pos Board B44 GPCR was low, but affinity was high for protonated S-ketamine; this is Antibiotic Permeation across the Bacterial Outer Membrane Porin mediated by specific residues in the binding sites. This implies a strong 1,2 1,2 3 Nandan Haloi , Mrinal Shekhar , Bryon S. Drown , pH dependence for ketamine binding to GPCRs. As mu and kappa receptors Paul J. Hergenrother3,4, Emad Tajkhorshid1,2. 1 are important in chronic pain and serotonin receptor has been implicated in Center for Biophysics and Quantitative Biology, University of Illinois at antidepressant activity as well as hallucinogenic effects, these findings may Urbana Champaign, Champaign, IL, USA, 2Beckman Institute of Advanced 3 serve to generate further hypotheses about the molecular mechanisms of Science and Technology, Urbana, IL, USA, Department of Chemistry, ketamine’s clinical effects. University of Illinois at Urbana Champaign, Champaign, IL, USA, 4Institute for Genomic Biology, Urbana, IL, USA. 1138-Pos Board B47 Antibiotic resistance has become a pressing concern for treating bacteria in- Amyloid Aggregation of hIAPP, Ab, and Calcitonin Altered by a Curcu- fections. Limited knowledge on physicochemical properties that enable min Derivative permeation of antibiotic in Gram-negative bacteria has severely limited the Sarah J. Cox1, Diana C. Rodriguez Camargo2, Young-Ho Lee3, Bernd Reif2, development of new antibiotics. To better understand the features which Magdalena Ivanova4, Ayyalusamy Ramamoorthy1. enable molecules to enter cells, we have directly measured the accumulation 1Chemistry, University of Michigan, Ann Arbor, MI, USA, 2Chemistry, of a collection of 180 diverse compounds in Escherichia coli (E. coli). From Technical University of Munich, Munich, Germany, 3Institute for Protein this dataset, we identified the presence of a primary amine, low flexibility, and Reseach, Osaka University, Osaka, Japan, 4Neurology, University of low three-dimensionality as key determinants for accumulation. These guide- Michigan, Ann Arbor, MI, USA. lines were then applied to convert deoxynybomycin (DNM), a natural product Small molecules have largely been studied for their ability to modulate dis- exclusively active against Gram-positive bacteria, into an antibiotic with þ ease related amyloidogenic peptides and proteins through inhibition of am- broad-spectrum activity that contains an amine (DNM-NH3 ). Having discov- yloid fibril growth, promotion of aggregation, or disaggregation of fully ered properties that confer accumulating ability, we sought to understand the mature fibers. Many small molecules that are known to interfere with amy- mechanism of penetrance. We used all-atom molecular dynamics (MD) sim- loid aggregation are considered pan assay inference compounds (PAINS) ulations combined with free-energy methods to study the permeation of DNM þ meaning they nonspecifically interact with many biological targets. A com- and DNM-NH3 across the bacterial outer membrane porin F (OmpF) of E. mon example is curcumin, the bulk polyphenol found in turmeric that is coli. Because of the unavailability of force field parameters for novel com- known to interact with many different proteins and peptides including amy- pounds, we developed the parameters for the two antibiotics, which can accu- loidogenic proteins. Despite this, curcumin is limited by poor water solubi- rately model the natural chemical behavior in our simulations. Thereafter, lity and fast degradation. CurDAc, a previously described water-soluble steered molecular dynamics is used to explore the potential permeation stable curcumin derivative, has been shown to inhibit hIAPP aggregation pathway of the drugs along the porin. Then, we employed bias-exchange um- in the presence and absence of lipid membranes. In this study, we show brella sampling to refine the permeation pathway and calculate the free-energy the effects of CurDAc on three different amyloidogenic peptides: human landscape. This intensive investigation enabled us to characterize the specific þ islet amyloid polypeptide (hIAPP), amyloid beta (Ab) and human calcitonin electrostatic interactions of DNM-NH3 with the charged residues at the (hCT). Using commonly employed biophysical techniques including constriction region of the porin, facilitating its permeation, which was absent thioflavin-T fluorescence assays, circular dichroism, isothermal calorimetry, in the case of DNM. and transmission electron microscopy, we show that CurDAc has an increased effect on hIAPP over Ab and hCT in both inhibition of growth 1136-Pos Board B45 and disaggregation of fibers. NMR spectroscopy was used to characterize Measurements of Enzyme Activity with Field-Effect Transistors hIAPP monomers and disaggregated fibers in the presence of CurDAc. Nicholas Guros1, Son T. Le1, Antonio Cardone1, Brent Sperling1, Cell toxicity experiments show that CurDAc is a non-toxic compound but Curt Richter1, Jeffery Klauda2, Harish Pant3, Arvind Balijepalli1. that both monomers and fibers treated with CurDAc are more toxic than hI- 1NIST, Gaithersburg, MD, USA, 2University of Maryland, College Park, APP monomers and fibers alone, suggesting that the small molecule stabi- MD, USA, 3NIH, Bethesda, MD, USA. lizes some sort of toxic species. This work shows the potential of We measure the activity of the cyclin dependent kinase, Cdk5, in the presence CurDAc to be used as a chemical probe to study hIAPP and suggests the of therapeutic inhibitors using field-effect transistors (FET). The technique mechanism of action is sequence specific.

BPJ 8613_8616 Monday, February 19, 2018 227a

1139-Pos Board B48 curli that are the major protein component of their extracellular matrix. Curli Binding Behavior and Energetics between Curcumin and Amyloid-b mediate cell-cell and cell-surface interactions and tie the bacteria together Aggregates at the Molecular Scale into multicellular communities called biofilms. Biofilms exhibit increased Tye D. Martin1, Angelina J. Malagodi2, Eva Y. Chi3, Deborah G. Evans4. resistance to antibiotics and host defenses and are implicated in persistent in- 1Center for Biomedical Engineering, University of New Mexico, fectious diseases including urinary tract infection (UTI) and cystic fibrosis. Albuquerque, NM, USA, 2Department of Chemistry, Macalester College, Interestingly, functional and disease-associated amyloids share several impor- Saint Paul, MN, USA, 3Department of Chemical and Biological Engineering, tant characteristics including an ordered beta-sheet repeat structure and the University of New Mexico, Albuquerque, NM, USA, 4Department of ability to self-assemble into amyloid fibers. In Parkinson’s disease, it is thought Chemistry and Chemical Biology, University of New Mexico, Albuquerque, that amyloid toxicity can be attributed not to the fibers themselves, but to sol- NM, USA. uble intermediate species. It is not known if functional amyloids go through Misfolding and aggregation of the amyloid-b (Ab) peptide into b-sheet en- similar aggregation pathways as disease associated amyloids, or if organisms riched fibrils is a central pathogenic event that leads to severe cognitive impair- that actively form functional amyloids have evolved a separate mechanism to ment in neurodegenerative disorders such as Alzheimer’s disease. Polyphenols avoid the toxic intermediates during fiber formation. and derivatives, many of which occur naturally, have been known for their ther- I will examine the amyloid assembly process by using small molecules to alter apeutic antioxidant and anti-inflammatory properties. More recently, their neu- the aggregation pathway. I will compare the effects of these molecules on the roprotective and anti-amyloidogenic properties are also becoming clear. We Parkinson’s disease associated amyloidogenic protein alpha-synuclein and the have shown that a particular polyphenol, curcumin found in turmeric, amelio- functional amyloid curli subunit, CsgA. I will delineate the inhibitory and rates toxicity of Ab40 oligomers and modulates the growth of Ab fibrils. Addi- accelerating effects of these molecules amyloid assembly based on Thioflavin tionally, when Ab40 is coincubated with curcumin, curcumin induces the T (ThT) fluorescence studies. My initial results illustrate that these molecules formation of off-pathway oligomers that are non-toxic. Herein, we investigate can bind selectively to different oligomeric species of alpha-synuclein, which the molecular scale interaction between curcumin and oligomeric Ab using can arrest the fiber formation at distinct steps. I will characterize the mechanism classical all-atom molecular dynamics (MD). Free energy calculations were of binding and action for each of these molecules and use this knowledge to carried out to obtain free energies of binding between curcumin complexes gain a deeper understanding of the amyloid assembly process. and the profibril (DGbind) and free energies of curcumin complexation (DGcur-cur). DGbind values were large and negative (up to 27 kcal/mol), indi- Posters: Protein Assemblies II cating favorable binding of curcumin complexes to the fibril. DGcur-cur values were approximately 11 kcal/mol. Curcumin also binds to the ends of the fi- 1142-Pos Board B51 brils with high binding energies and as single molecules. At these binding sites, Effect of Hyperglycemic Conditions on the Early Self-Assembly of the curcumin is embedded into the fibril ends. The locations of these bound curcu- Alzheimer’s Amyloid Beta Peptide: Implications for Neurotoxicity min suggest that they could affect fibril growth which involves the addition of Sneha Menon1, Neelanjana Sengupta2. 1 Ab monomers to ends of the fibrils. To investigate the effect of curcumin on Physical Chemistry Division, CSIR-National ChemicalLaboratory, Pune, 2 fibril growth, we are using steered MD simulations to pull Ab monomers to- India, Biological Sciences, Indian Institute of Science Education and ward the protofibril ends in presence and absence of bound curcumin. This Research Kolkata, Mohanpur, India. approach will illuminate curcumin impact pertaining to changes in b-sheet con- Clinical observations suggest correlations between type-2 diabetes mellitus and tent of the monomer and behavior at the protofibril growth axis to support our cognitive decrements associated with Alzheimer’s disease (AD) onset. Interest- experimental findings of altered fibril morphology in presence of curcumin. ingly, post-translational modifications of the amyloid beta (Ab& ) peptide un- der hyperglycemic conditions, in particular the process of glycation, is noted to 1140-Pos Board B49 exacerbate its neurotoxicity and accelerate AD progression. However, it re- NDGA Inhibits Functional Amyloid Biosynthesis and Biofilm Formation mains to be understood if these observations are independent of each other. by Uropathogenic E. coli Further, in view of the assertion that macromolecular crowding has an altering Joshua A. Visser, Lynette Cegelski. effect on protein self-assembly, it is crucial to characterize the effects of hyper- Chemistry, Stanford, Menlo Park, CA, USA. glycemic conditions on Ab& oligomerization, and therefore the potential ef- Uropathagenic Echerichia coli (UPEC) are a leading cause of community- fects of such conditions on the peptide’s neurotoxicity. In order to delineate acquired urinary tract infections (UTI). Recurrent and chronic UTIs serve as the physical effects of hyperglycemic conditions from the effects of chemical a particular challenge to treatment and can lead to antibiotic resistance and modifications, fully atomistic molecular dynamics simulations were employed sepsis. Biofilm formation is one mechanism used by bacterial to survive harsh to understand the effects of glucose crowding on early Ab& self-assembly. The insults in the urinary tract. UPEC form biofilms on catheters and also in the Ab& dimers formed in the glucose crowded environment are found to have intracellular niche of bladder epithelial cells, where cells can remain protected weaker associations as compared to those formed in pure aqueous environment. from antibiotics and other host defenses. Biofilms are a general mechanism Binding free energy calculations indicate an overall weakening of the disper- across bacterial species to assemble bacterial communities through the assem- sion interactions associated with substantial loss of inter-peptide hydrophobic bly of a self-secreted polymeric matrix surrounding bacterial cells. The extra- contacts. Comparison of the solvent-protein interaction patterns reveals that cellular matrix of most UPEC strains is comprised of two major components: glucose molecules cluster around the peptide units and thereby trap water mol- curli, a functional amyloid fiber, and a modified form of cellulose. Curli makes ecules near the surface, resulting in a screening effect on the overall hydropho- up 85% of the extracellular matrix in the UPEC strain UTI89 and is required for bic interactions. Interestingly, this screening manifests in noticeable biofilm formation on agar, at the air-liquid interface and attached to plastics. differential solvation effects in the dimers formed in pure aqueous and in the Thus, curli biosynthesis and curli-based adhesion represent ripe targets to glucose crowded environments. Our results strongly suggest that solely phys- inhibit biofilm formation. Using transmission electron microscopy (TEM), ical effects attributed to crowded hyperglycemic environments are incapable absorbance and fluorescence spectroscopy, and protein profiling by western of promoting Ab& self-assembly, and hence, the observed enhancement in blotting, we discovered that a small molecule inhibitor, nordihydroguaiaretic neurotoxicity is likely to arise from post-translational chemical modifications. acid (NDGA), is a potent inhibitor of curli assembly. Additionally, biofilm for- mation at the solid-air and liquid-air interfaces in the presence of compound 1143-Pos Board B52 was inhibited. NDGA, derived from the creosote bush, has been reported pre- Oligomerization and Fibrillization Dynamics of Amyloid Peptides and viously to have antioxidant activity, but its effects in bacterial adhesion and BETA-Barrel Oligomer Intermediates biofilm formation have not been explored. This work provides the potential Yunxiang Sun, Xinwei Ge, Feng Ding. for this natural product to be used therapeutically in an anti-virulence approach Department of Physics and Astronomy, Clemson University, Clemson, SC, for the prevention and treatment of UTI. USA. Aggregation of proteins and peptides into amyloid fibrils is associated 1141-Pos Board B50 with more than 25 degenerative diseases, including amyloid-b (Ab)in Interrogating Functional Amyloid Formation using Small Molecules Alzheimer’s disease, islet amyloid polypeptide (IAPP) in type 2 diabetes. Elizabeth Gichana. The amyloid fibrils formed by various amyloid peptides share the same char- University of Michigan, Ann Arbor, MI, USA. acteristic cross-b structures and soluble low molecular weight oligomers are Amyloids are most commonly recognized as misfolded, fibrous, protein aggre- more cytotoxic than mature fibrils. Structural characterization of the inter- gates that are associated with cellular toxicity and implicated in neurodegener- mediate oligomers is hindered by their highly transient and metastable prop- ative diseases such as Alzheimer’s and Parkinson’s. However, many enteric erties. Here, I will present our recent computational results about the bacteria such as E. coli produce extracellular ‘‘functional amyloids’’ called oligomerization and fibrillization dynamics of amyloid peptides, including

BPJ 8613_8616 228a Monday, February 19, 2018 the amyloid core of amyloid-beta (Ab16-22) and islet amyloid polypeptide ture provides insights into the PrPC to PrPSc conversion mechanism. The (IAPP22-28). Our all-atom discrete molecular dynamics simulations pro- folding properties of regular beta-solenoid proteins revealed a nearly universal vided a complete picture of amyloid peptide oligomerization, nucleation occurrence of ‘‘capping structures’’ that prevent the propagation of the beta- of b-sheets, and the formation of cross-b aggregates. Ab16-22 is highly solenoid conformation. These caps consist of short alpha-helices, loops, or aggregation-prone with a funneled free energy basin toward multi-layer other structures and their removal resulted in aggregation and amyloid forma- b-sheet aggregates. IAPP22-28, on the other hand, features the accumulation tion. The absence of capping structures in PrPSc allows the propagation of its of unstructured oligomers before the nucleation of b-sheets and growth into beta-solenoid conformation to neighboring PrPC molecules. The putative double-layer b-sheet aggregates. Oligomer size and structure analysis re- head-to-head arrangement of the PrPSc dimers implies a role for matching se- veals that the critical nucleus size for Ab16-22 is less than 2, and for quences in the conversion mechanism. The fidelity of the templating process, IAPP22-28 is approximately between 6 and 8. In a separate study of the which is a hallmark of the PrPSc propagation, can thus be attributed to molec- aggregation dynamics of hIAPP15-25 and hIAPP19-29 with contrasting ular properties of the PrPSc structure. cytotoxicity, we found a correlation between amyloid toxicity and the pro- pensity to form b-barrel oligomers as aggregation intermediates. The same 1146-Pos Board B55 b-barrel oligomers were also observed as the intermediate states in the Effect of Bio-molecules on Human Islet Amyloid Polypeptide Aggregation, Ab16-22 and hIAPP22-28 aggregation process. Therefore, our results sup- Fibril Remodeling and Cytoxicity b Yanting Xing1,2, Feng Ding1, Pu Chun Ke3. port the hypothesis of -barrel oligomers as the toxic species of amyloid 1 aggregation. Department of Physics and Astronomy, Clemson University, Clemson, SC, USA, 2Materials Science and Engineering, Clemson University, Clemson, 1144-Pos Board B53 SC, USA, 3Monash Institute of Pharmaceutical Sciences, Monash University, The Glycolytic Enzyme Phosphofructokinase-1 Assembles into Filaments Parkville, Australia. Bradley Webb1, Annie Dosey2, Torsten Wittmann1, Justin Kollman2, Amyloid aggregation of human islet amyloid polypeptide (IAPP) is a hallmark of Diane Barber1. type 2 diabetes (T2D), a metabolic disease and a global epidemic. Although 1Cell and Tissue Biology, UCSF, San Francisco, CA, USA, 2University of IAPP is synthesized in pancreatic b-cells, its fibrils and plaques are found in Washington, Seattle, WA, USA. the extracellular space. In consideration of the changing conformation and hy- Despite abundant knowledge of the enzymology, regulation, and biochemistry drophobicity of IAPP during its fibrillation process, it is reasonable to postulate of glycolytic enzymes, we have limited understanding of how they are spatially that the molecular ligands encountered by IAPP from cradle to grave may exert organized in the cell. Emerging evidence indicates that non-glycolytic meta- effects on the physical and biological identities of the IAPP species. Using bolic enzymes regulating diverse pathways can assemble into polymers. We discrete molecular dynamics simulations (DMD) with a battery of biophysical show here the tetramer- and substrate-dependent filament assembly by characterizations, we examined the interaction of freshly-dissolved IAPP as phosphofructokinase-1 (PFK1). Purified, recombinant liver isoform of PFK1 well as pre-formed fibrils with two homologous serum proteins, namely cationic (PFKL) but not the platelet (PFKP) or muscle PFKM isoforms assembled lysozyme (Lys) and anionic alpha-lactalbumin (aLac), as well as the most abun- into filaments, initiated by the substrate fructose-6-phosphate (F6P), as deter- dant lysophospholipid in the blood, lysophosphatidylcholine (LPC) in mono- mined by electron microscopy and light scattering. Negative stain electron mi- meric and micellar conformations. Depending on the physicochemical crographs reveal that PFKL filaments are apolar and made of stacked tetramers properties of molecular ligands including net charges, hydrophobicity, and oriented with exposed catalytic sites positioned along the edge of the filament. morphology, these biomolecules have different effects on IAPP aggregation dy- The interface between PFKL tetramers is mediated by two mutually exclusive namics and cytotoxicity. For example, Lys stabilized toxic oligomers while aLac interaction surfaces, explaining the 130 kinks frequently observed in fila- promoted amorphous aggregation with reduced toxicity. Monomeric LPC accel- ments. We performed live-cell microscopy of EGFP-tagged PFK1 expressed erated IAPP aggregation but the micellar LPC inhibited the aggregation by sta- in MTLn3 rat mammary carcinoma cells to assess filament assembly, dy- bilizing IAPP monomers in helical conformations. This study facilitates our namics, and regulation. TIRF microscopy revealed that PFKL-EGFP formed understanding of the fate and transformation of IAPP in vivo, which are expected dynamic punctae throughout the cytoplasm. A small subset of PFKL-EGFP to have consequential bearings on IAPP glycemic control and T2D pathology. particles remained stationary near the ventral plasma membrane for several sec- onds, suggesting temporary docking events. Neither glucose starvation nor in- 1147-Pos Board B56 cubation of cells with 2-deoxyglucose, a glucose analogue that cannot be A Pharmacophore Approach for Protein Interface Design metabolized, had a discernable impact on punctae size, dynamics, or membrane Andras Fiser. docking. However, addition of the allosteric inhibitor citrate induced the Systems and Computational Biology, Albert Einstein College of Medicine, reversible formation of large PFKL-EGFP-positive structures resembling stress Bronx, NY, USA. granules. Our findings reveal a new behavior of a key glycolytic enzyme with Members of extracellular Immunoglobulin Superfamily (IgSFs) play a key insights on spatial organization and isoform-specific glucose metabolism in role in immune regulation through the control of the co-stimulatory pathway, cells, including possibly a mechanism for localized ATP production. Moreover, and have emerged as potent drug targets in cancers, infectious diseases, and our findings open a clinically relevant new direction for determining whether autoimmunity. Despite the difficult experimental access to this class of pro- PFKL filaments regulate metabolic programming in diseases such as cancer. teins, single structures of ectodomains of IgSF proteins are solved with reg- ularity. However, the most biologically critical challenge for this class of proteins is the identification of their cognate ligands that communicate inter- 1145-Pos Board B54 cellular signals. We describe a conceptually novel method, Protein Ligand The Structure of the Infectious Prion Protein Constrains Potential Prion Interface Design (ProtLID), for the problem of identifying cognate ligands Replication Mechanisms from a sub-proteome for a given target receptor protein. ProtLID designs 1 2 Holger Wille , Jesu´s R. Requena . an optimal protein interface for a given receptor by running extensive molec- 1Department of Biochemistry, University of Alberta, Edmonton, AB, 2 ular dynamics simulations of single residue probes. The type and location of Canada, Department of Medical Sciences, University of Santiago de residue preferences establish a residue-based pharmacophore, which is sub- Compostela, Santiago de Compostela, Spain. sequently used to find potential matches among candidate ligands within a X-ray fiber diffraction and cryo electron microscopy revealed that the infec- Sc sub-proteome. In a subsequent study we illustrate how PROTLID can be tious prion protein, PrP , contains a four-rung beta-solenoid structure as a used to redesign a known protein binding interface. A wild type antibody key element of its self-propagating conformation. In particular, amyloid fibrils is redesigned for a higher affinity binder using an example of an antibody with their regular and repetitive nature allowed important insights into the that binds Denge virus coat protein and neutralizes all four serotypes Sc ˚ structure of PrP . X-ray fiber diffraction revealed a height of 19.2 A per mole- successfully. cule of PrPSc along the fibril axis. Meridional diffraction signals at 4.8, 6.4, and 9.6 A˚ indicated four rungs of beta structure (4 x 4.8 A˚ = 19.2 A˚ ), while the lack 1148-Pos Board B57 of a 10 A˚ equatorial reflection implied a beta-solenoid configuration. Unpro- Both Cell-Associated and Secreted Forms of the P. aeruginosa Adhesin cessed cryo EM micrographs of PrPSc fibrils displayed 4.8 A˚ spacings, confirm- CDRA Promote Biofilm Formation ing the cross-beta structure. 3D reconstructions of individual PrPSc fibrils Courtney Reichhardt1, Cynthis Wong1, Daniel Passos da Silva1, indicated a molecular height of 17.7 A˚ , in good agreement with the X-ray fi- Daniel J. Wozniak2, Matthew R. Parsek1. ber diffraction results. Moreover, single particle averaging of short fibril seg- 1Microbiology, University of Washington, Seattle, WA, USA, ments revealed periodicities of 2 and 4 nm. The first measurement is 2Microbiology, The Ohio State University, Columbus, OH, USA. consistent with the molecular height of PrPSc, while the latter suggests a higher Most microbes assemble as biofilms, which are multicellular aggregates order such as a head-to-head dimerization. The four-rung beta-solenoid struc- that are encased in a biopolymer-rich matrix. The individual matrix

BPJ 8613_8616 Monday, February 19, 2018 229a components interact to maintain bacterial association with aggregates and binding and enhancing kinase activity. We also describe a NMR-based shield against harsh conditions such as antimicrobials or oxidative stress. method to distinguish between myristate-site allosteric inhibitors and activa- Additionally, the matrix must be able to readily disassemble to allow bac- tors of Abl. teria to disseminate and infect new sites. These seemingly incongruent traits, structural stability and plasticity, are key to the biofilm lifecycle. Us- 1151-Pos Board B60 ing a model biofilm system, Pseudomonas aeruginosa, we sought to answer Integrative Structural Biology of the Calcium Dependent Type 2 Secretion how bacteria and matrix molecules interact to lend these traits to biofilms. Pseudopilus 1 1 1 Specifically, we investigated the role of a key biofilm matrix protein called Aracelys Lopez-Castilla , Benjamin Bardiaux , Jenny-Lee Thomassin , 2 1 2 1 CdrA in the maintenance and regulation of P. aeruginosa biofilm structural Weili Zheng , Michael Nilges , Edward Egelman , Olivera Francetic , 1 integrity. CdrA can be both cell-associated and proteolytically processed to Nadia Izadi Pruneyre . 1Structural Biology and Chemistry, Institut Pasteur, CNRS, Paris, France, be secreted away from the bacterial surface. Using a combination of micro- 2 biological and biophysical techniques including biofilm culturing assays, Biochemistry and Molecular Genetics, University of Virginia, confocal scanning laser microscopy, and electron microscopy, we sought Charlottesville, VA, USA. to determine the roles of cell-associated and released forms of CdrA. We Gram-negative bacteria use trans-envelope nano-machines to secrete proteins discovered that both forms of CdrA play distinct roles in interactions or complexes essential for nutrient acquisition, adaptation and virulence. The with the exopolysaccharide Psl and overall biofilm matrix stability. Our type 2 secretion system (T2SS) assembles membrane anchored fibers called pseudopili that promote secretion of specific and folded proteins from the peri- improved understanding serves as a model for other biofilm systems, and 1 additionally it could provide a mechanism to control or treat biofilms pre- plasm to the extracellular medium . Pseudopili are polymers of protein subunits sent in abiotic surfaces as well as in infections such as airway infections in assembled in helical fibers similarly to type 4 pili and flagella. Medical, envi- patients with cystic fibrosis or in chronic wounds. ronmental and fundamental relevance of these molecular machines can explain the growing interest in their study. However, despite considerable advances in 1149-Pos Board B58 genetic, biochemical and structural analysis, the molecular mechanism of fibre Understanding the Endogenous Inhibition of IAPP Aggregation assembly remains unclear. Feng Ding. In this work, we determined the structure of the T2SS pseudopilus from Kleb- Physics and Astronomy, Clemson Unversity, Clemson, SC, USA. siella by combining the NMR structure of its subunit PulG in calcium bound Amyloid aggregation of islet amyloid polypeptide (IAPP) is associated with state with the 5 A˚ resolution cryo-electron microscopy (cryoEM) reconstruc- b-cell death in type-2 diabetes (T2D)[1]. IAPP readily forms amyloid fibrils tion of assembled fibers2. in vitro at mM concentrations. However, no apparent IAPP aggregates are The structural data together with mutagenesis and functional assays revealed observed in healthy individuals where IAPP is stored in b-cell granules at the key role of calcium on the folding and the stability of PulG as well as mM concentrations. Therefore, physiological conditions of b-cell granules the integrity of pseudopili. Our results suggest wider significance of the regu- endogenously inhibit the amyloid aggregation of IAPP. Disruption of the latory role of calcium in the pseudopilus length control, essential for protein inhibitive environment of b-cell granules may lead to the accumulation of secretion. toxic IAPP aggregates, causing b-cell death in T2D. The cellular environ- 1 : Thomassin et al., Mol Mic 2017. 2 : Lopez Castilla et al., Nature Microbi- ment of beta-cell granules is unique in its high concentrations of Zn2þ,in- ology, in press. sulin and proinsulin c-peptide. Combining discrete molecular dynamics simulations with experimental characterizations and validations, we have 1152-Pos Board B61 investigated both the effects of individual molecules and also their coopera- Self-Assembling ABETA(30-36) Peptides: A Combined All-Atom and tive effects on IAPP aggregation. For example, insulin inhibits IAPP aggre- Coarse-Grained Simulation Study Zhenyu Qian1, Qingwen Zhang1, Guanghong Wei2, Peijie Chen1. gation by competing for the amyloid core around residues 22-29 with its B- 1 2 chain[2]. Depending on relative concentrations, zinc either promotes or in- Shanghai University of Sport, Shanghai, China, Fudan University, hibits IAPP aggregation by changing distributions of zinc-coordinated Shanghai, China. b b IAPP oligomers with distinct aggregation propensities[3]. Zinc and C-peptide Aggregation of the -amyloid peptide (A ) leads to the formation of oligomers synergistically inhibits IAPP aggregation by stabilizing IAPP in an and fibrils involved in neurodegeneration in Alzheimer’s disease. The key hy- b b aggregation-incompetent state upon the formation of zinc-coordinated mo- drophobic region, A 30-36 favors -sheet formation and promote aggregation. b lecular complex with C-peptide[4]. These mechanistic insights to the endog- Here we investigate the structure and assembling pathway of A 30-36 peptides enous inhibition of IAPP aggregation may help design novel therapeutic at different peptide concentrations by performing replica-exchange and coarse- b strategies that either mimic or promote the native inhibition of IAPP grained molecular dynamics (MD) simulations. We find that A 30-36 oligomers b aggregation. mainly adopt -sheet-rich bilayers, with in-register antiparallel and one-residue References: 1. ‘‘Implications of Peptide Assemblies in Amyloid Diseases’’, out-of-register parallel alignment. With the peptide concentration increased, b Chem. Soc. Rev., in press, 2017 the A 30-36 peptides display different assembly pathways. The peptides can 2. ‘‘Inhibition of IAPP aggregation by insulin depends on the insulin oligomeric spontaneously assemble into micelles at low peptide concentrations, whereas state regulated by zinc ion concentration’’, Scientific Reports, 5 (2015) it occurs through the formation of small micelles at first, followed by fusing 3. ‘‘Promotion or Inhibition of IAPP Aggregation by Zinc Coordination De- into nanorods at high concentrations. Energetic analysis suggests that the side- pends on Its Relative Concentration’’, Biochemistry, 54:7335-44 (2015) chain hydrophobic interaction and interpeptide hydrogen-bonding play critical 4. ‘‘Zinc-coordination and C-peptide complexation: a potential mechanism for roles in the formation and stabilization of different nanostructures. We also the endogenous inhibition of IAPP aggregation’’, Chem. Comm., 53:9394- examine the influence of G33V and L34T mutations on the assembling struc- b 9397 (2017) ture. This study provides detailed structural information of A 30-36 oligomers, as well as the dynamic pathway and mechanism of Ab30-36 peptides self- assembly. 1150-Pos Board B59 Gleevec Can Act as an Allosteric Activator of ABL Kinase 1153-Pos Board B62 Tao Xie, Tamjeed Saleh, Charalampos G. Kalodimos. Amyloid Beta Proteins Modified by PUFA Oxidation Products in Alz- Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, heimer’s Disease Brain USA. Haralambos A. Mourelatos1, Hiroaki Komatsu1, Ran Furman1, Gleevec remains the first-line therapy for chronic myelogenous leukemia Giuseppe Grasso1,2, Paul H. Axelsen1. (CML) by targeting deregulated kinase activity of Bcr-Abl as a specific 1Pharmacology, University of Pennsylvania, Philadelphia, PA, USA, ATP-competitive inhibitor and its success has revolutionized targeted cancer 2Dipartimento di Scienze Chimiche, Universita` degli Studi di Catania, therapy. Using nuclear magnetic resonance (NMR) spectroscopy and other Catania, Italy. biophysical methods we show other than the ATP binding site, Gleevec binds Oxidatively damaged lipid membranes are known to promote the aggregation to the myristate pocket with a sub-micromolar affinity and unexpectedly acts of amyloid beta (Ab) proteins into fibrils. When lipid membranes contain as an allosteric activator of Abl by preventing the aI-helix from forming a u6 polyunsaturated fatty acyl (PUFA) chains such arachidonic acid (ARA), conformation which is required for assembly of Abl to its inhibited state. 4-hydroxy-2-nonenal (HNE), a highly reactive short chain alkenal, is typically It may lead to diminished concentration of active drug for binding to the produced. In u3 PUFA chains such docosahexaenoic acid (DHA), 4-hydroxy- active site and development of resistance. These results suggest administra- 2-hexenal (HHE) is produced. These oxidation products spontaneously form tion of high-dose of Gleevec may result in poor inhibition of Abl kinase ac- adducts with the side chains of three His residues in Ab proteins. Our in vitro tivity of some Gleevec-resistant mutants by competing ATP-site for Gleevec investigations have shown that HNE can greatly accelerate fibril formation by

BPJ 8613_8616 230a Monday, February 19, 2018 covalently modifying the His residues of Ab proteins. The acceleration is likely 1156-Pos Board B65 due to an increased affinity of the Ab protein for the membrane surface where Explicit Solvent Molecular Dynamics Simulations of Self-Assembling fibril formation is effectively nucleated. HHE, in contrast, has no such effect, Amyloidogenic Peptides most likely because the shorter hydrophobic chains of HHE are not effective Maksim Kouza1, Andrzej Kolinski1, Irina Alexandra Buhimschi2, at anchoring the protein to the membrane. We recently demonstrated that Kloczkowski Andrzej3. oxidative ARA degradation, and hence HNE production, was actively occur- 1Faculty of Chemistry, University of Warsaw, Warsaw, Poland, 2Center for ring only in regions of the human Alzheimer’s disease (AD) brain where am- Perinatal Research, The Research Institute at Nationwide Children’s yloid plaques were present. Hospital, Columbus, OH, USA, 3Battelle Center for Mathematical Medicine, In this study, LC/MS/MS in multiple reaction monitoring (MRM) mode was The Research Institute at Nationwide Children’s Hospital, Columbus, OH, utilized to analyze HNE/HHE-modified Ab proteins pulled-down from human USA. frontal cortex with anti-Ab antibodies and formic acid solubilization. MRM Preeclampsia, a pregnancy-specific disorder has been shown to share typical transition signals indicated that HHE-modified Ab protein appears to be abun- pathophysiological features with protein misfolding disorders including Alz- dant in normal human brain and to be relatively less abundant in AD brain. In heimer’s disease1. All-atom explicit solvent model employed previously for order to quantify the amounts of HNE/HHE-modified Ab proteins in human protein unfolding, folding and docking studies2-4, is used to study oligomeriza- brains, we have developed MRM-LC/MS/MS methods using 13C-Leu-contain- tion of KLVFF and FVFLM peptides which are known to be linked to Alz- ing HNE/HHE-modified Ab proteins as internal standards. These methods heimer’s disease and preeclampsia. We report that FVFLM peptide from show that Ab proteins with 1, 2 and 3 HNE/HHE modifications are abundant SERPINA1 can aggregate and its aggregation is spontaneous with a remarkably in human normal and AD brains. faster rate than that recorded for aggregation ‘‘hot-spot’’ from b-amyloid5, 6. The fast kinetics of FVFLM aggregation was found to be driven primarily by core-like aromatic interactions originating from the anti-parallel orientation 1154-Pos Board B63 of complementarily uncharged strands. We demonstrate a high propensity of Computational Studies of Alpha-Synuclein Fibril Formation and Stability FVFLM for KLVFF binding which suggests that FVFLM-like peptides might Andrew H. Beaven1, Tod D. Romo2, Andrew K. Lewis1, Anthony R. Braun1, be used to prevent the assembly of full-length Ab or other pro-amyloidogenic Alan Grossfield2, Jonathan N. Sachs1. 1 2 peptides into amyloid fibrils. We also show that fibril formation rate can be ac- University of Minnesota, Minneapolis, MN, USA, University of Rochester cessed via mechanical stability or the binding free-energy of a monomer to the Medical Center, Rochester, NY, USA. fibril template. Implications of our finding for relationships between fibril for- Study of alpha-synuclein, the protein whose aggregation causes Parkinson’s mation rates, mechanical stability and binding free energy are discussed. disease, requires an understanding of its complex interactions and equilib- 1. I. A. Buhimschi et al., Science translational medicine 6 (245), 245ra292 rium. Typically, the protein exists in an alpha-helical membrane-bound state (2014). or in solution as an intrinsically disordered protein. However, given circum- 2. M. Kouza, C. K. Hu, H. Zung and M. S. Li, J Chem Phys 131, 215103 (2009). stances that are poorly understood, alpha-synuclein can oligomerize. The first 3. J. Wabik, S. Kmiecik, D. Gront, M. Kouza and A. Kolinski, Int J Mol Sci 14 high-resolution structures of the alpha-synuclein core and entire fibril (i.e., (5), 9893-9905 (2013). Greek key) were recently published by experimental laboratories, making 4. M. Blaszczyk et al., Methods 93, 72-83 (2016). computational studies feasible. Using a large ensemble of short molecular dy- 5. M. Kouza, A. Banerji, A. Kolinski, I. A. Buhimschi and A. Kloczkowski, namics simulations and enhanced sampling techniques, we detail the physical Phys Chem Chem Phys 19 (4), 2990-2999 (2017). chemistry and thermodynamics of these assemblies. The simulation results 6. M. Kouza, N. T. Co, P. H. Nguyen, A. Kolinski and M. S. Li, J Chem Phys describe how fibril stability increases with the number of monomeric sub- 142, 145104 (2015). units, i.e., an 8-mer is the minimally stable structure, while 14- and 18- mers are stable for more than 100 ns. Additional replica exchange molecular 1157-Pos Board B66 dynamics (REMD) simulations of a 10-mer with and without structural water Membrane Interaction and Assembly Mechanism of Ab in Alzheimer’s show that the internal threonine-water network has profound effects on fibril Disease stability. Moving forward, we will combine coarse-grained and all-atom sim- Ya-Ling Chiang1, Hsien-Shun Liao1,2, Catherine Stark1,3, Andrew Liu1, ulations with advanced sampling techniques to explain the mechanism of Audrey Huang1, James Yao1, Paul D. Smith1, Curtis W. Meuse4, fibril elongation and stability. Albert J. Jin1. 1LCIMB, National Institute of Biomedical Imaging and Bioengineering/ NIBIB, Bethesda, MD, USA, 2Department of Mechanical Engineering, 1155-Pos Board B64 National Taiwan University, Taipei, Taiwan, 3Department of Biochemistry, HIV-TAT 32-62 Protein Fragment Forms Fibrillar Structures Stanford University, Stanford, CA, USA, 4Institute for Bioscience and Alina Popescu Hategan1, Joseph Steiner1, Elena Karnaukhova2, Biotechnology Research, National Institute of Standards and Technology and Emilios K. Dimitriadis3, Avindra Nath1. the University of Maryland, Rockville, MD, USA. 1NIH/NINDS, Bethesda, MD, USA, 2FDA/CBER, Silver Spring, MD, USA, 3 Alzheimer’s disease (AD) is characterized by progressive dementia and the loss NIH/NIBIB, Bethesda, MD, USA. of bodily functions that are associated with the formation of plaques in the brain. HIV-Tat protein, the first protein released extracellularly in large amount after These plaques are composed of amyloid beta (Ab), peptides of 36-43 amino HIV virus enters the cell, causes activation of lymphocytes, glial cells and acids. A complex assembly pathway involving many different species of Ab olig- neurotoxicity. Atomic force microscopy showed that HIV-Tat protein can omers and differential interactions with biological membrane is now appreciated. rupture to smaller pieces, some of them being of similar dimensions with the We have used atomic force microscopy (AFM), quartz crystal microbalance with theoretically predicted inteins of this protein, which can spontaneously excise dissipation (QCM-D), and optical spectroscopes including ATR-IR and CD to themselves out of the protein chain. The HIV-Tat 32-62 piece, which we found investigate Ab assembly dynamics on mica and on rafts-forming lipid vesicles to be neurotoxic by itself, was synthetized and used in our experiments. Theo- and supported bilayers. We have observed fibril morphology and fibril elongation retical prediction by PONDR suggested that this fragment is likely unstructured and compared nanomechanics in physiologically relevant buffers. We have espe- and according to AGGRESCAN, prone to aggregation. Atomic force micro- cially focused on resolving small Ab oligomers that may initiate deterioration of scopy showed, aside of round aggregates of various sizes, formation of long brain function in AD and be most relevant to drug interventions. fibrillar structures for this peptide. The fibrils are irregular along length and width, with dimensions in the range 0.4-1.3 nm for height and 3.6 - 8.7 nm 1158-Pos Board B67 for width, as measured at half height. The fibrils present branching and their A New Insight into the Molecular Mechanism of the Inhibition of Lyso- bending is characterized by an average persistence length of 80 nm. Conforma- zyme Fibrillation by Gallic Acid tional assessment of HIV-Tat 32-62 by circular dichroism indicated a second- Mouli Konar, Swagata Dasgupta. ary structure dominated by random coil and beta sheet, with minimal alpha Department of Chemistry, Indian Institute of Technology, Kharagpur, helix content, contrary to full length HIV-Tat. The replacement of C34, F38 Kharagpur, India. and I45 with A in the polypeptide chain of HIV-Tat 32-62 led to a similar sec- It is believed that several life-threatening neurological disorders are caused by ondary structure, but to significant reduction in aggregation capacity, no stable the deposition of some misfolded protein or peptide aggregates in the brain tis- fibril formation and preliminary tests indicated less neurotoxicity than for the sues. The antioxidant activity of plant derived polyphenols makes these com- original peptide. Our results suggest that the aggregation of HIV-Tat 32-62 pounds potential therapeutics that reduce the risks associated with these fragment leads to neurotoxicity, likely through a different mechanism then diseases. Despite extensive investigation on the inhibitory effects of these poly- the full length protein. phenols on amyloidogenesis, the detailed molecular mechanism is still unclear.

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Gallic acid (GA) has been employed to explore the mechanism of inhibition of 1161-Pos Board B70 protein fibrillation. Various spectroscopic techniques such as UV-vis, fluores- Protein Local Conformations at the Light of a Structural Alphabet cence, circular dichroism and dynamic light scattering along with microscopic Alexandre G. de Brevern. studies have been performed to investigate the anti-amyloidogenic property of DSIMB, Sorbonne Paris Cite, INSERM, Univ Paris Diderot, Paris, France. GA on hen egg white lysozyme (HEWL). Results indicate a dose dependent in- Protein structures are classically described in terms of secondary structures. hibition of HEWL fibrillation by GA. Gel electrophoresis studies suggested that Even if the regular secondary structures have relevant physical meaning, their the ability of o-dihydroxy moiety present in the chemical structure of GA to be recognition from atomic coordinates has some important limitations such as oxidized into the quinone moiety and H2O2 in the system plays a pivotal role in uncertainties in the assignment of boundaries of helical and b-strand regions. the inhibition. Covalent binding of quinones to the hydrophobic Trp residues of Further, on an average about 50% of all residues are assigned to an irregular HEWL is the reason for which the hydrophobic regions of HEWL are not state, i.e., the coil. Thus different research teams have focused on abstracting affected upon denaturing conditions. A new insight from this study using cyclic conformation of protein backbone in the localized short stretches. Using voltammetry revealed that GA imparts protection to the partially unfolded pro- different geometric measures, local stretches in protein structures are clus- teins by oxidizing the Met residues into highly polar sulfoxide-modified side tered in a chosen number of states. A prototype representative of the local chains through the in situ generation of H2O2. This helps to prevent self- asso- structures in each cluster is generally defined. These libraries of local struc- ciation by stabilizing the partially unfolded proteins by the solvent molecules. tures prototypes are named as ‘‘structural alphabets’’. We have developed a structural alphabet, named Protein Blocks, not only to approximate the pro- 1159-Pos Board B68 tein structure, but also to predict them from sequence. Since its development, Investigating Curli and Cellulose Interactions in the Spatial Context of we and other teams have explored numerous new research fields using this Bacterial Biofilms structural alphabet. I will review here some of the most interesting applica- Nicolette F. Goularte1, Lynette Cegelski2. 1 2 tions: (i) the most efficient protein superimposition methods, (ii) new ways Structural Biology, Stanford University, Stanford, CA, USA, Chemistry, to analyse protein structures, and (iii) new tool for analysis of protein dy- Stanford University, Stanford, CA, USA. namics and allostery. Uropathogenic E. coli (UPEC) are responsible for the majority of urinary tract infections (UTIs), one of the most common bacterial infections in humans. Half 1162-Pos Board B71 of all women will contract at least one UTI in their lifetime, and the infection Ancient Thioredoxins Evolved to Modern Day Stability-Function Require- frequently recurs after treatment. UPEC enter the urinary tract then spread to ment by Altering Native State Ensemble the bladder as they colonize into bacterial biofilms encapsulated by the protec- Tushar Modi1, Jonathan Huihui2, Kingshuk Ghosh2, Banu Ozkan1. tive structure of an extracellular matrix (ECM). Biofilm formation provides a 1Department of Physics, Arizona State University, Tempe, AZ, USA, bacterial community with more resistance to environmental stresses as well 2Department of Physics and Astronomy, University of Denver, Denver, CO, as antibiotics. The main ECM constituents of UPEC are the functional amyloid USA. protein curli, and bacterial cellulose, a b-1,4 linked glucose polymer, as well as Thioredoxins - small globular proteins that reduce other proteins - are a modified form of cellulose. Curli fibers mediate interaction, attachment, and ubiquitous in all forms of life, from archaea to mammals. Ancestral Thio- colonization on the host while cellulose seems to provide cohesion and highly redoxins (Thrxs), which date back to almost 4 billion years, share high ordered assemblies within the biofilm architecture. sequential and structural similarity with the modern day (extant) Thrxs, We set out to distinguish between curli and cellulose in the complex three- and yet they exhibit significantly different functional activity and stability. dimensional spatial context of bacterial biofilm. Utilizing microscopy, We investigate this by comparative studies of their (ancient and modern biochemistry, and solid-state NMR spectroscopy we probe the structural con- day Thrxs) folded state ensemble quantified by the Dynamic Flexibility In- tacts between curli and cellulose and investigate the machinery responsible dex (DFI). DFI - computed from well-converged (micro-seconds long) all- for production of these ECM components at the molecular level. Obtaining a atom simulations of the native state - measures the relative resilience of an higher resolution view of the order and interactions within the matrix encapsu- amino acid to perturbations in the rest of the chain when displaced out of lating these persistent bacteria will allow for new strategies to disrupt biofilm equilibrium. Clustering proteins based on DFI profiles leads to groups that formation. strongly resemble alternate classification scheme based on their activities and phylogeny. Despite the high structural similarity between the extant Posters: Protein Dynamics and Allostery II and its ancestor on each branch, the DFI profiles of the extant proteins are substantially different around alpha3, alpha 4 and catalytic regions. 1160-Pos Board B69 Moreover, the change in flexibility alters the allosteric coupling of the Antigen Processing at the Atomic Level: MD Simulations of MHCI and its active site with rest of the protein in modern enzymes that may be respon- Peptide-Loading Complex sible for their decreased catalytic activity. At a global level, we observed Olivier Fisette, Sebastian Wingbermuehle, Lars Schaefer. changes in DFI distribution profiles of Thrxs as they evolve. In particular, Theoretical Chemistry, Ruhr-University Bochum, Bochum, Germany. we note the population of low flexible (called hinges) and high flexible Antigens exposed at the cell surface by major histocompatibility complex class sites increases by minimal but subtle manipulations in the flexibility pro- I (MHCI) proteins enable self/non-self recognition by cytotoxic T cells, pro- files without changing the actual hinges. Broadly speaking, the heterogene- tecting the organism against viral infections and cancer-causing mutations. ity (quantified by the variance) in the DFI distribution increases with the To perform their role, MHCI must first be loaded with an antigenic peptide in- decrease in the melting temperature typically associated with evolution side the endoplasmic reticulum (ER), a process controlled by a multi-protein of ancient proteins to their modern-day counterparts. assembly called the peptide-loading complex (PLC). In the absence of any experimental structure of the PLC, we used molecular dynamics (MD) simula- 1163-Pos Board B72 tions to study its individual components, their assembly and their function. We Mechanisms of Protein-Protein Sliding: Coiled Coils as a Tool Model have predicted the structure of the tapasin MHCI interface, and shown how David Gomez, Yaakov Levy. tapasin both protects empty MHCI from unfolding and catalyses the selection Structural Biology, Weizman Institute of Science, Rehovot, Israel. of high-affinity peptides through a molecular tug-of-war mechanism: tapasin The alpha-helical coiled coil domain is a ubiquitous protein motif found in pulls on a region of the MHCI peptide binding groove to open it, while the pep- approximately 5% of all translated protein sequences. Due to the relative tide simultaneously tries to close the groove. Low-affinity contenders ‘‘lose’’ simplicity of the sequence/structure relationship, coiled coils have been this challenge and are exchanged until a high-affinity one binds to and closes extensively studied, leading the emergence of novel protein design and en- the groove, thereby initiating complex break-down. We have also shown gineering techniques. Nevertheless, a complete understanding of coiled how tapasin recruits the transporter associated with antigen processing (TAP) coils structure and their dynamics is still far from being achieved. For into the PLC via transmembrane interactions. In addition, by truncating anti- instance, it has been experimentally observed that coiled coils exhibit stag- gens or removing them from the MHCI binding groove, we made a spatially gered conformations that are generated after sliding events between two resolved map of MHCI plasticity, revealing how peptide loading status affects helixes. Using structure-based modeling techniques as well as coiled coil key structural regions contacting tapasin. Finally, we have integrated the previ- design tools, we study the mechanisms of sliding for parallel and antipar- ous elements to build a computational model of the full PLC. Our MD simula- allel homodimer coiled coil conformations. We are motivated at character- tions explain experimental kinetics and mutagenesis data, and represent the first izing the molecular determinants that govern the kinetics and dynamics of in-depth, atomic-level study of the mechanism underlying the PLC, an impor- sliding in coiled coils and to predict its prevalence in natural coiled-coil tant step towards a better understanding of adaptive immunity. proteins.

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1164-Pos Board B73 1167-Pos Board B76 Molecular Basis for the Functionality of g-Secretase Inferred From Atomistic Simulations Reveal a Hindered Transition of the B-Loop Structure-Based Modeling and Druggability Simulations Domain of Influenza Hemagglutinin Ji Young Lee1, Zhiwei Feng2, Xiang-Qun (Sean) Xie2, Ivet Bahar1. Xingcheng Lin1, Jeffrey K. Noel2, Qinghua Wang3, Jianpeng Ma3, 1Department of Computational and Systems Biology, University of Jose N. Onuchic1. Pittsburgh, Pittsburgh, PA, USA, 2Department of Pharmaceutical Sciences, 1Physics and Astronomy, Rice University / CTBP, Houston, TX, USA, 2Max University of Pittsburgh, Pittsburgh, PA, USA. Delbruck Center for Molecular Medicine, Berlin, Germany, 3Biochemistry g-secretase is involved in the cleavage of amyloid protein precursor (APP) to and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. form Ab fibrils implicated in Alzheimer’s disease (AD). Therefore, g-secretase Influenza Hemagglutinin (HA) is a trimeric membrane fusion protein, crucial is an attractive target for treating AD. Two subunits of g-secretase, nicastrin for the invasion of flu viruses into host cells. Its stem domain, HA2, guides (NCT) in the extracellular (EC) domain and presenilin-1 (PS1) in the transmem- the fusion of viral and endosomal membranes through a large-scale structural brane (TM) domain, play important roles in binding substrate peptides (APPs) as transition under lowered pH. Based on crystallographic and biochemical well as modulating PS1 catalytic activity. However, the molecular mechanisms of data, a spontaneous random-loop-to-coiled-coil structural change of the B- coupling between substrate/modulator binding and catalytic activity remain to be loop domain of HA2 has been proposed to drive the HA-induced membrane elucidated. Recent determination of intact human g-secretase cryo-EM structure fusion. However, microscopic picture of thisspring-loaded’’ motion remained [Bai et al., 2015] now opens the way to a detailed investigation of the structural elusive. To understand the molecular mechanism of this B-loop transition, we dynamics of this complex. Our studies, based on membrane-coupled anisotropic use full-scale explicit-solvent simulations of the B-loop in a low pH environ- network model (membrANM), reveal two types of NCT motions, bending and ment with the CHARMM36 force field. The free-energy profile constructed twisting, with respect to PS1. These movements underlie the fluctuations between from our simulations suggests that the C-terminal half of the B-loop can stably the ’open’ and ’closed’ states of the lid-like NCT with respect to a hydrophilic fold into the post-fusion coiled-coil structure, while further folding of the N-ter- loop (HL1) on PS1, consolidated by the concerted dynamics of the lipid bilayer. minal half is unfavorable. A buried polar residue, Thr59, is found to destabilize We propose a model that these two states allow or block access of substrate pep- the post-fusion structure. Interestingly, despite the evolutionary conservation of tide (EC portion) to HL1/TM2 and induce cleavage of substrate in different HA2 in its entirety, further study of HA sequences shows the biochemical char- length. Our druggability simulations show that the PS1 presents hot spots for acter of the residue 59 strictly differentiates between two major taxonomic both orthosteric and allosteric inhibition of catalytic activity, consistent with groups of HAs, while within each group it is conserved. Additional simulations experimental data. Such simulations further point to hot spots at hinge regions of the B-loop domain with mutated sequences reveal that its stability is near the EC-TM domains’ interface as well as the interlobe groove of NCT, different between these two HA groups, indicating that the transition dynamics allo-targeting of which would interfere with the coupling between HL1/TM2 of HA during the viral infection process is sequence-dependent. and catalytic pocket. These results open new avenues for structure-based design of novel allosteric modulators of g-secretase protease activity. 1168-Pos Board B77 [Ref] Bai XC, Rajendra E, Yang GH, Shi YG, and Scheres SHW. (2015). Dynamic Modulation of Binding as a Mechanism for Regulating Interferon Elife 4, e11182. Signaling Hongchun Li1, Nanaocha Sharma2, Ignacio J. General1, Joseph M. Salvino3, 1165-Pos Board B74 Gideon Schreiber2, Ivet Bahar1. Mechanism of Complex Force-Dependent Unfolding Dynamics of Titin 1Department of Computational and Systems Biology, University of Immunoglobulin Domain Revealed by Magnetic Tweezers Pittsburgh, Pittsburgh, PA, USA, 2Department of Biomolecular Sciences, Guohua Yuan1, Wenjun Chen1, Xin Zhou2, Jie Yan3, Hu Chen1. Weizmann Institute of Science, Rehovot, Israel, 3The Wistar Cancer Center 1Physics, Xiamen University, Xiamen, China, 2Physics, University of Molecular Screening, The Wistar Institute, Philadelphia, PA, USA. Chinese Academy of Sciences, Beijing, China, 3Physics, National University We focused on interferon (IFN) signaling. IFNa2, a Type-I interferon, forms a of Singapore, Singapore, Singapore. ternary complex with two receptors, IFNAR1 and IFNAR2. We have shown Mechanical stability of immunoglobulin (Ig) domains in protein titin plays a critical that the binding affinity of IFNa2 to these receptors, as well as downstream role in regulating the passive elasticity of muscles. The unfolding and folding dy- signaling strength, can be modulated by altering the dynamics of the IFNAR1. namics of Ig domain in titin regulates the flexibility of muscle. As an example, un- The global motions of the IFNAR1-IFNa2-IFNAR2 ternary complex were pre- folding dynamics of titin I27 has been studied extensively by atomic force dicted using the membrANM in which the lipid environment is included in the microscopy (AFM) and recently by magnetic tweezers. The force-dependent un- model and simulations. IFNa2 associated immune responses were shown to be folding rate shows complex behavior over big force range from several pN to hun- modulated by introducing Cys-Cys double mutants which form cross-links be- dreds of pN. Though different models have been proposed to explain the unfolding tween different subdomains of IFNAR1. The downstream activities (gene expres- behavior at different fore range, there is still lacking a comprehensive model to give sion) and binding affinity experiments carried out with mutants selected based on a complete picture of the unfolding behavior. Here, we construct a two-dimensional our computations, showed the changes in binding and activity consistent with the (2D) free energy landscape based on the detailed interaction information obtained predictions. Druggability simulations identified multiple druggable sites of IF- from molecular dynamics simulation and the entropic elasticity of peeled polypep- NAR1. We have constructed the corresponding pharmacophore models which tide chain. At low force range, entropic elasticity of the transition state contributes are currently screened against libraries of small compounds to identify hit com- to the catchbond unfolding behavior. At force >110 pN, an unfolding intermediate pounds that may be developed into modulators of IFNAR1 activity, in collabora- state slows down the unfolding rate. This 2D free energy landscape reproduces the tion with the Weizmann Institute (Israel) and Wistar Institute (USA). complex force-dependent unfolding dynamics of I27 quantitatively. 1169-Pos Board B78 1166-Pos Board B75 A Comparison of Collective Coordinates for Analyzing Protein Dynamics Structural Dynamics is a Determinant of the Functional Significance of Eric R. Beyerle, Marina Guenza. Missense Variants Chemistry, University of Oregon, Eugene, OR, USA. Luca Ponzoni, Ivet Bahar. The internal dynamics of a protein is commonly analyzed by examining the Computational and Systems Biology, University of Pittsburgh, School of fluctuations along the protein’s alpha-carbons. However, a vast majority of Medicine, Pittsburgh, PA, USA. an N-residue protein’s internal dynamics can be described successfully using Accurate evaluation of the effect of point mutations on protein function is essential less than the complete (3N-6)-dimensional space spanned by the protein’s in- to assessing the genesis and prognosis of many inherited diseases and cancer types. ternal fluctuations. This dimensionality reduction is achieved by transforming Currently, a wealth of computational tools has been developed for pathogenicity from the lab-frame coordinates to a set of judiciously-selected collective coor- prediction of non-synonymous single-nucleotide polymorphisms (nsSNPs). Two dinates. A common technique, called principal component analysis (PCA), is to major types of data are used to this aim: sequence conservation/evolution and select the collective coordinates along which the variance of the original (3N- structural properties. Here, we demonstrate in a systematic way that another deter- 6)-dimensional coordinates is maximized. These statistically-independent, col- minant of the functional impact of missense variants is the protein’s structural dy- lective coordinates are termed principal modes (PMs) and are ordered in terms namics. Measurable improvement is shown in the predictive ability of of descending variance, with the first PM possessing the maximum variance. pathogenicity analyses by taking into consideration the dynamical context and im- However, these PMs contain no inherent information regarding the timescale plications of the mutation. Our study suggests that the newly introduced class of over which the motion described by the PM occurs. We present an alternative descriptors may be used in conjunction with existing features to not only increase set of independent, collective coordinates generated using the Langevin Equa- the prediction accuracy of the impact of variants on biological function, but also tion for Protein Dynamics (LE4PD), a method for analyzing the internal fluc- gain insights into the physical basis of the effect of missense variants. tuations of proteins along its alpha-carbon backbone. The LE4PD accounts

BPJ 8613_8616 Monday, February 19, 2018 233a for the presence of hydrodynamic interactions between each residue and bar- FcR complex. As the conformation dynamics of antibodies are critical to riers on each mode’s free-energy surface. In contrast to PMs, the LE4PD antibody function, we chose 12 independent initial conformation to enhance normal modes are ordered by timescales instead of variance; thus, the first the sampling. We reproduced experimental domain distribution density for normal mode from the LE4PD describes longest timescale internal motion. the apo antibody, with the two Fabs move highly dynamic around the Fc region The fluctuations predicted by the corresponding PMs and LE4PD normal with Fab and Fc domains constantly forming and breaking contacts. When bind modes are in good agreement without including hydrodynamics or barriers in to antigen, the antibody conformation shift to two dominant conformation clus- the LE4PD, but inclusion of these phenomena have a significant impact on ters, with one cluster has Fc-receptor binding site widely open. The final bind- the location and magnitude of the predicted fluctuations along the protein’s ing of Fc-g Receptors leads to two conformation clusters, one corresponding backbone. The results suggest the LE4PD is an effective method for describing to the antigen-free antibody-FcR baseline binding, and one for stronger antigen the internal fluctuations of proteins along collective coordinates, and the inclu- enhanced antibody-FcR interaction. Structural analysis of the antigen- sion of hydrodynamic interactions and free-energy barriers significantly alters antibody-FcR complex suggested that the antigen binding signal can trans- the fluctuations predicted along each normal mode. ferred from the CDRs to the FcR by two main pathways, i.e. through hinge region and CH1/CL region. The results suggested that the stable contacts be- 1170-Pos Board B79 tween CH1/CL and FcR is important for the transfer of antigen binding signals. Investigating Phosphoantigen-Induced Conformational Change of Butyro- Our study revealed the atomic mechanisms of the allosteric communications philin 3A1 using All-Atom Molecular Dynamics Simulations connection the antibody-antigen recognition and following FcR activation. Christopher T. Boughter, Benoıˆt Roux, Erin J. Adams. During these processes, antigen binding shifts the antibody conformations Biophysical Sciences, University of Chicago, Chicago, IL, USA. which facilitate the FcR binding as well as allosteric signal transfer. Classical T cell mediated immunity requires the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) molecule on the opposing 1173-Pos Board B82 cell. For Vg9Vd2 T cells, however, this response is conferred independently of Lessons from 8 Milliseconds of Aggregated Kinase Molecular Dynamics MHC molecules, suggesting recognition instead occurs via a novel pathway. Simulations Vg9Vd2 T cells instead respond to the intracellular accumulation of small pyro- Mohammad M. Sultan, Vijay Pande. phosphate metabolites, collectively called phosphoantigens (pAgs), in cells in- Chemistry, Stanford University, Stanford, CA, USA. fected with Mycobacterium tuberculosis and certain types of cancer. The TCRs Kinases control a large number of biochemical pathways and their misregula- on these cells do not, however, directly interact with pAg, instead detection of tion is linked to a number of diseases including cancers. Here we present on the pAg in the target cell is mediated by a transmembrane protein, butyrophilin 3A1 results of an aggregate of 8 milliseconds of Molecular Dynamics (MD) simu- (BTN3A1), which directly binds pAg via an intracellular B30.2 domain. The lation data for 8 difference kinase catalytic domain in their apo and ATP bound sequence of events that connects pAg binding to BTN3A1 and activation of states. We then build consensus Markov state models (MSM) for the dynamics Vg9Vd2 remains unclear. Previous crystallographic and biochemical studies of these protein sequences that allows us to make interesting observations about have provided a foundation for further studies, revealing conformational changes kinases at the family scale. and membrane immobilization of BTN3A.To further probe this system and test these hypotheses, molecular dynamics (MD) simulations have been employed to 1174-Pos Board B83 elucidate the dynamics of the pAg binding pocket and the subsequent conforma- Molecular Analysis of Dengue NS3 Helicase Function 1 1 2 tional changes associated with this binding. These MD simulations with explicit Kelly E. Du Pont , Russell B. Davidson , Brian J. Geiss , 1 solvent focused on the intracellular ligand-binding domain in an attempt to char- Martin McCullagh . 1Department of Chemistry, Colorado State University, Fort Collins, CO, acterize the protein-ligand interaction. Upon ligand binding, we observe a distinct 2 change in flexibility of the binding pocket, which could be important for intracel- USA, Department of Microbiology, Immunology, and Pathology, Colorado lular dimerization of these domains that helps to convey this inside-out signaling. State University, Fort Collins, CO, USA. The unwinding of double-stranded RNA intermediates is a critical component 1171-Pos Board B80 for the replication of flavivirus RNA genomes. This function is achieved by the Understanding How Environmental Pressure Influences Evolution of C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of Enzymes the superfamily 2 (SF2) helicases, NS3 is known to require the binding and hy- Toshiko Ichiye. drolysis of ATP/NTP to translocate along and unwind double-stranded nucleo- Chemistry, Georgetown University, Washington, DC, USA. tides. However, the mechanism of energy transduction between the ATP and Studies of extremophiles, namely organisms that live under extreme conditions, RNA binding pockets is not well understood. In order to investigate this pro- have been used to understand the relationship between external conditions and cess, a study combining molecular dynamics, biochemistry and virology has protein structure/ function as well as to define characteristics needed for protein been employed. Molecular dynamics simulations were performed to identify structure/function. The most heavily studied extremophiles live under extremes NS3 residues (Glu285/Ala286/Arg387/Ser411) that appear to dynamically of temperature: thermophiles at high temperatures and psychrophiles at low tem- couple the ATPase and RNA binding sites. Mutants (Glu285Asp, Ala286Leu, peratures. However, there is growing evidence that life evolved under high tem- Arg387Ala, Arg387Met and Ser411Ala) of these residues were then isolated perature, high pressure conditions. Although pressure has been a neglected from E. coli and biochemically assessed for their NS3 helicase activity and variable compared to temperature because of difficulties in the field and in the ligand binding affinity. Our biochemical results indicate that Ala286Leu laboratory, new high-pressure instrumentation and collection apparatus make decreases RNA binding affinity to NS3h and ablates RNA unwinding, while studies of piezophilic (high-pressure loving) organisms and their proteins Ser411Ala enhances both RNA binding and helicase activity. The effects of possible. Our molecular dynamics simulations based on recent X-ray crystallo- each mutant on viral genome replication in cell culture were also assessed. graphic structures of piezophile enzymes are shedding light on the adaptations Additional simulations of each mutant were used to probe structural changes necessary for enzyme activity under pressure. Using our new quasi-harmonic within NS3 caused by each mutation. These data help define the linkage analysis method, we have defined differences between enzymes from piezophiles between ATP hydrolysis and helicase activity within NS3, and provide insight and mesophiles in terms of intrinsic compressibilities and thermal expansivities. into the biophysical mechanisms for NS3 helicase function. 1172-Pos Board B81 1175-Pos Board B84 Antigen Induced Dynamic Conformation Changes of Antibody to Protein Diffusion in a Dense Solution Studied by All-Atom Molecular Facilitate Recognition of Fc Receptors Dynamics Simulations Jun Zhao1, Ruth Nussinov2, Buyong Ma2. Grzegorz Nawrocki1, Po-hung Wang2, Isseki Yu2,3, Yuji Sugita2,3, 1CIP, NCI-Frederick, NIH, Frederick, MD, USA, 2Leidos Biomedical Michael Feig1,4. Research, NCI-Frederick, NIH, Frederick, MD, USA. 1Department of Biochemistry and Molecular Biology, Michigan State Fc-g Receptors efficiently internalize antigen-antibodies (Ag-Ab) complexes University, East Lansing, MI, USA, 2Riken Theoretical Molecular Science and thus induce processing of antigens into peptides presented by MHC class Laboratory, Saitama, Japan, 3Riken iTHES, Saitama, Japan, 4Riken II molecules. The recognition of p-MHC complex by T-cell receptor (TCR) Quantitative Biology Center, Kobe, Japan. trigger further immune reactions. It is still unclear how antigen binding in anti- Although a large part of the cell volume is occupied by macromolecules, for a body and p-MHC binding in TCR communicate allosterically with their recep- long time the cellular surrounding has been mostly neglected in studies of pro- tor bindings, respectively. The CDRs are responsible for Ag recognition, while tein dynamics. Recent in vivo experiments show that protein diffusion is slower the constant domains are crucial to activate effector function. To study the allo- in a living cell than in a diluted solution however, the degree of retardation steric effects of antigen binding, we simulated four complex structures of free varies significantly even within the same cell. Details of this effect are contro- antibody, antigen bound antibody, FcR bound antibody, and antigen-antibody- versial and the underlying processes remain not well understood. By means of

BPJ 8613_8616 234a Monday, February 19, 2018 all-atom molecular dynamics simulations we provide extensive insight into A recent experimental study reported that the naturally occurring G127V variant microsecond dynamics of a villin solution at different concentrations, as a sim- of human PrPC is intrinsically resistant to prion conversion and completely pre- ple model of cellular environment. Since inaccuracies of current force fields are vents prion disease. However, the structural basis of the protective effect of V127 known to result in exaggerated protein aggregation we increase protein-water variant remains mostly unknown. Herein we performed multiple microsecond- interactions to better reproduce available experimental results. The simulations scale molecular dynamics simulations on both wildtype and V127 variant of hu- show that protein rotational diffusion slows down more significantly than trans- man prion protein at neutral pH with the aim to understand why V127 variant can lational diffusion with increasing concentration, while protein internal dy- prevent prion conversion. Our simulations show that the G127V mutation not namics remain largely unaltered. These findings correlate with observed only increases the rigidity of the loop between strand-2 (S2) and helix-2 (H2) formation of sub-microsecond persisting protein clusters which size distribu- but also enhances the stability of H2 through the formation of hydrophobic inter- tion shifts toward larger clusters with increasing protein concentration. We actions between V127 and P165 and that of the salt bridge between R164 and show that diffusion coefficients estimated for simulation-derived cluster struc- D178. The increased rigidity of S2-H2 loop and the enhanced H2 stability in tures weighted by the cluster size distribution mostly reproduce the observed V127 variant may inhibit the prion conversion of human PrPC,thusprevents overall diffusion. It indicates the clusters formation as a primary determinant prion disease. Our hypothesis is supported by the fact that prions are poorly trans- of diffusion slow-down upon crowding. Finally, we shed light on protein diffu- missible to animals with their PrPC carrying a rigid loop and a recent finding sion in a heterogeneous protein solution, near a lipid membrane and in the pres- showing that stabilization of H2 of prion protein prevents its misfolding and olig- ence of metabolites. omerization. In addition, our MD simulations at acidic pH demonstrate that G127V mutation facilitates the helix-to-sheet transition of the C-terminal region 1176-Pos Board B85 of H2 that is critical to the misfolding of wildtype PrPC. Our findings offer struc- k Hidden Native State Ensembles of NF B Dimers Provide Insights into tural basis for understanding the role of human 127V variant in preventing prion their Different DNA-Binding Affinities conversion and may provide key mechanistic insights into prion propagation and Wei Chen, Dominic Narang, Clarisse G. Ricci, Elizabeth A. Komives. the development of rational therapeutics. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA. 1179-Pos Board B88 The most abundant NFkB family member, p65 (also called RelA), exerts its Drivers of Conformational Variability in Transthyretin Monomers under transcriptional regulatory function as a homo- or hetero-dimer with p50. The Amyloidogenic Conditions two dimers have different but overlapping DNA binding specificities, consid- Matthew C. Childers, Valerie Daggett. ered to be the key to fine control of gene regulation. Past crystallographic Bioengineering, University of Washington, Seattle, WA, USA. studies on NFkB-DNA complexes showed high structural homology among The unfolding, misfolding and subsequent oligomerization of the protein transthyr- different NFkB dimers and left a puzzle: how do NFkB dimers bind to DNA etin (TTR) are associated with two amyloid diseases: senile systemic amyloidosis with different affinities? In this study, we discovered drastically different and familial amyloid neuropathy. Dissociation of the native tetramer is the rate- conformational dynamics between two free NFkB dimers: p65p50 and p65 ho- limiting step in TTR amyloid formation; afterwards, monomers partially unfold modimer. Hydrogen-deuterium exchange mass spectrometry (HDXMS) re- and reassemble into toxic oligomeric species that eventually form mature amyloid vealed different exchange levels in the p65 DNA-binding domains (DBDs) fibrils. Recent studies have used both solution and solid state NMR to identify of the homodimer as compared to the heterodimer, suggesting a sub-global conformational changes associated with the misfolding of TTR monomers; how- conformational difference between p65 bound to p50 and p65 bound to another ever, the specific interactions that give rise to these conformational changes remain p65. Furthermore, molecular dynamics (MD) simulations elucidated the unclear. To investigate the early events in the unfolding of TTR monomers, we conformational ensembles that give rise to different HDX patterns. We found have performed multiple molecular dynamics simulations of wild type and mutant that when not bound to DNA, the two DBDs of the NFkB dimer can twist TTR under amyloidogenic conditions. By analyzing over 10 ms of simulation data, around the linker between the dimerization domain and the DBD with a large we find that in the absence of stabilizing interactions present in the native state, in- amplitude (30 A˚ ). Through MD simulations, we observed very distinct teractions with solvent molecules reshape the conformational landscape of TTR conformational relaxation processes from DNA-bound to free states for and give rise to a heterogeneous ensemble of partially folded structures. Specif- p65p50 and p65 homodimer. While the two DBDs in p65p50 move apart ically, we find that b-sheet secondary structure, b-sheet tertiary structure, and from each other, those in p65 homodimer come together and form new con- the packing of solvent-exposed side chains are destabilized when a natively buried tacts. The new protein interface captured by MD in the p65 homodimer ex- b-sheet is exposed to solvent. These findings highlight how non-native solvent-pro- plains the different HDX behaviors between NFkB dimers. Moreover, we tein interactions can drive amyloid formation and may aid in the development of suggest that the differences in DNA-binding affinities are a consequence of therapeutic strategies to mitigate oligomer toxicity. distinct conformational dynamics displayed by the free NFkB dimers. By combining HDX-MS and MD simulations, we here provide an example of un- 1180-Pos Board B89 derstanding how signaling proteins function from the standpoint of conforma- Quantifying Peptide Binding Affinities from Non-equilibrium Work tional dynamics. Onur Serc¸inoglu 1, Pemra Ozbek Sarica2. 1Bioengineering, Marmara University Institute of Pure and Applied Sciences, 1177-Pos Board B86 2 Correlated Motions in Several Variants of the DHFR-NADPH Complex Istanbul, Turkey, Bioengineering, Marmara University, Istanbul, Turkey. Annika Hirmke1, Malvika Dua1, Craig J. Early1, Paul F. Maxson1, Peptides have great potential as novel medicinal agents. Computational design of Muhammad Mujtaba1, Moataz Noureddine1, Arish Mudra Rakshasa1, peptides binding to a target protein has received great interest of researchers in the Heather A. Carlson2, Michael G. Lerner1. recent years. Structural prediction of relative binding affinities of peptides to pro- 1Physics and Astronomy, Earlham College, Richmond, IN, USA, teins is an important step in the design of potentially therapeutic peptides, yet 2Department of Medicinal Chemistry, College of Pharmacy, University of both peptide-protein structure prediction and methods for quantitative affinity Michigan, Ann Arbor, MI, USA. prediction are far from being accurate. In the past, methods based on single static Correlated motions are thought to be related to the catalytic step for dihydrofo- structures as well as Molecular Dynamics simulations have been used for peptide late reductase (DHFR). We performed several molecular dynamics studies of binding affinity prediction. End-point methods such as MM-PBSA/GBSA are the DHFR-NADPH complex, the analog of an apo structure existing prior to usually preferred for this purpose, however their accuracy for peptide ligands the binding of dihydrofolate. We compare correlated and anti-correlated mo- is usually limited. We investigate the applicability of steered MD (sMD) simula- tions and their timescale to motions previously observed in the catalytically tions as an alternative structural Free Energy estimation method for peptide bind- active state. This is done both via cross correlation matrices and via wavelet ing affinity prediction. The method is based on pulling the peptide away from the analysis. We also compare correlated motions across several DHFR mutants, binding region of protein and recording the pull force along the pulling trajectory. and at several pH levels. The work performed during this nonequilibrium process is then taken as an indi- cator of binding affinity. We applied this method to predict the ranking of 1178-Pos Board B87 different peptide ligands of a Major Histocompatibility Complex Class I protein Dissecting the Structural Mechanism of a Naturally Occuring Variant of in terms of experimentally-determined binding affinities. The peptide-MHC com- the Prion Protein in Preventing Prion Disease plexes had either crystal structures available or were modeled by docking or side- Yiming Tang, Guanghong Wei. chain conformation prediction based on a template peptide ligand. Several pulling Physics Department, Fudan University, Shanghai, China. velocities were tested. Our results indicate that, if the pulling velocity is suffi- Prion diseases are a group of fatal neurodegenerative disorders associated with ciently low and the peptide contacts are correctly modeled, good correlation be- the misfolding and aggregation of mostly alpha-helical cellular prion protein tween experimental and predicted binding affinities can be obtained by the sMD (PrPC) into the beta-rich infectious (scrapie) form of the prion protein (PrPSc). method.

BPJ 8613_8616 Monday, February 19, 2018 235a

Posters: Membrane Protein Structures I the incapability of exploring the whole mutational space with minor effort and the unambiguous physical origin of the enhanced or lowered stability. 1181-Pos Board B90 In general, after the identification is successfully made for a GPCR, Dissociation of the Heterotrimeric G Protein Complex by Nanobodies: Po- the whole procedure must be followed all over again for the identification tential uses in the Modulation of Diverse GPCR Signaling for another GPCR. Here we report a theoretical strategy by which many Krzysztof Palczewski. different GPCRs can be considered at the same time. The strategy is Pharmacology, Case Western University, Cleveland, OH, USA. illustrated for three GPCRs of Class A in the inactive state. We argue G protein-coupled receptors (GPCRs) comprise the most abundant family of that a mutation of the residue at a position of NBW=3.39(NBW is the Bal- cell membrane receptors and share a general mechanism of signal transduc- lesteros Weinstein number), a hot-spot residue, leads to substantially tion. GPCRs respond to a wide variety of extracellular signals, including pho- higher stability for significantly many GPCRs of Class A in the inactive tons, ions, lipids, small molecules, peptides, and proteins. Typically, GPCRs state. The most stabilizing mutations of the residues with NBW =3.39are activate heterotrimeric G proteins by mediating GDP to GTP exchange in then identified for two of the three GPCRs, using the improved version of the a subunit. This leads to the dissociation of the heterotrimeric into our free-energy function. These identifications are experimentally corrobo- Gaa-GTP and Gbg complexes. While Ga-GTP facilitates its own signaling rated, which is followed by the determination of new three-dimensional events, the bg-subunits can also autonomously exert their effects in cellular (3D) structures for the two GPCRs. We expect that on the basis of the strat- signaling. In this study, we used several nanobodies that bind tightly to the egy, the 3D structures of many GPCRs of Class A can be solved for the first bg-complex causing a receptor-independent dissociation of the G protein. time in succession. S. Yasuda et al., J. Phys. Chem. B, 121,6341 6350 These nanobodies respond to all combinations of b- and g-subunits. Further, (2017). biochemical, structural, and physiological data support a broad application of 1184-Pos Board B93 these genetically encodable nanobodies to modulate GPCR signaling as well Characterizing a New Mechanism in GPCR Signaling and Energy Balance as reveal their potential therapeutic applications. Valerie Chen, Ashley Tess Wong. Chemistry and Biochemistry, University of California, Santa Cruz, Santa 1182-Pos Board B91 Cruz, CA, USA. Structure and Dynamics of the Receptor-Bound Ghrelin Lipopeptide The melanocortin signaling system, composed of five G-protein coupled re- 1 2 2 1 Guillaume Ferre , Marjorie Damian ,Celine M’Kadmi , Olivier Saurel , ceptors (GPCRs) and endogenous peptide ligands, regulates a wide range of 1 1 2 2 Georges Czaplicki , Pascal Demange , Jacky Marie , Nicolas Floquet , essential physiological processes. The melanocortin 4 receptor (MC4R) is Jean-Alain Fehrentz2, Alain Milon1, Jean-Louis Bane`res2. 1 expressed predominantly in the central nervous system and is essential for Institut de Pharmacologie et de Biologie Structurale, Universitede maintaining feeding behavior and energy balance. Heterozygous mutations Toulouse, CNRS, UPS, Toulouse, France, 2Institut des Biomolecules Max in MC4R are the most common genetic cause of human obesity. A unique Mousseron, Universite de Montpellier, CNRS, ENSCM, Montpellier, France. feature of MC4R signaling is that it is partially regulated by its endogenous G-protein coupled receptors (GPCR) play an essential role in human phys- antagonist, the agouti-related peptide (AgRP). Activation of MC4R through iology. They are prominent pharmacological targets and the understanding the Gas pathway results in an increase in cAMP and negative energy bal- of molecular mechanisms underlying their activity is fundamental for the ance, whereas AgRP antagonism reduces levels of cAMP and results in pos- design of new drugs. Since a decade, a number of structures have been eluci- itive energy balance. New research shows that melanocortin signaling is dated, but sparse in complex with peptide agonists. NMR spectroscopy al- more complex than previously understood, and cannot be explained simply lows determining conformations of peptides bound to their receptors. by agonist/antagonist modulation of the Gas pathway. There are newly Here, we report our work on Ghrelin, a lipopeptide hormone involved in identified accessory proteins involved in the regulation of MC4R in an phenomena such as appetite, growth hormone secretion and reward- AgRP-dependent manner. AgRP is able to activate an inwardly-rectifying seeking behaviours. Our study led to the determination of Ghrelin’s structure potassium channels (Kir7.1) through MC4R signaling. AgRP-induced open- and dynamics when bound to the growth hormone secretagogue receptor ing of Kir7.1 hyperpolarizes MC4R neurons and decreases the frequency of (GHSR). The perdeuterated receptor reconstituted into lipid nanodiscs after action potential firing. This occurs in a G protein-independent fashion; how- expression in E. coli allowed studying Ghrelin in its bound state by liquid ever, the mechanism by with MC4R couples to Kir7.1 is unknown. Under- state NMR. We performed transferred 1H NOE experiments to determine 15 standing the molecular basis of this G-protein independent signaling the hormone’s conformation and we measured N transverse relaxation to pathway will help to redefine the current model of melanocortin and decipher the conformational flexibility along its sequence. Consistent with GPCR signaling, and may lead to new concepts in the development of ther- pharmacological data, Ghrelin amino-terminal part folds with its acyl chain apeutics aimed at treating metabolic diseases. to form a hydrophobic core essential for GHSR binding and activation. In contrast, the carboxy-terminal part remains flexible and may participate in 1185-Pos Board B94 the binding with the receptor through electrostatic interactions. Furthermore, Intracellular Effect of b3-Adrenoceptor agonist Carazolol on Skeletal we are combining NMR data with molecular dynamics simulations to Muscle, a Direct Interaction with SERCA construct a model of the Ghrelin-GHSR complex. This approach provides Ibrahim A. Ramirez, Eduardo Rodriquez, Rocı´o Alvarez, Eugenio Quiroz, the first active structure of Ghrelin and a novel insight into the molecular Alicia Ortega. mechanisms responsible for its activity with respect to the activation of Biochemistry, Unam, Mexico, Mexico. GHSR. Considering the pharmacological relevance of Ghrelin and its recep- Carazolol is an agonist of b3 and antagonist of b1andb2 adrenoceptors, tor, our results may lead to the design of new drugs with applications in the used in the animal production industry to improve meat quality by reducing treatment of obesity, diabetes and addiction. animal stress and muscle proteolysis and in the doping of athletic horses for strengthening muscle performance. We study Carazolol’ s effect on calcium 1183-Pos Board B92 regulation by enzymatic activity kinetics of the Ca2þ-ATPase (SERCA), in Hot-Spot Residues to Be Mutated Common in G Protein-Coupled Receptors isolated Sarcoplasmic Reticulum (SR) from skeletal muscle (SM) and on the of Class A: Identification of Thermostabilizing Mutations Followed by Deter- mechanical properties of isolated muscle. Isolated SR from SM previously mination of Three-Dimensional Structures for Two Example Receptors incubated with 0.03 mM Carazolol, but absent during SERCA activity is Satoshi Yasuda1,2, Yuta Kajiwara3, Yosuke Toyoda4, Kazushi Morimoto4, shown to be reduced by 45%. Thermal analysis of SERCA activity with Car- 4 4 4 1 Ryoji Suno , So Iwata , Takuya Kobayashi , Takeshi Murata , azolol shifted the transition temperature of inactivation (Ti) from Ti =47to Masahiro Kinoshita2. 44C. When isolated SR from fast and slow SM is exposed to Carazolol, in- 1Graduate School of Science, Chiba University, Chiba, Japan, 2Institute of hibition of SERCA occurs in a dose dependent manner. Slow and fast SM Advanced Energy, Kyoto University, Kyoto, Japan, 3Graduate School of shift to a lower temperature in the presence of Carazolol and a second tran- Energy Science, Kyoto University, Kyto, Japan, 4Graduate School of sition appears at < 40C. In isolated extensor digitorium longus and soleus Medicine, Kyoto University, Kyoto, Japan. muscles, Carazolol reduces the contraction force and increases propensity to G protein-coupled receptors (GPCRs), which are indispensable to life and fatigue. However, recovery force after fatigue in either muscle was higher. also implicated in a number of diseases, construct important drug targets. Our results indicate that Carazolol penetrates the plasma membrane and in- For the efficient structure-guided drug design, however, their structural sta- teracts with SERCA, thus having an important effect on skeletal muscle bilities must be enhanced. An amino-acid mutation is known to possibly lead function by inhibiting SR Ca2þ-reuptake and in consequence causing to the enhancement, but currently available experimental and theoretical adecrementinSRCa2þ-release promoting further failure in muscle methods for identifying stabilizing mutations suffer such drawbacks as contraction.

BPJ 8617_8620 236a Monday, February 19, 2018

1186-Pos Board B95 membrane mimetic-protein complexes. In our study, we show methods to suc- Spatially Constrained Water Molecules are Conserved in GPCR cessfully prepare protein-bicelle complexes of suitable size and homogeneity Activation for NMR structural studies. We have optimized the sample preparation and A.J. Venkatakrishnan, Ron Dror. protein reconstitution parameters for two bacterial ion channel domains: Stanford University, Stanford, CA, USA. firstly, the full-length tetrameric potassium channel KcsA and the isolated G protein-coupled receptors (GPCRs) have evolved to recognize incredibly voltage sensing domain of the sodium channel NaChBac. Both samples diverse extracellular ligands while sharing a common architecture and structur- gave well resolved NMR spectra and allowed us to assign a significant portion ally conserved intracellular signaling partners. It remains unclear how binding of the resonances for the two proteins, using a combination of standard triple- of diverse ligands brings about GPCR activation, the common structural resonance experiments and specifically labeled samples. These assignments change that enables intracellular signaling. Here, we identify a conserved will form the basis for the structural study of toxin interactions with both network of stable water molecules that plays a central role in activation. channels. Using atomic-level simulations of diverse GPCRs, we show that most of the water molecules in GPCR crystal structures are highly mobile. Several water 1189-Pos Board B98 molecules near the G protein-coupling interface, however, are stable. These Towards Truly Stealth Nanodiscs waters form a polar network that rearranges upon activation and that is Cheol Jeong1,2, Ryan Franklin3, Karen Edler4, Joseph E. Curtis1. highly conserved across class A GPCRs, suggesting that these receptors 1Center for Neutron Research, National Institute of Standards and evolved to exploit this network in the activation process. This conserved water Technology, Gaithersburg, MD, USA, 2Department of Chemistry, University network, along with diverse water-mediated interactions with extracellular li- of Tennessee, Knoxville, TN, USA, 3Department of Chemistry and Physics, gands, has direct implications for structure-based drug design and GPCR Hood College, Frederick, MD, USA, 4Department of Chemistry, University engineering. of Bath, Claverton Down, United Kingdom. Investigation of membrane proteins is one of the remaining challenges in bio- 1187-Pos Board B96 physical society. Lipid nanodiscs forming a self-assembly with membrane pro- Structure of a Phosphatidylinositol-Phosphate Synthase from Myco- teins allow the characterization of membrane proteins in solution. The low- bacteria resolution structural analysis of the membrane proteins would be enabled by Meagan L. Belcher Dufrisne1, Carla D. Jorge2, Oliver B. Clarke3, small-angle neutron scattering (SANS) measurements in dilute solution. Wayne A. Hendrickson3, Helena Santos2, Filippo Mancia1. Despite of recent successes in the SANS approach for multi-component protein 1Department of Physiology and Cellular Biophysics, Columbia University, systems in solution, it is difficult to extract the information of proteins from a New York, NY, USA, 2Instituto de Tecnologia Quı´mica e Biolo´gica, protein-nanodisc complex sample due to the nontrivial contribution of nano- Universidade Nova de Lisboa, Oeiras, Portugal, 3Department of discs to the measured SANS signal. To overcome this difficulty, Selma and co- Biochemistry and Molecular Biophysics, Columbia University, New York, workers proposed the usage of the fully deuterated nanodiscs, so call ‘‘stealth NY, USA. carriers’’ in 100 % D2O solution, but they actually succeeded in synthesizing a Phosphatidylinositol (PI) is critical for intracellular signaling and anchoring ‘‘near’’ stealth nanodisc if considered their SANS data. Here, we present our of carbohydrates and proteins to cellular membranes. In eukaryotes, recent efforts to design a nanodisc equipped with the ‘‘truly’’ stealth function phosphatidylinositol-based lipids play important roles in numerous aspects of by performing in-silico SANS measurements in combination with atomistic signal transduction and in the anchoring of glycosylphosphatidylinositol (GPI) molecular dynamics (MD) simulations on the nanodisc-protein complex sys- linked proteins to the membrane. In some prokaryotes, including mycobacteria, tems. From this computer simulation approach, it is found that the SANS sig- PI is required for the biosynthesis of key cell wall components, including glyco- nals from the fully deuterated nanodiscs cannot be matched out with D2O lipids lipomannan and lipoarabinomannan. Here, PI acts as a common anchor teth- solvent. We find that it is important to separately control the isotope conversion ering these glycolipids to the membrane. In Mycobacterium tuberculosis, these ratio (D/H) for lipid head groups, tails, and belts in order to make the nanodiscs glycolipids function as virulence factors and modulators of host immune response, signals matched out. making the enzyme responsible for PI synthesis in this organism a potential target for novel anti-tuberculosis drugs. 1190-Pos Board B99 The defining step in phosphatidylinositol biosynthesis is catalyzed by CDP- Quaternary Structure of Small Amino Acids Transporter OprG of Pseudo- alcohol phosphotransferases, transmembrane enzymes that use CDP- monas aeruginosa diacylglycerol as donor substrate and either inositol in eukaryotes or Raghavendar Reddy Sanganna Gari1, Patrick Seelheim1, Brendan Marsh2, inositol phosphate in prokaryotes as acceptor alcohol. In prokaryotes, Volker Kiessling1, Carl Creutz3, Lukas Tamm1. phosphatidylinositol-phosphate synthase (PIPS; a member of the CDP- 1Center for Membrane and Cell Physiology and Department of Molecular alcohol phosphotransferase family) catalyzes this reaction producing phospha- Physiology and Biological Physics, University of Virgina, Charlottesville, tidylinositol-phosphate, which is dephosphorylated by an uncharacterized VA, USA, 2Department of Physics, University of Missouri, Columbia, MO, enzyme to PI. USA, 3Center for Membrane and Cell Physiology and Department of Previously solved structures of PIPS from Renibacterium salmoninarum Pharmacology, University of Virgina, Charlottesville, VA, USA. (RsPIPS), with and without bound CDP-diacylglycerol, have revealed the Pseudomonas aeruginosa (PA) is an opportunistic human pathogen that is acceptor site location as well as molecular determinants of substrate specificity responsible for a growing number of nosocomial infections. The outer mem- and catalysis. However, RsPIPS has low activity compared to PIPS from Myco- brane (OM) of PA contains many specific porins that facilitate the uptake of bacterium tuberculosis (MtPIPS) and the two only share 40% sequence iden- different substrates. An outer membrane protein OprG has been shown to facil- tity. Therefore, these initial structures have limited potential for meaningful itate the transport of small amino acids such as glycine, alanine, valine, and homology modeling and drug design. Following a crystal engineering serine both in vitro and in vivo. In contrast, a critical proline (P92A) mutation approach, we have now determined the structure of PIPS from Mycobacterium hampered transport of these amino acids. According to structural data, the pore kansasii (MkPIPS), which shares 86% sequence identity with MtPIPS. It shows size of the channel is probably too narrow to accommodate even the smallest the same basic architecture, however may have important differences that amino acid, glycine. A possible hypothesis for transport is that wild-type affect enzyme activity. This work provides a structural and functional frame- OprG may function as an oligomer in the membrane where the pathway for work to understand the mechanism of phosphatidylinositol-phosphate biosyn- translocation might be through a pore formed by the oligomer, and that the thesis in context of mycobacterial pathogens. P92A mutant might disrupt oligomerizaton. We tested this hypothesis by deter- mining the oligomeric state of OprG in artificial liposomes and in native outer 1188-Pos Board B97 membrane vesicles. Atomic force microscopy (AFM) imaging of proteolipo- Bicelle Reconstitution of Ion Channel Domains for NMR Structural somes containing WT or P92A mutant OprG revealed similar volume distribu- Studies tions comprising monomers, dimers, and higher-order oligomers in the Jing Zhu1, Mangmang Zhu1, Sebastien F. Poget1,2. membrane. Counting subunits of OprG in membranes by observing stepwise 1Department of Chemistry, College of Staten Island, City University of New bleaching of Alexa-647 labelled WT-OprG also revealed a distribution of York, Staten Island, NY, USA, 2Ph.D. Programs in Chemistry and monomers, dimers, and trimers. Chemical crosslinking of double cysteine mu- Biochemistry, The Graduate Center, CUNY, New York, NY, USA. tants of WT- and P92A-OprG with bifunctional, thiol-reactive crosslinkers in Solution NMR is an excellent tool for the investigation of protein dynamics native outer membrane vesicles of PA further confirmed a population of mono- and protein-ligand interactions. However, NMR data for membrane proteins mers, dimers, trimers and higher-order oligomers. Taken together, our data often suffers from significantly reduced quality due to the large size of the show that OprG indeed forms oligomers in membranes, but that the P92A

BPJ 8617_8620 Monday, February 19, 2018 237a mutation does not disrupt oligomerization. We speculate that restricted dy- I FPs and TMs, such as those from influenza and HIV viruses, has been ob- namics of loop 3 in P92A mutant as observed by solution NMR likely contrib- tained in recent years, high-resolution structural information of fusion peptides utes to the transport mechanism in the oligomer. and transmembrane domains from coronaviruses is surprisingly scarce. The SARS-like respiratory illness caused by the MERS coronavirus highlights 1191-Pos Board B100 the need for further studies of functional FP and TM domains. Due to their NMR Structural Studies of the Yersinia Pestis Outer Membrane Protein sequence conservation among coronaviruses, they provide new targets for AIL in Lipid Bilayers drug development. Other classes of viral membrane proteins, such as the viro- Yong Yao, Lynn Fujimoto, Samit Dutta, Francesca Marassi. porins Vpu from HIV-1 and p7 from HCV, have also been regarded as drug tar- Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA. gets. Viroporins are small, hydrophobic proteins that can oligomerize in The outer membrane protein Ail (Adhesion invasion locus) is one of the most membranes and induce channel or pore activity. Here we report recent progress abundant proteins on the cell surface of Yersinia pestis during human infection. on the determination of high-resolution NMR structures of the SARS Its functions are expressed through interactions with a variety of human host membrane-active domains as well as viroporins reconstituted in detergent mi- proteins, and are essential for microbial virulence. Structures of Ail in micelles celles, isotropic bicelles, lipid nanodiscs, and in the more physiologically- have been determined by X-ray diffraction and solution NMR spectroscopy, relevant phospholipid bilayer environment. All of these proteins adopt primar- but those samples contained detergents that interfere with functionality, thus, ily a-helical structures in membrane environments. precluding analysis of the structural basis for Ail’s biological activity. Here, we present high-resolution magic angle spinning (MAS) solid-state NMR 1194-Pos Board B103 spectra of Ail in detergent-free phospholipid liposomes, prepared with a lipid Site-Directed Spin-Label EPR Spectroscopy of Influenza a M2 Protein to protein molar ratio of 100. The 13C-detected spectra enable resonance assign- Aaron Holmes, Kathleen Howard. ments for many nitrogen and carbon sites in the sequence of Ail, covering 80% Swarthmore College, Swarthmore, PA, USA. of the transmembrane region. The 1H-detected 1H/15N correlation solid-state M2 is a membrane protein critical to the life cycle of influenza A. We have NMR spectra of Ail in liposomes compare very favorably with the solution collected site-directed spin labeling electron paramagnetic resonance spectros- NMR TROSY spectra of Ail in nanodiscs, prepared with a similar lipid to pro- copy (SDSL-EPR) data on the homotetrameric M2 protein embedded in lipid tein molar ratio. We also acquired high resolution 15N-detection solid state bilayers. We have obtained data on full length M2 protein in membrane bila- NMR spectra of Ail in magnetically aligned bicelles. These static spectra yers with a range of different lipid compositions and established that M2’s contain rich anisotropic information complementary to the MAS data. Further- conformation and dynamics depend on the lipid environment. Furthermore, more, we studied the effect of lipopolysaccharide, a major component of the we have explored how the antiviral drug amantadine impacts the conformation bacteria outer membrane, on the structure and dynamics of Ail in membranes. and dynamics of membrane-bound M2 protein. Altogether, the ability to acquire high resolution NMR data under both MAS and static conditions allows us to obtain structural information about this mem- 1195-Pos Board B104 brane protein in a near native environment and to probe protein dynamics on a A Structural and Computational Study of Barium Blockade in the KcsA wide range of time scale. NMR studies also provide molecular information Channel Ahmed Rohaim1, LiDong Gong2, Jing Li1, Huan Rui1, Benoit Roux1. about the functional interactions of Ail with its protein partners from human 1 2 host cells, useful for the development of drugs targeting Ail. University of Chicago, Chicago, IL, USA, Liaoning Normal University, Dalian, China. 1192-Pos Board B101 Structural studies have revealed binding sites of Ba2þ in the selectivity filter of Membrane Interactions of the Protease MT1-MMP the KcsA channel. Knowledge of this information is critical to interpret func- þ Tara C. Marcink1,BoAn2, Barbara Brodsky2, Tommi White1, tional measurements of Ba2 blockade in terms of external and internal lock-in Steven R. Van Doren1. sites and determine the selectivity of specific sites along the selectivity filter. 1Biochemistry, University of Missouri, Columbia, MO, USA, 2School of Here, we used x-ray crystallography and molecular dynamic simulation to Engineering, Tufts University, Medford, MA, USA. study in details the most preferable Ba2þ lock-in sites in the KcsA channel. Membrane type-1 matrix metalloproteinase (MT1-MMP) is a membrane- X-ray data were collected for crystal of KcsA in 5 mM KCl soaked in 1, 2, 5 2þ anchored collagenase. Decreased MT1-MMP expression has been linked to and 10 mM BaCl2 solution. Difference Fourier maps show that Ba predom- attenuated tumor progression and increased survival after myocardial infarc- inantly binds to site ‘‘S4’’ of the selectivity filter; this was confirmed by anom- tion. MT1-MMP’s extracellular domain consists of a catalytic and a alous diffraction experiments. The location of the Ba2þin S4 is in good hemopexin-like (HPX) domain. The enzyme’s digestion of collagen triple- agreement with the previously reported structures, however, unlike previous helix mimics is enhanced by small unilamellar vesicles. Electron microscopy studies, no Ba2þ density was observed in S2. To complement this analysis, mo- further confirms the interaction of the extracellular domain with discoidal lecular simulation of Ba2þ blockade of KcsA were carried out with the Ba2þ membrane mimics known as Nanodiscs. NMR and FRET have found a salt- ion located at sites S0-S4 along the selectivity filter in the presence of 150 dependent affinity for membrane mimics. We have been testing the hypothesis mM KCl. The MD simulations indicate that the two most stable configurations that positively charged residues on the HPX interact with the phosphate moi- occur when Baþ2 is bound in S4 while Kþ is bound in S2, or when Baþ2 is eties of the lipid headgroups. Paramagnetic NMR measurement of proximity bound in S3 while Kþ is bound in S1. All other simulations with Ba2þ bound to Nanodiscs doped with nitroxide spin-labeled lipids confirms a membrane in other sites displayed instabilities, suggesting a lower likelihood of Ba2þ binding site in the HPX domain that was reported. However, the paramagnetic binding in the outermost sites (S0,S1,S2). The magnitude of electrostatic inter- relaxation enhancements suggest a second and more dominant binding site on actions in the narrow selectivity filter are examined in the context of the Drude the opposite side of the HPX domain. Rigid body docking using HADDOCK polarizable force field. Collectively, these results suggests that Baþ2 has a high suggests the orientations of approach. Understanding these membrane recogni- affinity to the site S4 and that S3 and S4 are the most probable Baþ2 lock-in tion surfaces may be relevant to the assembly of this protease on cell surfaces sites in KcsA. and to potential interference in that by some monoclonal antibodies. Supported by a subcontract NIH grant R01CA098799. 1196-Pos Board B105 Structural and Functional Studies Uncover Two Networks Stabilizing the 1193-Pos Board B102 Active Form of GLIC, a Bacterial Proton-Gated Pentameric Ion Channel Structures, Dynamics, and Functions of Viral Membrane Proteins by Haidai Hu. NMR Institut Pasteur, Paris, France. Luis G. Basso1, Sang H. Park2, Antonio J. Costa-Filho3, Stanley J. Opella2. The proton-activated pentameric ligand-gated ion channel from Gloeobacter 1University of Sao Paulo, Ribeirao Preto, Brazil, 2University of California violaceus GLIC can be used as a model system to study the structural and func- San Diego, San Diego, CA, USA, 3Physics Department, University of Sao tional properties of its eukaryotic counterparts, which mediate signaling trans- Paulo, Ribeirao Preto, Brazil. duction in animal neuron cells by controlling permeation of ions flux gated by Membrane proteins perform a multitude of tasks in a viral lifecycle. Fusion gly- neurotransmitter binding. Here we used the Finite Difference Poisson-Boltz- coproteins are specialized membrane-anchored biological machines that play mann/Debye-Huckel€ method to predict the pKas of all the Asp and Glu in critical roles in viral entry and infection. The membrane fusion mediated by GLIC both in the active and resting states. Those residues with a high deviation SARS glycoprotein requires an orchestrated participation of functionally rele- of predicted pKas in both forms were titrated by Fourier-transform infra-red vant domains such as the fusion peptide (FP) and the transmembrane domain spectroscopy after reconstitution in lipid bilayers. The results showed that (TM). Interaction of these segments with membranes drives large conforma- E35 is the main key proton-sensor residue. Examination of the active form tional changes in the viral glycoprotein that ultimately lead to the merge of structure shows that E35 interacts with T158 from Loop F. Here we verified the virus envelope with the cell membrane. Although a wealth of data of class that breaking this interaction hinders the proton-elicited currents. We next

BPJ 8617_8620 238a Monday, February 19, 2018 probed the interfacial crevice immediately below E35, shaped by Loop F, Q193 in close proximity. This allowed us to see how SP-B amino and carboxyl ter- (pre-M1) and the neighboring M2-M3 loop, where a previously unnoticed minal peptides and KL4 interact with surface lipids and, therefore, lower sur- hydrogen bond network may help maintaining the open channel. We show face tension. Through this study, SP-B’s complex function can be further that breaking this hydrophilic network originating from Q193 favors the ion elucidated. channel in a nonconductive conformation in the crystal state. Replacing side- chains of residues just below Q193, such as Y197 and I201, with other hydro- 1200-Pos Board B109 phobic side-chains blocks the receptor in a locally closed form. Two signal Molecular Dynamics Simulations Reveal the Role of Membrane Choles- transduction networks are proposed, both originating from the key proton- terol during Pore Forming Pathway of Cytolysin A 1 1,2 sensing residue E35, loop F and the pre-M1 region: i) one hydrophilic network Amit Behera , K. Ganapathy Ayappa . 1Chemical Engineering, Indian Institute of Science, Bengaluru, India, extends to the M2-M3 loop from the neighboring subunit and ii) one hydropho- 2 bic network interacts with the Cys-loop and the M2-M3 loop of the same sub- Center for Biosystems Science and Engineering, Indian Institute of Science, unit. Due to the strong conservation of these loop regions, this model could be Bengaluru, India. a generalized to the entire pLGIC family. Cytolysin A (ClyA), an pore-forming toxin expressed by E. Coli as a water- soluble monomer, undergoes a large conformational change during pore forma- 1197-Pos Board B106 tion. Although cholesterol is known to enhance the lytic activity of ClyA, mo- Novel Mechanism of Channel Gating by a Ring of RCK Domains lecular aspects of the interactions between cholesterol and ClyA during pore Hanzhi Zhang, Yaping Pan, Zhao Wang, Ming Zhou. formation are not well understood. Using all-atom molecular dynamics simula- Baylor College of Medicine, Houston, TX, USA. tions ranging from 0.5 - 0.9 ms, we study a single membrane-inserted protomer, The superfamily of Kþ transporters (SKT) is ubiquitous in bacteria, fungi a dimer (two protomers) and the dodecameric ClyA pore embedded in a and plants. SKT proteins are required for survival of bacteria in low Kþ con- DOPCþ30% cholesterol bilayer. In the single membrane-inserted protomer, ditions and are involved in salt regulation in fungi and plants. Bacterial high cholesterol occupancy was observed around the transmembrane residues SKTs have two components, a membrane embedded protein TrkH that forms of N-terminus which form part of a CRAC motif and also around residues of a homodimer with each protomer resembling an ion channel, and a cytosolic the b-tongue. Although high cholesterol occupancy sites were not observed protein TrkA that forms a homotetramer with each protomer containing two near the N-terminus in the dimer simulations, a cholesterol molecule was pref- RCK domains. Single-channel activities of the TrkH-TrkA complex were re- erentially located in the pocket formed between two adjacent b-tongues of the corded and analyzed: ATP or ATP analogs such as AMPPNP and ATPgS dimer. Cholesterol spent 97% of the simulation time (600 ns) inside this pocket activate the channel while ADP closes it. In order to understand how sampling two major orientations. Energies of two conformations were reported ADP and ATP regulate the ion channel, we solved the structures of TrkH- from docking simulations. Formation of transmembrane water channels were TrkA in the presence of ADP, AMPPNP, ATPgS or ATP by either x-ray observed in both single membrane inserted and dimer ClyA simulations. crystallography or cryo-electron microscopy in atomic resolution. These From the dodecameric pore simulations, density map showed regions of high structures show different conformations of TrkH and different shapes of cholesterol population between the b-tongue pockets and mobility map indi- the TrkA tetramer, and suggest a novel mechanism of gating. We then cated slower cholesterol in the vicinity of the pore as compared to bulk. Our applied mutational analyses to validate key components of the gating simulations elucidate specific interactions with cholesterol that could stabilize mechanism. both the single membrane inserted protomeric state as well as the dodecameric pore. 1198-Pos Board B107 NMR Structure of the Human KCNQ1 Voltage-Sensing Domain 1201-Pos Board B110 Keenan C. Taylor, Georg Kuenze, Hui Huang, Chuck R. Sanders. Improved Purification and Crystal Formation of Native Muscle-Type Biochemistry, Vanderbilt, Nashville, TN, USA. nAChR Using mAbs Voltage-gated ion channels are integral membrane proteins that mediate ion Rafael Maldonado-Herna´ndez, Claudia Silva, Adriana Pastrana, transport across membranes. KCNQ1 is a human voltage-gated potassium Claude Maysonet, Jose Lasalde. channel that is expressed in both epithelial and cardiac tissues. KCNQ1 as- Biology, University of Puerto Rico, San Juan, PR, USA. sociates with KCNE1 and mediates the Iks current responsible for the repo- The nicotinic acetylcholine receptors (nAChR) are part of the Cys-loop larization of the cardiac action potential. Mutations of KCNQ1 that cause a ligand-gated ion channel family and one of the most widely studied, but loss-of-function result in congenital long-QT syndrome (LQTS), which pre- their molecular structure is still unknown. These receptors are important disposes individuals to cardiac arrhythmia and can result in sudden as the biological target for the development of therapies for a great number death. We have previously described the high level expression and pu- of diseases, including Myasthenia gravis, Parkinson’s disease, and nicotine rification of the S1-S4 voltage sensor domain (VSD) of human KCNQ1 addiction. Our aim is to obtain high-quality protein crystals and determine (Peng-D et al., Biochemistry 53:2032-42, 2014). Here we describe the the structure of the native nAChR via X-ray crystallography. We extracted NMR-determined structure of this domain, which appears to adopt an muscle-type nAChRs from the tissue of Torpedo californica,usingLFC- intermediate-active conformation under the LMPG detergent micelle condi- 16 as a detergent, followed by several steps of purification. Four monoclonal tions of the NMR experiments. Our results can be compared and contrasted antibodies (mAbs) with a high affinity against the receptor and the impu- with the open-state CryoEM structure of this domain reported by Sun and rities were assayed via western blot, and BioLayer interferometry on the MacKinnon (Cell, 2017). This work was supported by RO1 HL122010 Octet platform for kinetic characterization. One of these mAbs yielded a 12 and by F32 GM117770. KD <10 which suggests strong mAbs-nAChR interactions with no disso- ciation occurring. Furthermore, we developed a method of immunodepletion 1199-Pos Board B108 to remove impurities from the nAChR-detergent complex, increasing sample Using FRET to Elucidate the Lipid Trafficking Mechanism of SP-B N and purity. Fluorescence recovery after photobleaching (FRAP) assays displayed C Terminal Peptides in Comparison with KL4 a lower mobile fraction with mAbs incorporation compared to control, sug- Kayla Kroning1, Otonye Braide-Moncoeur2. 1 2 gesting significant protein-protein interactions. Our next goal is to identify Gordon College, Wenham, MA, USA, Chemistry, Gordon College, by western blot and protein fingerprinting which proteins are identified by Wenham, MA, USA. the mAbs. These mAbs will be used to obtain high-quality nAChR crystals Infant Respiratory Distress Syndrome (IRDS) is a disorder which commonly and also high-resolution X-ray data. This research was supported by the NIH affects premature babies. It is caused by a complete or partial deficiency of NIGMS grants 1R01GM098343 (JALD); COBRE NIEF 1P20GM103642 lung surfactant (LS), a film that lowers the surface tension of the alveoli; (J.R and JALD) and Research Initiative for Scientific Enhancement permitting inflation and oxygen exchange at ambient pressure and preventing (RISE) program. collapse during respiration. Specifically, surfactant protein B (SP-B) has been shown to play an essential role in surface tension reduction, though Posters: Membrane Protein Dynamics II how it functions is largely unknown. A structurally simpler synthetic peptide, called KL4, is used for IRDS treatment. It mimics the carboxyl-terminus of 1202-Pos Board B111 SP-B and has been shown to lower the alveolar surface tension at the air- Characterizing GPCR Allostery by NMR Spectroscopy fluid interface. We compared the functions of SP-B’s two functional units, Shuya K. Huang, Libin Ye, Robert S. Prosser. its carboxyl-terminus and amino-terminus, with KL4 by individually studying Chemistry, University of Toronto, Mississauga, ON, Canada. the proteins’ interactions in a liposomal environment. Specifically, we studied Forty percent of current pharmaceuticals target G protein-coupled receptors how the peptides mediated membrane fusion of liposomes by observing the (GPCRs), a class of membrane proteins that regulate diverse biological FRET phenomenon when the two probes NBD-PE and Rhodamine-PE were processes including sensory perception, hormonal regulation, and immune

BPJ 8617_8620 Monday, February 19, 2018 239a response. Structurally, GPCRs exist in a dynamic ensemble of conformations 1205-Pos Board B114 spanning inactive, intermediate, and active states capable of engaging G pro- Identification of GPCR Transition Pathways using Go Models teins. The adenosine A2A receptor (A2AR) is a prototypical GPCR and a drug Leslie A. Salas-Estrada, Stephen J. Constable, Anthony Pane, target for the treatment of inflammation, cancer, diabetes, and Parkinson’s dis- Alan Grossfield. ease. While X-ray crystal structures reveal only an inactive and an active University of Rochester, Rochester, NY, USA. conformation, nuclear magnetic resonance (NMR) spectroscopy show that One caveat of standard all-atom molecular dynamics (MD) simulations is that detergent-reconstituted A2AR exhibits at least four functional states spanning they are resource-intensive, particularly when inspecting state transition path- inactive, intermediate, and active states capable of engaging G proteins. ways. To date, the timescales involved in processes such as G protein-coupled Though mechanistically poorly understood, the lipid bilayer plays an important receptor (GPCR) activation make it largely impossible to study them using role in GPCR regulation. Several GPCRs are known to require membrane standard all-atom MD under equilibrium conditions. Indeed, the nature of their 19 cholesterol for function, including A2AR. Here, we employ fluorine ( F) activation/deactivation pathway(s) is not yet fully characterized. To overcome NMR to characterize A2AR activity and drug-response in a synthetic lipid this issue, our group previously showed that simpler structure-based (Go-like) bilayer (nanodisc). We show that cholesterol is an allosteric effector of A2AR potentials could be used to study GPCR state transitions, yielding results in through modulation of its functional states. This knowledge furthers our under- qualitative agreement with those obtained from standard all-atom simulations standing of GPCR allostery and protein-membrane interactions. but with negligible computational cost. These potentials are energetically smooth by design with few non-native traps and a single global energy mini- 1203-Pos Board B112 mum corresponding to the end-point of the transition of interest. In the case Ligand Modulation of Sidechain Dynamics in a Wild-Type Human GPCR of rhodopsin, a prototypical GPCR, two distinct but reproducible pathways Lindsay D. Clark1,2, Igor Dikiy3, Karen Chapman1, Karin E. Rodstrom4, were observed to be populated during activation and deactivation. While it is James Aramini3, Michael V. LeVine5,6, George Khelashvili5,6, possible that a separation of pathways is mechanistically required for function, Soren G.F. Rasmussen4, Kevin H. Gardner3,7, Daniel M. Rosenbaum1,2. it could also be the result of simulating state transitions occurring in two sepa- 1Department of Biophysics, University of Texas Southwestern Medical 2 rate energy surfaces out of equilibrium (one for activation and one for deacti- Center, Dallas, TX, USA, Molecular Biophysics Graduate Program, vation). To address this potential source of error, we implemented a strategy for University of Texas Southwestern Medical Center, Dallas, TX, USA, 3 modeling Go-like energy landscapes with multiple energy minima at all-heavy Structural Biology Initiative, CUNY Advanced Science Research Center, atom resolution. This approach allows the study of state interconversion New York City, NY, USA, 4Department of Neuroscience and Pharmacology, 5 in equilibrium and extensive sampling, still at a low computational cost. More- University of Copenhagen, Copenhagen, Denmark, Department of over, it can also be used for quick hypothesis testing, to characterize the effects Physiology and Biophysics, Weill Cornell Medical College, New York City, of different ligands, model point mutations, study substate fluctuations and NY, USA, 6Institute for Computational Bioscience, Weill Cornell Medical 7 guide all-atom simulations by quickly generating reasonable reaction coordi- College, New York City, NY, USA, Chemistry and Biochemistry, City nates. As long as the end states are known, this strategy can be extrapolated College of New York, New York City, NY, USA. to almost any biomolecular system in a straight forward manner, complement- GPCRs regulate all aspects of human physiology, and biophysical studies have ing current standard all-atom MD approaches. deepened our understanding of GPCR conformational regulation by different ligands. Yet there is no experimental evidence for how sidechain dynamics 1206-Pos Board B115 control allosteric transitions between GPCR conformations. To address this Diverse Diffusion Regimes of Individual M2 Muscarinic Receptors and Gi deficit, we generated samples of a wild-type GPCR (A2AR) that are deuterated Proteins in Live Cells apart from 1H/13C NMR probes at isoleucine d1 methyl groups, which facili- Claudiu Gradinaru. tated 1H/13C methyl TROSY NMR measurements with opposing ligands. Physics, University of Toronto, Toronto, ON, Canada. Our data indicate that low [Naþ] is required to allow large agonist-induced GPCRs are known for high signal transduction efficiencies. One of the key as- structural changes in A2AR, and that patterns of sidechain dynamics substan- pects of the GPCR signaling mechanism is the coupling interaction between the tially differ between agonist (NECA) and inverse agonist (ZM241385) bound receptor and the G protein in response to external stimuli. We examined the receptors, with the inverse agonist suppressing fast ps-ns timescale motions pre-stimulus receptor - G protein coupling state by tracking the movement of at the G protein binding site. Our approach to GPCR NMR creates a framework individual particles of M2 muscarinic receptors and Gi proteins in the mem- for exploring how different regions of a receptor respond to different ligands or brane of live cells. signaling proteins through modulation of fast ps-ns sidechain dynamics. M2 receptors and Gi proteins were genetically fused with fluorescent proteins (GFP and mCherry) and then expressed in CHO cells. TIRF image sequences 1204-Pos Board B113 of the fluorescently labeled particles were acquired and then analyzed with the Distinct Dynamics of Biased Agonists Bound AT1R TrackMate single-particle tracking software. Mean-squared displacement Sangbae Lee1, Anita K. Nivedha1, HyunDeok Song2, Nagarajan Vaidehi1. (MSD) functions were computed for all single-particle trajectories. The diffu- 1City of Hope, Duarte, CA, USA, 2Chemistry, Vanderbilt University, sion properties of the M2 receptors and of the Gi proteins were retrieved by Nashville, TN, USA. fitting the MSD functions to appropriate diffusion models. Recent studies on a large number of GPCRs (G-protein coupled receptors) When G proteins were co-expressed with M receptors, the motion of G b i 2 i have shown that -arrestin transmit unique signals to catalytically active proteins was found to be highly distinctive from that of the receptors, but b proteins, in addition to a traditional G protein-signaling pathway. These -ar- then became similar to that of the receptors when the receptor agonist was restin based signaling mechanisms in cell are promoted independent of G-pro- added. Both the M2 receptors and the Gi proteins exhibited significant frac- tein and are known to form a distinct and different ensemble of conformations tions of confined diffusion (compatible with the membrane compartment signaling though the G-protein. The angiotensin-II type 1 receptor (AT1R), a formed by actin microfilament-based meshwork) and active transportation prototypical class A GPCR, is known to activate various effectors with the (compatible with the rate of myosin trafficking along actin microfilaments). b endogenous angiotensin II (AngII) including the Gq protein or -arrestin. Corroborated with dual-color fluorescence correlation spectroscopy mea- To verify this pharmacological evidence, microseconds of molecular dy- surements performed on the same samples, the results favor the receptor- namics (MD) simulations were computed to explore the conformational G protein transient recruitment interaction model and disfavor the pre- changes sampled by wild type AT1R caused by G-protein biased agonists stimulus coupling model. (TRV055 and TRV056) and b-arrestin biased agonists (TRV023, TRV034, TRV044, TRV045, TRV026, TRV027, and SII). In addition to biased 1207-Pos Board B116 signaling dependent on the ligand, we also performed several MD simulations Molecular Basis of Class B GPCRS Revealed by Multiscale Modeling for receptor mutated systems (D74N, DRY to AAY, and N298A mutation) for Chenyi Liao, Jianing Li. various efficacies depending on mutations on the receptor. Through conven- Department of Chemistry, University of Vermont, Burlington, VT, USA. tional MD simulations, we could be able to monitor the conformational tran- Despite considerable interest in Class B G protein-coupled receptors (GPCRs) sition within the microsecond time scale between G-protein biased and and their physiological roles in nervous systems, there is still a dearth of infor- b-arrestin biased agonists, showing that distinct conformational states are sta- mation with respect to the molecular structures and dynamics, which has hin- bilized by each biased agonist. Also, we found that the MD simulations of dered advances in understandings of receptor specifity, biased signaling and the mutated receptors revealed the specific structural rearrangement compared development of small-molecule ligands. With limited known receptor struc- with neutral wild type. Therefore, the understanding of specific conforma- tures and state-of-the-art protein modeling and simulation technologies, we tional transitions induced by biased agonists and receptor mutations will have studied the pituitary adenylate cyclase-activating polypeptide receptor lead to development of more efficient drugs. (PAC1R) and its variants, which are a potential target for endocrine, metabolic

BPJ 8617_8620 240a Monday, February 19, 2018 and stress-related disorders. Using large-scale microsecond simulations, we minated rhodopsin effectively captures the state of the excited protein while un- examined the open and closed PAC1R conformations interconnected within dergoing the conformational changes leading to activation. These discoveries an ensemble of transitional states. The open-to-closed transition can be initiated create new avenues towards understanding the central role of water in GPCR by ‘‘unzipping’’ the extracellular domain (ECD) and the transmembrane activation and unlocking the mechanisms of the biologically important domain (7TM), mediated by a unique segment within the beta3-beta4 loop. signaling process of rhodopsin. [1] S.M.D.C. Perera et al. (2016) J. Phys. Transitions between the open and closed states are 10-30 times longer than Chem. Lett.7, 4239-4235. [2] U.R. Shresta et al. (2016) J. Phys. Chem. those within the closed states, which clearly implicate allosteric effects propa- Lett. 7, 4130-4136. gating from the extracellular face of the receptor to the intracellular G protein- binding site. By simulating the agonist PACAP1-38 and the antagonist 1210-Pos Board B119 PACAP6-38 bound to the PAC1R orthosteric site, we observed the ligand- Exploring the Rhodopsin Dimer Interface in Live Cells Donald P. Mallory1, Adam Smith1, Beata Jastrzebska2, Elizabeth Gutierrez2. induced conformational changes that are associated with activation and deacti- 1 2 vation. In particular, our data demonstrate evidence for the key role of trans- Chemistry, The University of Akron, Cuyahoga Falls, OH, USA, School of membrane helix 6 (TM6) in connecting the ligand-receptor interactions with Medicine, Case Western Reserve University, Cleveland, OH, USA. the opening/closing of the intracellular G protein-binding site. Such allosteric In low-light environments, the G protein-coupled receptor (GPCR), rhodopsin, dynamics provides structural and mechanistic insights for the activation and is responsible for detection of light within the retina. Several studies have modulation of PAC1R and related Class B receptors, which will serve as the shown evidence for dimeric, or oligomeric, conformations of rhodopsin within essential molecular basis for the discovery novel anti-stress therapeutic agents. native disc membranes. However, while many oligomeric packing structures have been proposed, the exact dimerization interface of rhodopsin has yet to 1208-Pos Board B117 be firmly establish. Here we investigate rhodopsin dimerization in live cells Dynamic Behaviors of Various Conformational States of A2A Receptor with fluorescence correlation spectroscopy (FCS) and pulsed-interleaved exci- Sangbae Lee1, Anita K. Nivedha1, Christopher Tate2, Nagarajan Vaidehi1. tation fluorescence cross-correlation spectroscopy (PIE-FCCS). One of the pro- 1 2 City of Hope, Duarte, CA, USA, Cambridge University, Cambridge, United posed dimerization interfaces is between TM4/5 of rhodopsin. Recently we Kingdom. found evidence for a similar interface in red cone opsin. Based on those previ- G protein-coupled receptors (GPCRs) are integral membrane proteins encoded ous findings, we have made point mutations along rhodopsin’s TM 5, and found by numerous genes in the human genome and play essential role in cellular significant disruption of the dimerization affinity compared to the wild-type physiology and are targeted by approximately 40% of all modern medical construct. Our results provide new chemical details for the previously proposed drugs. Even though the extracellular loops of GPCRs play a key role in early packing structure between TM 4 and 5 of rhodopsin. Moreover, our findings stage on ligand recognition, the complete understanding of the extracellular further support the idea of conserved dimerization interface motifs shared loops for ligand binding in the GPCR is still largely missing, and it remains un- among class A GPCRs. Such a discovery may provide more general conceptu- clear how ligands and extracellular loops modulate a GPCR’s activity in the alization of self-association among one of the largest classes of transmembrane presence and absence of G-protein binding site. To elucidate the accurately receptors, which is essential for properly gauging the potential relationship be- quantifying the structural properties and the energetics of ligand binding and tween dimerization and function. extracellular region of the human adenosine A2A receptor (A2AR) with and without G-protein, we performed the microsecond time scale of molecular dy- 1211-Pos Board B120 namics simulations and binding free energy calculation for the fully active Substrate Interactions in the LacY Membrane Protein Transporter states, active intermediate state, and inactive state of receptors with two ago- Lutimba Stuart1, Stephen H. White2, Ronald H. Kaback3, nists bound, respectively. We have observed distinct structural states from fully Magnus Andersson1. 1 2 active state, active-intermediate state, and inactive state in the agonist NECA KTH Royal Institute of Technology, Solna, Sweden, UCI, Irvine, CA, USA, 3 and adenosine separately. We noticed the calculated binding affinity of agonist UCLA, Los Angeles, CA, USA. in the three different states of A2AR, and inter-molecular contacts between Membrane protein transporters govern important cellular processes and are agonist and receptor as well as the role of water molecules in the ligand bind- therefore central to human health. To accomplish transport, these proteins ing. Further, volumetric comparison between ligand binding pocket and G-pro- rearrange their structures to alternatively expose an internal binding site to tein binding cavity and entropic contribution toward extracellular loops provide either side of the membrane. Recent advances in protein structural determi- quantitative evidence for activation and deactivation mechanism. Our analyses nation methods have resulted in a steadily increasing number of high- suggest that the fully active, active-intermediate and inactive state of A2A re- resolution structures of membrane transporters trapped in different interme- ceptor can be affected by ligand binding pocket that have three distinct regions. diate states. However, to understand the underlying transport mechanism, the Overall, our atomic level of description of the binding process suggests oppor- molecular details of the interactions between the protein and the transported tunities for allosteric modulation and provides in the discovery of more effec- compound need to be determined. We have used specialized simulation tive and selective ligands of GPCRs. hardware and algorithms to simulate Lactose permease (LacY) of Escheri- chia coli interactions to the natural galactoside substrate lactose in the 1209-Pos Board B118 context of the outward-open and occluded crystal structures. We observed Hydration Thermodynamics of a Powdered G-Protein-Coupled Receptor protonation-dependent differences in the structural dynamics of the Andres M. Salinas, Suchithranga M.D.C. Perera, Michael F. Brown. protein-substrate interactions. Together, our simulations seek to answer the Department of Chemistry and Biochemistry, University of Arizona, Tucson, question why the galactoside sugar can bind only to a protonated state of AZ, USA. the LacY transporter. Membrane proteins such as G protein-coupled receptors (GPCRs) are promi- nent targets for novel pharmaceutical drugs. Preparation and storage of fully 1212-Pos Board B121 functional GPCRs is crucial to the processes of drug delivery and discovery. Proton Stabilization and Conduction Pathway in the Matrix Protein M2 Here we describe a novel method of preparing powdered GPCRs using Huong T. Kratochvil1, Jessica L. Thomaston1, Feng Gai2, rhodopsin as the prototype. Our new method for generating powdered samples, William F. DeGrado1. notable for exceptionally high protein content while retaining photochemical 1Pharmaceutical Chemistry, University of California-San Francisco, San functionality, paves the way for conducting functional and biophysical exper- Francisco, CA, USA, 2University of Pennsylvania, Philadelphia, PA, USA. iments on the dynamic process of receptor activation. As an illustrative appli- The transmembrane channel M2 of Influenza A virus plays a critical role in cation, powdered rhodopsin is prepared with and without the cofactor 11-cis its lifecycle, and serves primarily to acidify the viral interior for the release 2 retinal enabling the partial hydration of the protein with H2O in a controlled of ribonucleoproteins prior to viral-endosomal membrane fusion. Protons manner [1]. These samples were used to study the changes in hydrogen-atom enter the channel via a narrow opening from the viral exterior and diffuse dynamics using the quasi-elastic neutron scattering (QENS) technique. Evalu- to the His37 tetrad, where multiple protonation events ultimately lead to ation of QENS data using the interpretations of both spatial motion and energy conformational changes in the protein and dissociation of proton to the viral landscape models offer key insights into the thermodynamics of GPCR activa- interior. The slow rate of proton transport in M2 suggests that conforma- tion. The QENS studies reveal a broadly distributed relaxation of the hydrogen tional change of the protein is the rate-limiting step of the conduction pro- atom dynamics of rhodopsin on a picosecond-nanosecond time scale, essential cess. However, it remains unclear whether these conformational changes in for protein function, previously observed for only globular proteins [2]. Inter- the protein are intimately correlated for the conduction of protons. We estingly, the results suggest significant differences in the intrinsic protein dy- address this question by correlating conformational changes as measured namics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. by PET FCS with proton transport rates of full-length M2 measured using These differences can be attributed to the influence of the covalently bound a well-established vesicle flux assay. Furthermore, recent computational retinal ligand. Furthermore, preparation of powdered samples from photoillu- models and high-resolution X-ray structures have shown that protons diffuse

BPJ 8617_8620 Monday, February 19, 2018 241a along Grotthuss water wires that line the inside of the pore leading to the 1215-Pos Board B124 His37 tetrad. While these studies offer insight into the proton conduction Conformational Transitions in YddG Bacterial Transporter: A Mecha- pathway through the channel, little is understood about the distribution nistic Picture and dynamics of the water within the pore. Using two-dimensional infrared Shashank Pant1,2, Emad Tajkhorshid1,2. (2D IR) spectroscopy, we aim to probe the dynamics of water and pore- 1Center for Biophysics and Quantitative Biology, University of Illinois, lining carbonyls in the M2 channel. In applying 2D IR spectroscopy on mi- Urbana-Champaign, Urbana, IL, USA, 2Beckman Institure of Advanced crocrystals of isotopically labeled M2, we can probe the pH-dependent Science and technology, Urbana, IL, USA. changes in the distribution and dynamics of the water independent of YddG is a DMT (Drug and Metabolite Transporter) protein found in the inner changes to the population of conformational states of the protein. This bacterial membrane and helps in maintaining cell homeostasis by expelling methodology will allow us to dissect the effects of pH and the protonation amino acids and exogenous toxic compounds. During the transport cycle it un- state of the His37 tetrad on the hydrogen-bonding network inside the dergoes a large scale structural transitions from outward-(OF) to inward-facing channel. (IF) state. We have used Steered Molecular Dynamics to drive the transitions and based on the non-equilibrium work we came up with the optimal reaction 1213-Pos Board B122 co-ordinate, which was then used to obtain the transition free energy landscape The NapA Antiporter Undergoes Rocking-Bundle Alternation between using well-tempered metadynamics. We were able to capture the occluded state Oppositely-Facing Conformations: A Simulation Study which act like an intermediate between OF-IF transition. The transition relies Gal Masrati. on the movement of TM1, TM3, TM4 and TM9 in the outward facing confor- Tel Aviv University, Tel Aviv, Israel. mation and TM4, TM6, TM8 and TM9 in the inward facing conformation. We Membrane-spanning cation/proton antiporters (CPAs) mediate a vast variety have also explored the translocation of the substrate threonine in WT and of critical biological processes, and are associated with pathologies ranging mutant (H79A) YddG by performing 2D well-tempered metadynamics. The from hypertension to autism spectrum disorders, affecting millions world- free energy landscape highlights the importance of hydrophilic nature of the wide. Though CPAs are widely studied, there is an ongoing debate regarding lumen in threonine transport. whether these antiporters alternate between their outward- and inward-facing conformations through an elevator or a rocking-bundle mechanism. The two 1216-Pos Board B125 mechanisms differ in the type of relative motion between the two domains Rotational Diffusion of Membrane Proteins Probed by Anisotropic T2 and composing the transporter. To address this question, we used molecular dy- T1r NMR Relaxation in Aligned Lipid Bilayers namics simulations to study the Naþ/Hþ antiporter NapA from Thermus Alexander Nevzorov, Emmanuel Awosanya. thermophilus (TtNapA) embedded in a lipid bilayer; TtNapA’s structure is Chemistry, North Carolina State University, Raleigh, NC, USA. known in both inward- and outward-facing states. Specifically, we applied Membrane proteins are known to undergo fast uniaxial rotational diffusion an external potential to TtNapA, to bias the conformational sampling along about the membrane normal on the microsecond scale. Accurate determination two axes of motion between the transporter domains. The first was a vertical of local bilayer viscosity can provide quantitative information about the envi- translation along the membrane normal, consistent with an elevator mecha- ronment surrounding membrane-embedded proteins. Using Pf1 coat protein re- nism; and the second was a tilting movement of one domain relative to the constituted in aligned DMPC/DHPC bicelles as a probe, rotational diffusion other, consistent with a rocking-bundle mechanism. By applying the bias po- coefficients and membrane viscosity were assessed. The diffusion coefficients tential for the two axes simultaneously, as well as for each axis separately, were independently measured by T2 and T1r relaxation rates, which depend on we reproduced each of the known states of TtNapA when starting from the the heteronuclear dipolar couplings. A model for interpreting anisotropic relax- 15 opposite state, with an accuracy of up to 1.7 Angstrom. Importantly, our re- ation was developed based on uniaxial diffusion on a cone. The N T1r relax- sults suggest that the change between the inward- and the outward-open ation rates have been determined by fitting individually the decay of NMR states depends mainly on the tilting movement rather than on the vertical peaks for the Pf1 transmembrane domain using a modified SAMPI4 sequence. 4 translation. These results favor the rocking-bundle over the elevator mecha- The model yields a linear correlation with respect to the bond factor sin qB, nism of the ion/proton antiporting process. Furthermore, applying this where the NH bond angle qB can be independently measured from the SAMPI4 5 1 approach to the inward-facing conformation of NhaA from Escherichia spectrum. The diffusion coefficient D|| = 8.010 s was determined from the coli, we sampled what could be its long-sought-after outward-facing linear regression of the experimental T1r data even without any spectroscopic conformation. assignment. Remarkably, the assigned R1r relaxation data for the helical domain of Pf1 can be fitted to a wave-like pattern having a periodicity of 1214-Pos Board B123 5 1 3.6, which yields a close value of D|| = 7.710 s . A reasonably close value Microscopic View of the Outward- to Inward-Facing Transition Pathway 5 1 (D|| =510 s ) has been obtained from the fitting of T2 relaxation data, of the Human Dopamine Transporter though the latter has additional contributions arising from magnetic field inho- Zhiyu Zhao, Emad Tajkhorshid. mogeneity, incomplete dipolar decoupling, and inhomogeneous contribution UIUC, Champaign, IL, USA. from the sample mosaicity. The experimentally obtained diffusion coefficients The dopamine transporter (DAT) belongs to the family of neurotransmitter are consistent with simple estimates using the Stokes-Einstein equation if the sodium symporters (NSSs), which harnesses transmembrane electrochemical protein is modeled as a rigid cylinder having the radius of 10 A˚ and local gradient of ions for uphill transport of their substrate. The flow of ions across bilayer viscosity of 1 Poise, i.e. similar to the classical value of Saffman and the membrane drives the movement of neurotransmitters, such as dopamine, Delbruck. serotonin, GABA, and norepinephrine into the cell against their chemical gradients. DAT-substrate coupling induces large-scale conformational 1217-Pos Board B126 change between outward-facing (OF) and inward-facing (IF) states, the de- Modeling and Simulation of Outer Membrane Proteins in Pseudomonas tails of which remain unknown. In this study, we characterized the OF -IF Aeruginosa Outer Membranes conformational transition pathway of human dopamine transporter Joonseong Lee, Wonpil Im. (hDAT). Due to the lack of crystal structure of hDAT, an OF homology Lehigh University, Bethlehem, PA, USA. model of hDAT was generated using crystal structure of Drosophila mela- The Gram-negative bacteria have both the inner and outer membranes. The nogaster DAT as a template, and an IF hDAT based on a equilibrated struc- outer membrane (OM) is a unique asymmetric lipid bilayer containing lipo- ture of LeuT, both in the context of a lipid bilayer, which provided structure polysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. targets for simulating the full transport cycle. A detailed exploration of prac- Pseudomonas aeruginosa is one of the most notorious pathogenic Gram- tical collective variables highlights the importance of TM1 and TM8 in the negative bacteria and lacks general, large porins for small molecule transport. transition. Relative motion between TM1 and TM8 opens the inner vesti- This acts as a selective barrier and makes P. aeruginosa resistant to many an- bule, upon which Na2 diffuses into cytosol, followed by a displacement of tibiotics. Instead of general porins, P. aeruginosa utilizes substrate-specific dopamine towards the Na2 binding site. Advanced path refinement algo- outer membrane proteins (OMP) for the uptake of small molecules. Both rithms including bias-exchange umbrella sampling (BEUS) and the string (anion-selective) OccK5 and (cation-selective) OprD in P. aeruginosa are method with swarms of trajectories (SMwST) were used to characterize a known as specific channel proteins that require a carboxyl group in the sub- potential transition pathway between the OF and IF states, elucidating the strate for efficient transport. In this study, we performed all-atom molecular dy- mechanism of the closure of the extracellular ‘thin gate’ and the opening namics simulations of OccK5 and OprD in P. aeruginosa outer membrane of the intracellular ‘thick gate’. Our investigation into the structural dy- environments. We will present the influences of LPS compositions on OMP namics of hDAT provides a deeper understanding of the functional mecha- structure and dynamics, ion selectivity, ion mobility along the membrane nism of hDAT, applicable to other NSSs. normal, and interactions of OMPs with the OM.

BPJ 8617_8620 242a Monday, February 19, 2018

1218-Pos Board B127 the structural basis for the membrane-disrupting function of hBD is still Local and Global Dynamics in Klebsiella pneumoniae Outer Membrane unknown. Protein a in Lipid Bilayers Probed at Atomic Resolution In order to understand the interaction mechanism of hBD-3 with zwitter- Olivier Saurel. ionic (but charge-neutral) lipid membrane, umbrella-sampling simulations CNRS - IPBS, Toulouse, France. were performed on the hBD-3 wildtype as well as the linear analog, both The role of membrane proteins in cellular mechanisms strongly depends on in the monomer and dimer forms. Based on the free energy calculation, their dynamics; herein we make use of advances in 1H-detected MAS NMR both hBD-3 wildtype and linear analog need to overcome a very high energy to describe the dynamics of the membrane domain of the Outer membrane pro- barrier in order to translocate through the neutrally charged lipid bilayer. tein A of Klebsiella pneumoniae (KpOmpA). By measuring 1H-15N dipolar- hBD-3 without disulfides is much more flexible than the wildtype, and 15 coupling as well as NR1 and R1r relaxation rates at fast (60 kHz) MAS thus the hBD-3 analog can pass through the lipid bilayer relatively easier and high magnetic field (1 GHz), we were able to describe the motion of the energetically. Furthermore, forming a dimer can also help to decrease the b-barrel as a collective rocking of low amplitude and of hundreds of nano- energy barrier significantly. Based on this simple model, forming a higher seconds time scale. Residual local motions at the edges of the strands, under- order oligomer at high concentration is necessary for hBD-3 to transpass 15 scored by enhanced NR1r relaxation rates, report on the mobility of the con- the normal cell lipid membrane. nected loops. In agreement with MAS NMR data, proteolysis experiments performed on the full length KpOmpA as well as on its membrane domain, re- 1221-Pos Board B130 constituted in liposomes or in detergent micelles, revealed in all cases the ex- Microsecond-Level Simulations Reveal Membrane Protein Insertion istence of a unique Trypsin cleavage site within the membrane domain (out of Mechanism of Insertase YidC 16 potential Lys and Arg sites). This site is located in the extracellular loop L3, Thomas Harkey, Mahmoud Moradi, Jeevapani Hettige. indicating that this loop may be highly accessible to protein-protein interac- Chemistry and Biochemistry, University of Arkansas, Little Rock, AR, USA. tions. KpOmpA is involved in cell-cell recognition, adhesion and immune YidC, a member of the YidC/Alb3/Oxa1 insertase family, mediates mem- response mechanisms. The L3 region may therefore play a key role in brane protein assembly and insertion both with and without the involvement pathogenicity. of Sec machinery. The mechanistic details of the insertion process, however, remain elusive at the molecular level partly due to experimental limitations 1219-Pos Board B128 associated with structural studies. Here microsecond-level all-atom molecu- Dynamics of Membrane Proteins Studied by Solid State 2H NMR lar dynamics (MD) simulations are employed to investigate the structural Relaxation dynamics of YidC both in its apo form and bound to a Pf3 coat protein in Xiaolin Xu1, Andrey V. Struts2,3, Aswini Kumar Giri2, order to characterize the Sec-independent protein insertion mechanism of Trivikram R. Molugu2, Charitha Guruge4, Samira Faylough5, YidC. Carolina L. Nascimento5, Nasri Nesnas5, Victor J. Hruby2, Structural studies suggest that the cooperative interaction between the cyto- Michael F. Brown1,2. plasmic loops (C1 and C2), the conserved hydrophilic groove of YidC, and 1Physics, University of Arizona, Tucson, AZ, USA, 2Chemistry and the incoming peptide are the framework for the binding/insertion mechanism Biochemistry, University of Arizona, Tucson, AZ, USA, 3Laboratory of of YidC. Our simulations provide a dynamic picture of protein structure at Biomolecular NMR, St. Petersburg State University, St. Petersburg, Russian atomic resolution that complements the limited structural data. We have Federation, 4Chemistry and Biochemistry, Florida Institute of Technology, modeled YidC both without the C2 loop, which is missing in the crystal Melbourne, FL, USA, 5Chemistry, Florida Institute of Technology, structure, and with a modeled C2 loop. Both systems were modeled in the Melbourne, FL, USA. natural environment of the protein involving lipids, water, and ions, fol- Solid-state 2H NMR relaxation was applied to study the dynamics of the G- lowed by microsecond-level simulations. We have also modeled the same protein-coupled-receptor rhodopsin in the dark, preactive Meta-I, and active systems with a docked Pf3 coat protein in different poses and performed Meta-II states, as well as a high-affinity transducin peptide (GaCT2) bound microsecond-level simulations. to opsin. To observe how the NMR relaxation rates and molecular dynamics The data provided by our extensive set of simulations suggest a key role not depend on the local environment, 2H-labeled methyl groups were introduced only for the C1 loop, which has already been suggested, but also for the missing at carbons C5, C9, or C13 of retinal [1], and leucine positions 2, 5, or 10 in C2 loop that stabilizes YidC according to our model. Importantly, the simula- the GaCT2 peptide (ILENLKDVGLF). Solid-state Zeeman (T1Z) and quad- tions illustrate the role of water dynamics in the insertion process of the Pf3 rupolar order (T1Q) NMR relaxation times were measured in a wide temper- coat protein, which involves several stages of entering and exiting the waters ature range from 15 to 120 C. The T1Z and T1Q temperature to the core region of the protein while the substrate is being inserted in the dependences were analyzed using either a model-free approach [2] or membrane. various models of molecular motion (e.g., 3-site jumps and rotational diffu- sion). Calculated dynamical parameters (correlation times, activation bar- 1222-Pos Board B131 riers) for the methyl groups of retinal showed site-specific differences [1] Lipid-Dependent Modulation of Conformational Switching by Protonation attributed to intra-retinal torsional barriers and interactions with surrounding during Membrane Protein Insertion amino acid side chains. Surprisingly we found that unlike retinal, the dy- Victor Vasquez-Montes, Mykola V. Rodnin, Alexey S. Ladokhin. namics of deuterated methyl groups of the transducin peptide do not depend KUMC, Kansas City, KS, USA. on the leucine position. Furthermore the residual quadrupolar couplings of The conversion of a protein structure from a water-soluble to membrane- the NMR spectra indicate fast spinning of methyl groups about their symme- inserted form is one of the least understood cellular processes. In many cases try axis, whereas the side chain dynamics are much slower than the NMR this transition is triggered by the changes in side-chain protonation. Examples time scale. Activation energies were relatively high (similar to C5-methyl include cellular action of various bacterial toxins and of multiple proteins of of retinal), indicating that in the peptide even the methyl rotations are the Bcl-2 family of apoptotic regulators. Here we use an array of tools of fluo- restricted. Methyl dynamics of the peptide leucines were the same in a rescence spectroscopy to compare and contrast membrane insertion of the two dry powder, frozen water, and peptide bound to the opsin apoprotein sug- structurally similar, but functionally unrelated, representatives from these gesting tight packing of the transducin peptide in the rhodopsin binding families: (a) the diphtheria toxin translocation (T) domain and (b) anti- pocket.[1] A.V. Struts et al. (2011) PNAS 108, 8263. [2] X. Xu et al. apoptotic regulator Bcl-xL. Our results indicate that insertion pathway of (2014) eMagRes. 3, 275. the T-domain contains two staggered pH-dependent transitions and that several key protonatable residues (e.g., H223, H257, H322, E362) play impor- 1220-Pos Board B129 tant roles in conformational switching. The redundancy of conformational Free Energy Study on HBD-3 Translocation on Lipid Membrane switching in T-domain correlates well with its physiological function to Liqun Zhang. ensure robust translocation of the catalytic moiety of the toxin across the Tennessee Technological University, Cookeville, TN, USA. bilayer in response to acidification of the endosome. In contrast, bilayer- Human defensins are a class of antimicrobial peptides that are crucial compo- mediated interactions of the Bcl-2 family are tightly regulated to produce in- nents of the innate immune system. Human b defensin type 3 (hBD-3) has been hibition of mitochondrial outer membrane permeation (MOMP) under normal discovered recently, and has a charge density of þ11. It includes 3 pairs of in- conditions and trigger MOMP in apoptosis. We hypothesize that changes in tramolecular disulfide bonds, which can break and converts hBD-3 into the lipid composition modulate the balance of cell death and survival by causing linear analog form under reducing conditions. hBD-3 can selectively disrupt conformational switching in Bcl-2 proteins. For Bcl-xL, this modulation bacterial lipid membrane, cause cell leakage thus kill the bacterial cell. But can be seen by the effects of physicochemical properties of the lipid bilayer

BPJ 8617_8620 Monday, February 19, 2018 243a interface on the switching between anchored and inserted conformations contribution of specific residues in hV2 links inversely to its functional impor- involved in different modes of MOMP inhibition. Our results indicate that tance. We conclude that the evolutionary selection of hV2 sequence for channel while T-domain of the diphtheria toxin and Bcl-xL share some architectural function and regulation is at the expense of innate barrel stability. To our similarities along the insertion pathway, their modes of conformational knowledge, this is the first study to establish stability-function trade-off in a hu- switching are distinctly different: T-domain is switched via actual changes man b-barrel membrane protein. We propose that the antagonistic behavior of in pH, while Bcl-xL is switched via lipid-dependent changes in pKa at con- the three VDAC isoforms resides in subtle differences in the molecular ele- stant pH. NIH:P30-GM110761. ments that we have identified. These molecular elements decide thermody- namic stability, function, and the association network of the VDAC channels 1223-Pos Board B132 in the mitochondrial outer membrane. Characterization of Collective Protein-Water-Membrane Dynamics Christopher Paeslack1,2, Lars Schaefer1, Matthias Heyden2. 1226-Pos Board B135 1Theoretical Chemistry, Ruhr-University Bochum, Bochum, Germany, Folding Proteins Outside Their Native Environment: Folding the Innards 2Theoretical Chemistry, Max-Planck Institut fuer Kohlenforschung, of an Outer Membrane Transporter Muelheim, Germany. Adam M. Zmyslowski. Hydration water has been shown to play a key role in various biomolecular pro- Biochemistry and Molecular Biophysics, University of Chicago, Chicago, IL, cesses, such as molecular recognition, mediation of protein-protein interac- USA. tions, folding, and enzymatic reactions. In fact, the motions of proteins and During metabolite transport, TonB dependent receptors, found in the outer their hydration water are coupled via collective vibrations on the picosecond membranes of many bacterial species, are hypothesized to unfold their N- time scale. This collective motion is still observable at 10 A˚ distance from terminal ‘‘plug’’ domain. The 130 aa plug normally is folded inside a the protein surface and can be described as acoustic longitudinal modes prop- 450-550 aa, 22-strand b barrel, and must undergo significant rearrange- agating through the solvent with wave velocities similar to so-called ’’fast ment to allow passage of large (1kDa) substrates such as vitamin B12. sound’’ in bulk water. In this work, we use molecular dynamics (MD) simula- In the crystal structures, the interface between the plugs and the inside sur- tions to characterize collective motions in a protein-membrane system. Cross- face of the barrels is well hydrated and is composed primarily of H-bonded, correlation spectra of atomic velocities reveal that the dynamics of a peripheral charged, and polar contacts, despite being located inside the membrane membrane protein and a lipid bilayer are correlated with each other up to sep- plane. Here, we present CD, NMR, and SAXS results indicating that several aration distances of at least 20 A˚ . We further demonstrate that the hydration such plug domains are unfolded in solution whereas those from BtuB and shell overlap upon protein binding to the lipid bilayer changes the characteris- CirA adopt a partial globular fold consistent with their crystal structures. tics of both protein-water and membrane-water collective vibrations, an indica- In the crystal structure, the plugs have internal void volumes, suggesting tion that collective motions between protein and membrane are mediated by that they can unfold non-cooperatively, as has been shown in urea denatur- their shared hydration shells. ation EPR as well as recent AFM experiments. This is supported by our ki- netic folding data and pressure denaturation NMR experiments, which has 1224-Pos Board B133 implications for folding pathways as well as the transport function. NMR Measuring the Bilayer Depth Dependence of the SCN Infrared Probe studies of the BtuB & CirA plug domains will be presented, in which we Group using Poly-L Transmembrane Peptides take a divide and conquer approach to finding the minimal folding subunit Julia C. Fortier, Sara T. Gebre, Casey H. Londergan. of the plug. Haverford College, Haverford, PA, USA. Most conventional protein characterization techniques cannot be applied to 1227-Pos Board B136 membrane proteins due to their large size, complicated structures, and Slow Interconversion in a Conformationally Heterogeneous Unfolded- dependence on their natural environments. Vibrational probe groups are State Ensemble of Outer Membrane Phospholipase A one new method that can provide site-specific structural information with Georg Krainer1,2, Pablo Gracia1, Erik Frotscher2, Neharika Chamachi1, a fast intrinsic time scale in membrane proteins. The SCN group has a Andreas Hartmann1, Philip Gro¨ger1, Sandro Keller2, Michael Schlierf1. unique CN stretching band whose frequency reports on membrane burial. 1B CUBE, TU Dresden, Dresden, Germany, 2Molecular Biophysics, TU Four poly-L transmembrane peptides were synthesized with cysteines at Kaiserslautern, Kaiserslautern, Germany. varying positions so that the SCN vibrational probe group could be inserted Structural and dynamic investigations of unfolded proteins are important for at four different known burial depths. The four variants were cyanylated us- understanding protein-folding mechanisms as well as the interactions of ing 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) with at unfolded polypeptide chains with other cell components. In the case of outer least an 80% yield determined by liquid chromatography-mass spectrometry membrane proteins (OMPs), unfolded-state properties are of particular phys- (LCMS). Size exclusion chromatography on a Superdex Peptide column was iological relevance because these proteins remain unfolded for extended pe- used to purify the peptides, which were then mixed with POPC lipids and riods of time during their biogenesis and rely on interactions with binding inserted into rehydrated bilayers and vesicle samples via a sequence of partners to support proper folding. Using a combination of ensemble and freeze-thaw and sonication cycles. Infrared spectroscopy (IR) spectra were single-molecule spectroscopy, we have scrutinized the unfolded state of collected to observe the stretching frequencies and bandwidths of the in- Outer Membrane Phospholipase A (OmpLA) to provide a detailed view of serted SCN vibrational probes. its structural dynamics from nanoseconds to milliseconds. We find that, even under strongly denaturing conditions and in the absence of residual Posters: Membrane Protein Folding secondary structure, OmpLA populates an ensemble of slowly (>100 ms) interconverting and conformationally heterogeneous unfolded states that 1225-Pos Board B134 lack the fast chain reconfiguration motions expected for an unstructured, Human Mitochondrial VDAC Functionality Governs Scaffold Stability fully unfolded chain. The drastically slowed sampling of potentially Radhakrishnan Mahalakshmi. folding-competent states as compared with a random-coil polypeptide may Indian Institute of Science Education and Research, Bhopal, India. contribute to the slow in vitro folding kinetics observed for many OMPs. ATP transport across the mitochondrial outer membrane occurs through In vivo, however, slow intramolecular long-range dynamics might be advan- voltage-dependent anion channels (VDACs). The three human VDAC isoforms tageous for entropically favored binding of unfolded OMPs to chaperones share a similar primary sequence but differ in their interactome, and thereby, and, by facilitating conformational selection after release from chaperones, show antagonistic physiological functions. The anti-apoptotic and regulatory for preserving binding-competent conformations before insertion into the role of human VDAC2 (hV2) isoform must therefore originate from subtle var- outer membrane. iations in its primary sequence. However, the molecular details of how stability and function are regulated in hV2, and its impact on protein-protein interaction, 1228-Pos Board B137 are unknown. Here, we show that the 19-stranded hV2 barrel has evolutionarily Interactions of the Membrane Protein Chaperone Skp with BamD of the retained an energetically suboptimal sequence for superior channel gating and b-Barrel Assembly Machinery Complex from E. coli voltage sensing. We find that residues in the N- and C-terminal zones, Meenakshi Sharma, Jo¨rg H. Kleinschmidt. comprising strands b2-b8 and b17-b18, lower the scaffold stability by Dept. of Biophysics, University of Kassel, Kassel, Germany. 1.0-3.0 kcal/mol. Furthermore, strands b5-b10 and b17 possess sequences Insertion and folding of b-barrel membrane proteins (b-MPs) is mediated by that are intrinsically prone to association and aggregation. However, hV2 the essential b-barrel assembly machinery (BAM) complex. The BAM com- trades-off stability and aggregation for function. Residues in the N-terminal plex from the outer membrane of Escherichia coli is composed of the inte- zone are important for voltage-dependent channel gating characteristics that gral membrane protein BamA and 4 peripherally bound lipoproteins, BamB, are essential for metabolite transport. Our results demonstrate how energetic C, D, and E. We have previously examined the effect of BamA on insertion

BPJ 8617_8620 244a Monday, February 19, 2018 and folding of outer membrane protein A (OmpA) from E. coli into lipid bi- 1231-Pos Board B140 layers [1]. BamA facilitated folding of OmpA from an unfolded form bound Structure and Function of the Transporter NKCC1 to the periplasmic chaperone Skp, which is a trimer in solution. Here, BamD Corinne Portioli1,2, Marco De Vivo3, Ming Zhou2, Laura Cancedda1,4. and several single tryptophan mutants of BamD were isolated from mem- 1Neuroscience and Brain Technologies Department, Istituto Italiano di brane fractions of E. coli to investigate interactions of BamD with Skp. Tecnologia (IIT), Genova, Italy, 2Verna and Marrs McLean Department of For site-directed spectroscopy of Skp, we first prepared a plasmid for the Biochemistry and Molecular Biology, Baylor College of Medicine (BCM), expression of a synthetic variant of Skp. In this variant named Scp, the Houston, TX, USA, 3Molecular Modeling and Drug Discovery Laboratory, redundancy of the genetic code was used to connect three synthetic skp Istituto Italiano di Tecnologia (IIT), Genova, Italy, 4Dulbecco Telethon genes with different codon usage with two short DNA sequences encoding Institute, Rome, Italy. short and flexible linkers. Scp was over-expressed and isolated. It was pre- Neurodevelopmental disorders affect millions of children in Europe and world- sent as a monomer in solution with very similar secondary structure as the wide. Although a large body of literature indicates inhibitory GABAergic trans- trimeric Skp and functioned as a chaperone for folding of OmpA. Scp bound mission thorough Cl-permeable GABAA receptors as defective in numerous of to BamD in solution with high affinity and at a stoichiometry of 1 BamD/ those disorders, an effective pharmaceutical treatment is still missing. In this Scp. Fluorescence energy transfer of single Trp mutants of BamD bound regard, increasing scientific evidence has demonstrated that one of the more to lipid bilayers to single cysteine mutants of Scp labeled with IAEDANS physiological and effective ways to modulate GABAAergic transmission is indicated that the periplasmic chaperone Skp directly interacts with BamD by varying intracellular Cl concentration. This concentration is mainly estab- in a membrane environment. lished by the activity of the importer NKCC1 and the exporter KCC2. Impor- [1] G. Patel, J.H. Kleinschmidt, Biochemistry, 52 (2013) 3974-3986. tantly, NKCC1/KCC2 ratio is defective in a number of brain disorders and NKCC1 inhibition by the FDA-approved diuretic bumetanide rescues many 1229-Pos Board B138 symptoms in animal models. These findings have already motivated clinical Partition Coefficient of Arginine between Translocon Interior and Lipid studies for bumetanide in a broad range of brain disorders. Nevertheless, since Phase chronic treatments seem to be required, the diuretic effect of bumetanide - Denis G. Knyazev, Mirjam Zimmermann, Roland Kuttner, Peter Pohl. caused by the inhibition of the kidney-specific transporter NKCC2 - poses Institute of Biophysics, Johannes Kepler University, Linz, Austria. serious issues for drug compliance. These issues could be solved by selective Depending on its hydrophobicity, the protein translocation complex sorts NKCC1 inhibitors that avoid the diuretic effect. However, the NKCC1 trans- polypeptide segments into the membrane or into the aqueous solution. Its porter’s structure is still unknown, nor is how the transporter coordinates the short residence time in the channel limits polypeptide sampling between the three ions, Naþ,Kþ and Cl-, and shuttles them across the cell membrane. aqueous lumen and the membrane interior, giving rise to an out-of- We take a combined structural, functional and computational approach to equilibrium condition. Moreover, reduced mobility and orientational restric- address key questions on the mechanism of transport and its selective inhibition tions of confined intraluminal water molecules are bound to further bias pep- by small molecules. These studies will provide insights on the structure- tide partition into the membrane, rendering good agreement between the function relationships for Cl transportation and will accelerate the discovery physico-chemical and biological hydrophobicity scales all the more surpris- of new drugs urgently needed for brain disorders. ing. Here we report the equilibrium partition coefficient of arginine derived on the single molecule level by monitoring -repetitive lateral movement of 1232-Pos Board B141 a stalled polypeptide segment between the SecYEG lumen and the lipid inte- Iron Mediated Interaction of Alpha Synuclein with Lipid Rafts in Model rior. Polypeptide movements in the direction normal to the membrane were Membrane Systems inhibited by sandwiching the transmembrane sequence between a SecA bind- Loredana Casalis1, Fabio Perissinotto2, Denis Scaini3. ing sequence and the sequence of the calmodulin binding protein. With 1Elettra Sincrotrone Trieste, Trieste, Italy, 2University of Trieste, Trieste, calmodulin and SecA bound to opposite sides of the membrane, polypeptide Italy, 3International School for Advanced Studies (SISSA), Trieste, Italy. residence within the SecYEG pore was indicated by channel opening, whereas The aberrant misfolding and aggregation of alpha synuclein into toxic oligo- episodes of residence in the hydrophobic membrane interior corresponded to mers is one of the key hallmark involved in the pathogenesis of Parkinson’s channel closings. Translocon purification and reconstitution into planar lipid disease. It is well known that its physiological function is to bind membrane bilayers were performed as described previously.1-2 Comparison to amino presynaptic vesicles and to modulate vesicle trafficking at neurons terminals, acid partitioning in a biphasic system (physico-chemical hydrophobicity and that alpha synuclein in membrane is associated with lipid raft domains. scale) allowed us to address the effects of water confinement by the SecYEG We investigate here how environmental factors, such as lipid membrane bind- pore, while comparison to the biological hydrophobicity scale reveals out-of- ing and interactions with heavy metal ions, which have been shown to be a equilibrium effects. 1. Knyazev, D. G.; /; Pohl, P., The bacterial translocon risk factor for Parkinson’s disease, can promote alpha synuclein aggregations. SecYEG opens upon ribosome binding. J. Biol. Chem. 2013, 288 (25), 17941- Iron in particularly is a component of Lewy bodies, cytoplasmic protein inclu- 6. 2. Knyazev, D. G.;/ ; Pohl, P., Ion conductivity of the bacterial transloca- sions of alpha synuclein aggregates in dopaminergic neurons of substantia tion channel SecYEG engaged in translocation. J. Biol. Chem. 2014, 289 (35), nigra. By atomic force microscopy, we investigated the role of iron (II) in 24611-6. the interaction of the wild type alpha synuclein and the mutated A53T species with model membranes mimicking lipid raft domains. Iron strongly promotes 1230-Pos Board B139 formation of globular protein aggregates, resembling oligomers, with much How CLC Chloride Transporter Folds greater effect on the mutated A53T protein. The protein conformational Duyoung Min1, Robert E. Jefferson1, Yifei Qi2, Jing Yang Wang1, change induced by iron leads to different interactions of the proteins with Mark A. Arbing1, Wonpil Im3, James U. Bowie1. 1 model membranes. In absence of iron, protein monomers produce membrane Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, defects which can be associated to an a-helix conformation of the protein. On USA, 2College of Chemistry and Molecular Engineering, East China Normal 3 the contrary, iron-mediated oligomers are targeted to lipid raft domains, University, Shanghai, China, Departments of Biological Sciences and assembling ring-shape clusters and impairing those functional domains. The Bioengineering, Lehigh University, Bethlehem, PA, USA. results highlight the strong connection between iron and the aggregation ClC family members of chloride channels and transporters are important for of alpha synuclein, underlying a new mechanism of membrane-protein inter- chloride homeostasis in species from bacteria to man. Mutations in six different action which might be at the basis of the neurodegenerative disease family members cause genetically inherited diseases and some of the mutated development. proteins are likely to have folding defects. The ClC proteins present a chal- lenging and unusual biological folding problem because they are large mem- 1233-Pos Board B142 brane proteins possessing a complex architecture with many re-entrant Mechanism of Assembly of a Transmembrane Helix Dimer from All-Atom helices that go only part way through the membrane and loop back out. Here Simulation we were able to examine the folding of the E. coli ClC transporter, ClC-ec1 us- Robert B. Best1, Jan Domanski1, Mark S.P. Sansom2, Phillip Stansfeld2. ing single-molecule forced unfolding methods. We find that the protein can be 1Laboratory of Chemical Physics, NIH, Bethesda, MD, USA, 2Department of separated into two stable halves that unfold independently. The independence Biochemistry, University of Oxford, Oxford, United Kingdom. of the two domains is consistent with an evolutionary model in which the two Studying the folding and assembly of globular proteins using all-atom force halves may have arisen from independent folding subunits that later fused fields is now well established. However, doing so for membrane proteins is together. Maintaining smaller folding domains of lesser complexity within much more challenging, not least because of the high viscosity of the mem- large membrane proteins may be an advantageous strategy to avoid misfolding brane. Nonetheless, simulations could help to fill in important details which traps. are hard to obtain experimentally. In this work, we have studied the assembly

BPJ 8617_8620 Monday, February 19, 2018 245a of the transmembrane helix dimer Glycophorin A, the simplest prototype for (VX-809) reverses hairpin opening to restore a compact state as in the WT. membrane protein folding. We find that although the native state is a local min- The observed membrane escape of TM4 in the V232D hairpin recapitulates imum in the force field used, it is only metastable. After a simple force field cell-based mutational analyses of full-length CFTR, which suggest that a modification to improved dimer stability, we have studied the assembly rate loss of local nonpolar interactions propagates among several TM domains and mechanism using transition-path sampling. We find that the overall rate to produce misfolding of the V232D mutant CFTR. Thus, our findings provide of dimer assembly is comparable to that for similar transmembrane helix di- in vivo/in vitro correlates and molecular-level insights into the structural ef- mers, and that assembly proceeds first by formation of a non-native dimer, fol- fects of a disease-causing CFTR mutation and its pharmacological rescue. lowed by a transition to the fully native state with formation of the We anticipate our approach to be a promising tool for studying misfolding characteristic GXXXG packing motif. and drug rescue of other CFTR mutants and for facilitating the development of mechanism-based therapies of other misfolding-prone helical membrane 1234-Pos Board B143 proteins. Molecular Mechanism of Polypeptide Insertion into Bilayer and Exit Gregory Slaybaugh, Dhammika Weerakkody, Oleg Andreev, 1237-Pos Board B146 Yana Reshetnyak. Functional Stabilization of Purified Human CFTR by NBD1 Mutations Physics, University of Rhode Island, Kingston, RI, USA. and by Phosphatidylserine Using the pH-triggered insertion of the pHLIP (pH Low Insertion Peptide) Ina Urbatsch1, Zhengrong Yang2, Ellen Hildebrandt1, Fan Jiang3, peptide to enable kinetic analysis, previously we showed that insertion oc- Qingxian Zhou2, Jiangli An2, Bala M. Xavier1, Netaly Khazanov4, curs in several steps, with a rapid interfacial helix formation followed by Hanoch Senderowitz4, John C. Kappes3, Christie G. Brouillette2. a much slower insertion pathway to give a transmembrane helix. The reverse 1Cell Biology and Biochemistry, Texas Tech University Health Sciences process of unfolding and peptide exit from the bilayer core, which can be Center, Lubbock, TX, USA, 2Department of Chemistry, University of induced by a rapid rise of the pH from acidic to basic, proceeds through Alabama at Birmingham, Birmingham, AL, USA, 3Department of Medicine, different intermediate states. To gain insights into molecule mechanism of University of Alabama at Birmingham, Birmingham, AL, USA, 4Department membrane-associated folding and unfolding we designed and investigated of Chemistry, Bar-Ilan University, Ramat-Gan, Israel. three single-Trp variants, were tryptophan fluorophores were placed at the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chlo- positions 6 (W6-pHLIP), 17 (W17-pHLIP) and 30 (W30-pHLIP) of 32-res- ride channel of the ABC transporter superfamily. It contains two transmem- idues pHLIP. Kinetics of membrane insertion and exit of various parts of the brane domains that form the ion pore, and two nucleotide binding domains polypeptide were investigated in great details. The obtained results allowed (NBD1 and NBD2) that bind and hydrolyze ATP to gate the channel, regu- us to complete molecular model of pHLIP propagation into bilayer and exit lated by phosphorylation. This complex and nonrobust integral membrane triggered by pH changes. protein is a key target for cystic fibrosis drug development. Here we used 1235-Pos Board B144 a systematic and sequential approach to compare the effect of single and Mechanisms by Which Lipids Shape the Reaction Coordinate of GlpG multiple site NBD1 mutations on stability of both the NBD1 domain alone, Protease and on purified full length human CFTR. We demonstrate that mutations Ana-Nicoleta Bondar. that stabilize NBD1 structure, quantified by calorimetry, also improve the Department of Physics, Freie Universit€at Berlin, Berlin, Germany. structural stability of purified, full-length CFTR in a predictable manner. Rhomboid proteases are membrane-embedded enzymes whose catalytic activ- Moreover, in either the NBD1 domain or in full length CFTR, the effect ity depends on the composition of the lipid membrane that surrounds them. This of single mutations is additive when combined. Combinations of up to six makes rhomboids an excellent model system to dissect mechanisms by which mutations S492P/A534P/I539T/M470V/S495P/R555K yielded a super stabi- lipids shape the reaction mechanisms of membrane proteins. Extensive all- lized CFTR variant (6SS-CFTR) with highly improved structural stability, > o atom molecular dynamics simulations of the Escherichia coli rhomboid, exhibiting a thermal unfolding 20 ChigherthanwildtypeCFTR. GlpG, indicate that the orientation of GlpG and lipid interactions at the sub- NBD1 stabilizing mutations also improved biogenesis and cell surface strate docking site, which are essential for substrate binding, depend on the expression in a mammalian expression system. These stabilized proteins lipid membrane composition. Different protein conformations can associate all displayed robust ATPase activity, and better retained functional stability with different orientations in the membrane, suggesting that protein orientation after heat treatment. Replica exchange molecular dynamics simulations per- is a dynamic parameter that changes during the reaction coordinate. The com- formed on the most stable CFTR construct suggested better packing of the putations provide a comprehensive view of the molecular picture of GlpG/lipid mutated side chain. Further stabilization of purified CFTR could be achieved interactions in different lipid membrane environments. This research was sup- specifically with addition of phosphatidylserine, complementing the stabiliz- ported in part by funding from the Excellence Initiative of the German Federal ing effects of introduced mutations. A practical outcome of these studies is and State Governments provided via the Freie Universit€at Berlin, and by an the observation that structurally stabilized CFTR has improved solution allocation of computing time from HLRN, the North-German Supercomputing properties and maintains enzymatic function, allowing for the purification Alliance (bec00076). of implicitly properly folded protein in quantities sufficient for future bio- physical and structural studies. 1236-Pos Board B145 A Minimal Helical Hairpin Motif Recapitulates Misfolding and Pharmaco- Posters: Transcription logical Rescue of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) 1238-Pos Board B147 Georg Krainer1,2, Antoine Treff1, Andreas Hartmann1, Tracy A. Stone3,4, Roles of Upstream Promoter DNA in Bacterial Transcription Initiation Sandro Keller2, Charles M. Deber3,4, Michael Schlierf1. Munish Chhabra1, Christina Mcnerney2, Katelyn Callies3, 1BCUBE - Center for Molecular Bioengineering, TU Dresden, Dresden, Claire Cimperman2, Andrew Xue4, Irina Shkel4, Tom Record5. Germany, 2Molecular Biophysics, TU Kaiserslautern, Kaiserslautern, 1Biophysics, University of Wisconsin Madison, Madison, WI, USA, Germany, 3Division of Molecular Medicine, Hospital for Sick Children, 2Biochemistry, University of Wisconsin Madison, Madison, WI, USA, Toronto, ON, Canada, 4Department of Biochemistry, University of Toronto, 3Chemistry, University of Wisconsin Madison, Madison, WI, USA, Toronto, ON, Canada. 4University of Wisconsin Madison, Madison, WI, USA, 5Biophysics, A major challenge in the development of cystic fibrosis (CF) disease Biochemistry and Chemistry, University of Wisconsin Madison, Madison, mechanism-based therapies is the incomplete understanding of the molecular WI, USA. basis of CFTR misfolding and its drug rescue by pharmacological correctors. Far-upstream promoter DNA plays multiple roles in bacterial transcription Here we use a helical hairpin construct—essentially the simplest in vitro initiation. a-CTD of RNA polymerase (RNAP) and transcription factors regu- model of membrane-protein tertiary contacts—containing transmembrane late initiation rate by binding to this upstream region (1). Even without factor (TM) helices 3 and 4 of CFTR TM domain 1 and its corresponding binding, upstream truncations between 100 and 40 greatly reduce the rate of CF-phenotypic mutant (V232D in TM4) to recapitulate the misfolding and open complex formation at lPR (2) and lacUV5 (3) promoters. Both closed and drug-rescue effects observed for full-length CFTR in vivo. Employing a open promoter complexes (CC, OC) exhibit strong FRET between cyanine single-molecule Fo¨rster resonance energy transfer spectroscopy approach to dyes at 100 (upstream) and þ14 (downstream) positions of lPR DNA (4), study hairpin conformations in phosphatidylcholine lipid bilayers, we find demonstrating bending and wrapping of upstream DNA around RNAP. These that the WT hairpin is well-folded, while the V232D mutant assumes an results confirm and extend what was known from real-time footprinting of CC open hairpin conformation in bilayer thicknesses mimicking the endoplasmic and OC (5) and AFM on OC (6). Here we use stopped-flow fluorescence FRET reticulum. Titration of V232D hairpin with the CFTR corrector Lumacaftor and PIFE assays to determine the kinetics of bending and wrapping the initial

BPJ 8617_8620 246a Monday, February 19, 2018 early CC to form an advanced CC in which the upstream promoter DNA is 1241-Pos Board B150 bent/wrapped on RNAP, allowing the downstream DNA to be bent into the Single-Molecule Analysis of the Influenza Virus Replication Initiation active site cleft to initiate DNA opening. After NTP-addition, we detect two ki- Mechanism netic steps of unwrapping of promoter DNA during initiation and promoter Nicole C. Robb1, Aartjan J.W. te Velthuis2, Ervin Fodor3, escape. In OC dissociation by high-salt upshift, unwrapping occurs in or prior Achillefs N. Kapanidis1. to the DNA closing step. We obtain mechanistic and structural information 1Physics, University of Oxford, Oxford, United Kingdom, 2Pathology, about key intermediates in the mechanisms of OC formation and initiation University of Cambridge, Cambridge, United Kingdom, 3Pathology, and about differences between the OC and the elongation complex. Reference: University of Oxford, Oxford, United Kingdom. 1. Gourse R.L. et al. (2000) Mol. Microbiol. 37(4):687-95. 2. Davis C.A. et al. Influenza viruses have a segmented viral RNA (vRNA) genome, which is repli- (2005) PNAS USA 102(2):285-90. 3. Ross, W et al. (2005) PNAS USA 102 , cated by the viral RNA-dependent RNA polymerase (RNAP). Replication ini- 291-296. 4. Sreenivasan et al. (2016) Biochem. 55(14):2174-86. 5. Davis C.A. tiates on the vRNA 3’ terminus, producing a complementary RNA (cRNA) et al. (2007) PNAS May 8;104(19):7833-8. 6. Rivetti, C. et al. (1999) EMBO J. intermediate, which serves as a template for the synthesis of new vRNA. 18 , 4464-4475. RNAP structures show that the 3’ vRNA template can adopt more than one configuration; it can be bound in a pre-initiation state on the surface of the 1239-Pos Board B148 RNAP, as well as in an initiation state in the active site of the RNAP. No infor- Electron Microscopy and Integrative Modeling Shed Light on the Mecha- mation is available on 3’ cRNA binding, although a crystal structure of the nisms of Transcription Initiation RNAP with a short 5’ cRNA terminus shows that the conformation of the first Chunli Yan1, Yuan He2, Eva Nogales3, Ivaylo Ivanov1. 1 twelve residues of the 5´ cRNA is virtually identical to that of the vRNA 5´ ter- Department of Chemistry, Georgia State University, Atlanta, GA, USA, minus. We have used single-molecule Fo¨rster resonance energy transfer 2Department of Molecular Biosciences, Northwestern University, Evanston, 3 (smFRET) on surface-immobilised initiation complexes to probe the RNA con- IL, USA, University of California, Berkeley, Berkeley, CA, USA. formations adopted during RNAP binding and initial replication. We show that Advances in cryo-electron microscopy have opened up new avenues to struc- in the absence of nucleotides, the RNAP-bound 3’ terminus of the vRNA pro- turally define biomolecular assemblies. To arrive at detailed psedoatomic moter exists in dynamic equilibrium between the pre-initiation and initiation models, it is necessary to employ integrative computational modeling. Here, conformations. Analysing the dynamics of immobilised RNAP/RNA com- we illustrate the success of this hybrid structural approach in describing pre- plexes in real-time has provided dwell times for the two states. Nucleotide addi- initiation complexes of eukaryotic RNA Polymerases I and II (Pol I and Pol tion stabilises the 3’ vRNA in the active site and results in unwinding of the II) at near atomic resolution. During initiation Pol II and general transcription duplexed region of the promoter. Intriguingly, and in contrast to the vRNA pro- factors (GTFs) assemble into a pre-initiation complex (PIC). After assembly, moter, the cRNA promoter is stably bound, with limited dynamics, suggestive the initial closed promoter complex (CC) transitions into an open complex of differences in the initiation mechanisms for the two promoters. Our data pro- (OC), in which the melted template DNA is inserted into the Pol II active vide novel insights into the dynamic motions of RNA that occur during initial site. The OC is eventually converted into an initial transcribing complex influenza replication and has implications for our understanding of the replica- (ITC), competent to synthesize RNA. Atomic resolution description of this tion mechanisms of similar pathogenic viruses. complex machinery had proved elusive. Combining cryo-EM with state-of- the-art computation allowed visualization of the Pol II PIC at all major stages 1242-Pos Board B151 of transcription initiation. Comparisons of the functional states provided new How does a Viral RNA Polymerase Ratchet along DNA without Back- mechanistic insights into the processes of DNA engagement, promoter melting tracking? and transcription bubble stabilization. By contrast, Pol I transcription initiation Lin-Tai Da1, E. Chao2, Yao Shuai3, Shaogui Wu4, Xiao-Dong Su3, Jin Yu2. depends on the interaction of Pol I with the GTF Core Factor (CF). Visualizing 1Shanghai Jiaotong University, Shanghai, China, 2Beijing Computational the Pol I PIC showed that Pol I and promoter DNA are a preconditioned in an Science Research Center, Beijing, China, 3Peking University, Beijing, China, elongation competent form. The structures also revealed a bipartite architecture 4Sichuan Normal University, Chengdu, China. of the Core Factor and its specific recognition of the promoter DNA. In this The single-subunit viral RNA polymerase (RNAP) from bacteriophage T7 is study the Pol I PIC was captured in three distinct functional states. Comparison widely utilized in lab gene expression system and synthetic circuit. T7 of these states with the Pol II system suggests that a ratchet motion of the CF RNAP is a smallest transcription machine, working self-sufficiently without bound to the upstream DNA facilitates promoter melting in an ATP- transcription factors. We have studied T7 RNAP on both its mechano- independent manner. chemical coupling and fidelity control during transcription elongation, combining physical modeling with structural dynamics simulations. In partic- 1240-Pos Board B149 ular, we have implemented extensive atomistic molecular dynamics simula- Transcription Enhancement by Nuclear Speckle Association tions and constructed the Markov state model to investigate product release Jiah Kim, Nimish Khanna, Andrew S. Belmont. (Da et al PLoS Comp Biol 2015) and translocation mechanisms of T7 RNAP University of Illinois at Urbana-Champaign, Urbana, IL, USA. (Da and E et al, Nuc Acids Res 2017), demonstrating its Brownian ratchet na- The textbook view of nuclear speckles is that they serve primarily as storage ture moving along DNA while synthesizing RNA. Notably, we found that an O- sites for RNA processing factors. A long-standing competing model has been helix on the fingers domain of the hand-like structure of T7 RNAP could open that nuclear speckles act as a transcriptional hub for a subset of active genes; occasionally in the pre-translocation state, after the PPi product release. The O- however, this hub model was based on analysis of just a few genes. The assign- helix opening would not only facilitate the translocation, but also appeared to ment by TSA-Seq of the Hi-C A1 active subcompartment as corresponding to prevent backtracking, which might explain why T7 RNAP had not been de- genomic regions positioned close to nuclear speckles, now greatly bolsters this tected with backtracking. To test our hypothesis, we computationally designed model of nuclear speckles as a transcriptional hub, yet this TSA-Seq mapping mutants of T7 RNAP by replacing several amino acids on the O-helix with remains correlative. Here we demonstrate a tight temporal relationship between counterpart residues from a mitochondrial RNAP that is supposed to be able speckle association and heat-shock induced transcriptional activation using a to backtrack. Preliminary experimental results show supports for the hypothe- BAC transgene system that closely recapitulates the transcriptional induction sis. The mechanistic insight revealed in our studies can also be general for a kinetics of the endogenous Hsp70 locus. smRNA FISH shows that both class of transcription core engines with compact molecular structures. Hsp70 BAC transgenes and the endogenous Hsp70 locus synchronously acti- vate transcription between 3-5 minutes after heat shock. However, speckle- 1243-Pos Board B152 associated transgenes have 4, 12, and 50-fold increased levels of nascent tran- Abortive and Productive Transcription Initiation by E. coli RNA scripts compared to non-associated ones at 30 mins, 1 hr, and 2 hrs after heat Polymerase shock, respectively; a 13-fold difference at 2 hrs is observed for the endogenous Kate Henderson, Cristen M. Molzahn, Lindsey C. Felth, Claire Evensen, Hsp70 loci. Moreover, live-cell imaging reveals several minute delays in robust Sarah Dyke, Guanyu Liao, Jack Prazich, M. Thomas Record. transcriptional activation correlate with the time required for a BAC transgene Department of Biochemistry, University of Wisconsin-Madison, Madison, to move to a nuclear speckle. Further analysis of live-cell data reveals examples WI, USA. 70 where transcription decreases after BAC transgenes move away from speckles, E. coli RNA polymerase (RNAP; 2 ) binds, bends, and opens PR promoter and even increase again or restart after the BAC transgene forms a new speckle DNA to form a highly stable, transcription-ready open complex (OC) with a association. This strong temporal correlation between speckle association and lifetime in vitro of 13 hours. In order to escape from this promoter and syn- changes in gene expression suggests that stochastic variations in gene expres- thesize a full-length RNA transcript, RNAP must first break contacts with the sion may be related to spatial positioning relative to nuclear speckles for a sub- promoter that stabilize its OC by 10 kcal/mol relative to the ribosomal rrnBP1 set of active genes. promoter. It does so through DNA scrunching (Revyakin A., et al. Science

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2006; 314 (5802):1139-1143), which we find destabilizes OC by 1 kcal/mol Critical contacts made between the RNA polymerase (RNAP) holoenzyme o per step of RNA hybrid extension. In initiation from PR at 37 C, OCs that can and promoter DNA modulate not only the strength of promoter binding, escape the promoter do so in <10 s. Those OCs that cannot escape slowly and but also the frequency and timing of promoter escape during transcription. reiteratively synthesize short (3-10 nucleotide) RNAs. Here, we describe a single-molecule optical-trapping assay to study tran- Using Rapid Quench Flow techniques and quantitative single-round transcrip- scription initiation in real time, and use it to map contacts formed between tion assays, we are investigating the mechanism of productive and abortive s70 RNAP holoenzyme from E. coli and the T7A1 promoter, as well as to o transcription initiation at the PR promoter. At 37 C, no initiation intermediates observe the remodeling of those contacts during the transition to the elonga- are detected even on a millisecond time scale and amounts of short and long tion phase. The strong binding contacts identified in certain well-known pro- RNAs increase rapidly at early times. By reducing the temperature and varying moter regions, such as the 35 and 10 elements, do not necessarily nucleoside triphosphate (NTP) concentrations, we slow the process and observe coincide with the most highly conserved portions of these sequences. Strong the progression of transiently- paused intermediate lengths in productive syn- contacts formed within the spacer region (10 to 35) and with the 10 thesis on a ms time scale. At low temperature, these paused intermediates on element are essential for initiation and promoter escape, respectively, and the pathway to escape and long RNA synthesis are resolved from stalled com- the holoenzyme releases contacts with promoter elements in a non- plexes that will not escape. From the NTP concentration dependence of these sequential fashion during escape (Meng et al., Nature Communications kinetics, we are obtaining a quantitative mechanistic description of productive 2017). and abortive transcription initiation for the multi-subunit enzyme. We gratefully acknowledge the NIH for support of this research (GM118100 to 1247-Pos Board B156 M.T.R. and GM122303 to K.L.H.). Effects of TEFM on Mitochondrial RNA Polymerase Transcription Elongation 1244-Pos Board B153 Cheng Xue. The Thermal Mobility of Nucleosomes: A Versatile Tool for the Regulation HKUST, Hong Kong, Hong Kong. of Gene Expression Transcription elongation regulation is one of the key mechanisms employed Ariel Kaplan. to control gene expression. Mitochondrial DNA is transcribed by the single- Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel. subunit mitochondrial RNA polymerase (mtRNAP). TEFM (transcription Nucleosomes at the promoters of genes regulate the accessibility of the tran- elongation factor mitochondria) is reported as a stimulator of transcription scription machinery to DNA, and function as a basic layer in the complex regu- elongation by directly interacting with mtRNAP. We used optical tweezers lation of gene expression. However, our understanding of the role of the to perform in vitro single molecular transcription elongation assay and quan- nucleosome’s spontaneous, thermally driven position changes in modulating titatively analyzed the function of TEFM in the mtRNAP transcription pro- expression is lacking, due to the paucity of experimental data on these dy- cess on normal DNA template and the conserved sequence block II (CSBII) namics, at high-resolution, and for DNA sequences that belong to real, tran- transcription termination site, which plays a crucial role in the switch of scribed genes. Using the nucleosomes at the promoters of two model genes, transcription and DNA replication. We observed that TEFM enhances tran- Cga and Lhb, and optical tweezers to repeatedly probe their position over scription elongation and modulates mtRNAP pausing step. Our findings time, we show that the mobility of nucleosomes is modulated by the sequence explain how TEFM is involved in mitochondrial transcription elongation of DNA and by the use of alternative histone variants. We then show that mo- regulation. bile nucleosomes are able to modulate the ability of transcription factors to bind their target, and to facilitate elongation by RNA polymerase. Taken together, 1248-Pos Board B157 our findings reveal that nucleosome mobility is a potent and versatile tool for MFD Dynamically Regulates Transcription 1 1 1 2 gene regulation. Tung T. Le , Yi Yang , Chuang Tan , Margaret Suhanovsky , Robert M. Fulbright Jr.1, James T. Inman1, Ming Li1, Jaeyoon Lee1, 1245-Pos Board B154 Jeffrey W. Roberts1, Alexandra M. Deaconescu2, Michelle D. Wang1. Transcription Dynamics at the HIV-1 Reporter Locus 1Cornell University, Ithaca, NY, USA, 2Brown University, Providence, RI, Linda Stephanie Forero-Quintero1, Michael May2, Edouard Bertrand3, USA. Eugenia Basyuk3, Hiroshi Kimura4, Brian Munsky2, Timothy Stasevich1. Fundamental processes that occur along DNA, such as transcription and 1Biochemistry and Molecular Biology, Colorado State University, Fort replication, commonly encounter obstacles or ’roadblocks’ which may Collins, CO, USA, 2Biomedical Engineering, Colorado State University, Fort impede progress and ultimately result in mutations and/or DNA damage. Collins, CO, USA, 3Institut de Genetique Moleculaire de Montpellier, Thus, spatiotemporal control of these factors can have far-reaching conse- Montpellier, France, 4Biological Sciences, Tokyo Institute of Technology, quences. In bacteria, the mutation frequency decline (Mfd) protein has Tokyo, Japan. been widely recognized for dealing with transcriptional roadblock resolu- Transcription is a centerpiece in the regulation of gene expression and cell tion through either facilitating transcription through a roadblock or termi- metabolism. It can be divided in three stages: initiation, elongation and termi- nating transcription altogether. Despite this importance, it is unclear how nation. The molecular mechanisms and steps involved in the regulation of tran- Mfd interacts with RNAP and DNA and how such interactions lead scription are of a dynamic nature. Decades ago, biochemistry techniques could to obstacle resolution. Using novel assays, we discovered that Mfd not tell us much about the kinetics of transcription. However, recent advances interacts with RNAP in a dynamic and highly regulated fashion. Our find- in microscopy make it feasible to visualize and quantify transcription in live ings demonstrate a remarkably delicate coordination between Mfd and cells at the single-cell and single-molecule levels. With state-of-the art live RNAP, allowing efficient targeting and recycling of Mfd, and expedient cell microscopy, it is now possible to observe fluctuations in transcription ki- conflict resolution. netics at single copy genes. Using an established cell line and our Fabs (Frag- ment Antigen-Binding) for live imaging of RNAPII, we have imaged 1249-Pos Board B158 transcription dynamics at the locus of a reporter gene controlled by the HIV- Highly Specific Circular RNA Quantification in Single Yeast Cells 1 promoter and tagged with 128x MS2 cassette, when transcribed in RNA, Gable M. Wadsworth, Harold D. Kim. the MS2 repeats are bound by MCP-GFP. The MS2 bright spot at the reporter School of Physics, Georgia Institute of Technology, Atlanta, GA, USA. locus provides information about the gene location and transcription readout, Circular RNA, ubiquitously expressed in eukaryotes, is emerging as an while our Fabs, C13 and Pa57 tell us about the recruitment of the RNAPII to important regulatory ncRNA with clinical applications in cancer detection; the promoter and initiation of transcription, respectively. Here, simultaneous however, the function and localization of these transcripts is poorly charac- imaging of transcription (MS2), RNAPII recruitment (C13) and initiation terized. Ensemble methods such as nuclease resistance have shown the pres- (Pa57) dynamics show temporal correlations at the reporter locus. ence of circular RNA in budding yeast, but methods that can quantify the abundance of circular RNA at the single cell level are lacking. We demon- 1246-Pos Board B155 strate a new in situ fluorescence technique based on hybridization chain re- Real-Time Observation of Polymerase- Promoter Contact Remodeling action, which enables discrimination of a circular transcript from precursor during Transcription Initiation linear transcripts in single budding yeast cells. Further, we report the spatial Furqan M. Fazal1,2, Cong A. Meng3, Steven M. Block1,4. distribution of circular RNA for the gene SUS1 inside single cells. We 1Department of Applied Physics, Stanford University, Stanford, CA, USA, observe high intrinsic noise in log phase cells due to the low copy number 2Stanford University School of Medicine, Stanford, CA, USA, 3Department of SUS1 circular RNA. Our method represents a highly specific means to of Chemistry, Stanford University, Stanford, CA, USA, 4Department of quantify splicing variation and other short polymorphisms in RNA sequence Biology, Stanford University, Stanford, CA, USA. at the single cell level.

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1250-Pos Board B159 clusters in the nucleus of live stem cells. We also observed stable and large In Vivo Effects of Discriminator Sequences on Transcription Initiation in Mediator clusters. A rank-ordered size distribution of Mediator clusters was E. coli similar to a rank-ordered distribution of Mediator ChIP-seq data. By orthogonal Kevin Lauterjung1, Xiangyang Liu1, Kate Henderson2, Vatsan Raman2,3, labeling of Pol II we could observe that stable Mediator clusters co-localize Tom Record2,4. with stable Pol II clusters, and the enhancer associated transcription factors 1Program in Biophysics, University of Wisconsin Madison, Madison, WI, Sox2 and Oct3/4 in live mESCs. Using Lattice Light Sheet Microscopy we USA, 2Department of Biochemistry, University of Wisconsin Madison, directly observed Mediator and Pol II clusters at high temporal resolution Madison, WI, USA, 3Department of Bacteriology, University of Wisconsin and followed their 3D dynamics over extended periods of time. Upon incuba- Madison, Madison, WI, USA, 4Department of Chemistry, University of tion with the BRD4 inhibitor JQ1, known to dissolve super-enhancer signatures Wisconsin Madison, Madison, WI, USA. in ChIP-seq data, stable Mediator clusters disappeared. Based on our data we A major unresolved question in the mechanism of transcription initiation by the hypothesize that super-enhancer domains with high Mediator signal observed ’ 70 multi-subunit Escherichia coli RNA polymerase (RNAP, subunits a2bb us ) in ChIP-seq result from interactions of chromatin stretches with the large pro- is the relevance of the wide range of open complex (OC) lifetimes exhibited by tein clusters observed in this study. Co-localization of many other key tran- different promoters. OC lifetimes for promoters lPR, T7A1, and rrnB P1 show scription factors, namely Pol II, Sox2, and Oct3/4, suggests that the entities 5 OC lifetimes range from >10 s(lPR) to less than 1 s (rrnB P1). An elaborate observed here play an important role in the transcriptional program of mESC. network of in-cleft and downstream interactions directed by the discriminator region—just upstream of the transcription start site—is involved in lifetime 1253-Pos Board B162 determination. We recently presented evidence that longer-lived, more stable Microarray Analysis Reveals Deregulated LNCRNAS and MRNAS in DB/ OC synthesize a longer RNA-DNA hybrid before escape. Consequently, DB Mice Plasma and Heart: Diagnostic Biomarkers of Diabetic Cardiomy- we’ve seen a wider length range of short (abortive) RNA from longer-lived, opathy more stable open complexes, while few short RNA of any length are synthe- Tarun Pant. sized from short lived OC under conditions investigated. It is unclear if these Anesthesiology, Research, Medical College of Wisconsin, Milwaukee, WI, differences in short RNA distribution are the only consequence of the large dif- USA. ferences in OC lifetime and stability, and the roles of these short RNAs in regu- Introduction: Diabetic cardiomyopathy (DCM) is one of the independent risk lation of gene expression are also largely unknown. factors for cardiovascular diseases and heart failure. LncRNA are 200 or more My research focuses on in vivo effects of discriminator sequence and length nucleotide long with no protein coding capacity. In this study, we evaluated variation by utilizing synthetic plasmid libraries, promoting a super folder differentially expressed LncRNAs and mRNAs in both plasma and heart sam- green fluorescent protein (sfGFP) gene, and with upstream promoter sequences ples Spectrum of LncRNA appears to depict different level of expression in tis- sue and blood of patients with diabetes having biomarker potential. homologous to either lPR, T7A1, or rrnB P1. E. coli cells transformed with these libraries are measured for sfGFP expression via flow cytometry, sorted Methods: Microarray profiling was performed on total RNA isolated from by expression level using fluorescence-activated cell sorting (FACS), and cells heart and plasma of biological replicates (n=12) of 10-12 week male Wild þ of each expression level queried for discriminator consensuses using Illumina Type Young (C57BLKS/J- db/ )vs diabetic-Young (C57BLKS/J-db/db) and þ sequencing. Our sequence findings, with in vitro studies using consensus se- 18-20 week age male Wild Type old (C57BLKS/J- db/ )vs diabetic quences, will enable us to elucidate regulatory effects of the discriminator re- old(C57BLKS/J-db/db) mice. Left ventricular (LV) parameters were measured gion, getting us closer to the mechanistic explanation for the large range of OC by a Visual Sonic echocardiography. formation in bacteria. Results: Total numbers of LncRNAs and mRNA identified in heart sample profiling were 23,578 and 20,124, respectively, whereas plasma samples pro- 1251-Pos Board B160 filed 17,124 LncRNA and 13,333 mRNA genes. Wild Type Young Using Minimal Synthetic Enhancers to Reach a Predictive Understanding (C57BLKS/J- db/þ) mice vs diabetic-Young (C57BLKS/J-db/db) total differ- of Transcriptional Regulation in Development entially expressed (DE; adjusted p –value<0.05) LncRNAs in heart and plasma Myron Child1, Armando Reimer2, Emma Luu3, Hernan Garcia1,3. were 663 and 152 whereas the mRNA expressions in the same order were 447 1Molecular and Cell Biology, University of California Berkeley, Berkeley, and 127. In the Wild Type old (C57BLKS/J- db/þ) mice vs diabetic CA, USA, 2Biophysics Graduate Program, University of California Berkeley, old(C57BLKS/J-db/db) total LncRNA and mRNA deregulated in heart were Berkeley, CA, USA, 3Physics, University of California Berkeley, Berkeley, 816 and 662 whereas the mRNA expression in the same order were 662 and CA, USA. 2580. In the past few decades, developmental biologists have made tremendous prog- Conclusion: Our comprehensive profiling of heart and plasma samples reveals ress identifying enhancers and binding motifs for transcription factors that drive high overlap with LncRNAs and mRNAs DE in heart and plasma within each the expression of genes responsible for cell fate decisions. Despite this prog- group indicating that DE LncRNA and mRNA within the plasma reflects ress, we are still incapable of determining mRNA transcript copy numbers LncRNA and mRNA changes in the heart tissue. Further studies will be given a regulatory DNA architecture and transcription factor concentrations. required to resolve which of these overlapping biomarkers can serve as a potent Recently, several genetic and microscopy tools have been developed that therapeutic target for attenuating DCM. make it possible to engage in such dialogue between theory and experiment in the context of living embryos of the fruit fly Drosophila melanogaster. 1254-Pos Board B163 We use thermodynamic models to predict how the placement, number, and af- The Role of the CBP Transcriptional Activator in the Circadian Repres- finity of binding sites for transcription factors govern transcriptional dynamics sive Complex in the embryo. To experimentally test the predictions stemming from these Efrain H. Ceh Pavia, Jennifer Fribourgh, Leslee Nguyen, Carrie L. Partch. models, we designed a minimal gene cassette that serves as a scaffold for sys- University of Santa Cruz, Santa Cruz, CA, USA. tematically modulating regulatory parameters. Our gene construct includes Circadian clocks are endogenous oscillators that control our 24-hour physio- MS2 bacteriophage stem loop sequences to report on gene expression from logical processes. Nearly every biological function, from hormone release to an enhancer with one or more copies of the Bicoid activator and Zelda co- metabolic homeostasis, is linked to these internal clocks. As an autoregulatory activator binding motifs. Our experiments show that the number and affinity system with a negative feedback loop, the circadian clock goes through of binding motifs modulate the probability that nuclei initiate transcription different activator/repressor complexes throughout its 24-hour cycle. Active and their rate of polymerase recruitment in a manner consistent with equilib- transcription occurs during the day, while gene expression is generally rium thermodynamic models. repressed at nighttime. There are two repressive complexes, the early complex (after dusk) composed of Per1, Per2, Cry1 and Cry2 bound to the Clock- 1252-Pos Board B161 Bmal1 transcription factor, and the late complex (dawn) that has only Cry1 Mediator Forms Clusters with RNA Polymerase II in Live Stem Cells interacting with Clock-Bmal1. In the transition between these two repressive Won-Ki Cho, Jan-Hendrik Spille, Micca Hecht, Choongman Lee, states, the CREB-binding protein (CBP) can also found at complexes of clock Ibrahim Cisse. proteins through an interaction with Per2. CBP is generally considered a tran- MIT, Cambridge, MA, USA. scriptional activator that acetylates histones, giving them a tag that signals the Transcription in eukaryotic cells is regulated not only by promoters but also start of transcription. So what is a transcriptional activator doing as part of a enhancer elements. Mediator is the key transcription factor that links enhancers repressive complex? Here we corroborate the Per2-CBP interaction and take it and promoters. Here, we labelled endogenous Mediator using CRISPR/Cas9 a step further by identifying the Bromo-PHD-HAT domains of CBP as impor- gene editing in mouse embryonic stem cells (mESCs). Using live cell super- tant for binding. Furthermore, we identified the Per2 C-terminal domain as resolution imaging we observed that Mediator forms transient sub-diffractive essential for its interaction with CBP. This study opens the door to further

BPJ 8617_8620 Monday, February 19, 2018 249a analyze the role of the CBP-Per2 interaction in keeping the circadian clock affect the strength of a roadblock, we compare LacI roadblocks with those ticking. of l bacteriophage CI. The results provide insight into how transcription fac- tors generate complex responses in genomic contexts. 1255-Pos Board B164 Transcriptional Dynamics and the Regulation of Chromatin Accessibility 1257-Pos Board B166 in Drosophila Embryonic Development Uncovering Dynamical Mechanisms of Transcription Factor Readout in Elizabeth Eck, Jonathan Liu, Hernan Garcia. Development using Optogenetics University of California, Berkeley, Berkeley, CA, USA. Simon Alamos1, Yang Joon Kim2, Jordan Y. Xiao3, Elizabeth Earley4, The genetic regulatory code is determined by more than the arrangement of Hernan G. Garcia5. transcription factor binding sites on the genome. Epigenetic regulation is 1PMB, UC Berkeley, Union City, CA, USA, 2Biophysics, UC Berkeley, known to play a key role in determining whether binding sites are accessible Union City, CA, USA, 3Physics, UC Berkeley, Berkeley, CA, USA, 4MCB, to transcription factors. Therefore, both cis regulatory architecture and accessi- UC Berkeley, Berkeley, CA, USA, 5MCB, Physics, UC Berkeley, Berkeley, bility must be considered to achieve a predictive understanding of transcrip- CA, USA. tional regulation. Embryonic development depends on the precise regulation of gene expression In the early embryo of the fruit fly Drosophila melanogaster, the transcription by transcription factors (TFs), whose concentration changes rapidly in space factor Zelda has been proposed to mediate transcriptional activation by facili- and time as they lay out the adult body plan. A widespread assumption is tating chromatin accessibility. Zelda’s role in the activation of hunchback, one that instantaneous TF input concentration dictates output mRNA production. of the most widely studied genes in developmental biology, provides an oppor- However, recent studies in various organisms have challenged this readout tunity to uncover the quantitative interplay between the regulation of accessi- mechanism by demonstrating that the frequency, derivative, and fold-change bility and gene expression dynamics in development. Currently, the of TF concentration can also provide regulatory information. To put these consequences of Zelda’s regulatory actions remain unclear, as does its effects different scenarios to test, it is necessary to manipulate the dynamics of the on gene expression dynamics. For example, does Zelda-mediated access to input TF at will. We established the optogenetic control of Bicoid activator DNA modulate the rate of polymerase loading at promoters or dictate the prob- concentration dynamics. Bicoid, one of the most widely studied transcription ability of promoters turning on? factors in development, was fused to the LEXY optogenetic tag and the mCi- To reveal the molecular consequences of the regulation of chromatin accessi- trine fluorescent protein using CRISPR/Cas9-mediated homologous recombi- bility on development, we use novel technologies to image real time transcrip- nation. Upon illumination with blue light, LEXY is activated, leading to the tional dynamics of a hunchback reporter in living embryos. By examining zelda nuclear export of Bicoid molecules. By combining this optogenetic system mutant flies, we find that the loss of Zelda has no effect on polymerase loading with the MS2 system to measure transcriptional activity, we show that we rates at the promoter. Contrastingly, these mutants presented a substantial delay can independently control and measure Bicoid concentration dynamics and in transcriptional onset after mitosis. Thus, while Zelda does not affect the rate its resulting effect on the transcription of a reporter gene. We utilized this of transcriptional initiation, it does play a role in determining the proper timing tool to study how Bicoid concentration is read out in time by the hunchback of transcription. We present a theoretical Monod-Wyman-Changeux based P2 enhancer. Here, we present our progress towards understanding the temporal model aimed at explaining the interplay between chromatin accessibility, tran- readout mechanism of TF concentration in development, with the ultimate scription factor binding, and output gene expression. Our model makes exper- objective of deriving theoretical models that predict output mRNA dynamics imentally testable predictions about how the number, placement, and affinity of from knowledge of the input TF concentration. Zelda binding sites within enhancers dictate transcriptional dynamics. 1258-Pos Board B167 1256-Pos Board B165 Determination of Muscle-Specific Structural Variations of Titin Based on Roadblocks by Protein-Mediated DNA Loops mRNA Transcript Analysis Zsuzsanna Vo¨ro¨s, Cristin R. Hendrickson, David Dunlap, Laura Finzi. Pabodha Hettige1, Uzma Tahir2, Kiisa C. Nishikawa3, Matthew J. Gage1. Department of Physics, Emory University, Atlanta, GA, USA. 1Department of Chemistry, University of Massachusetts Lowell, Lowell, RNA polymerase elongation during transcription may be hindered by the MA, USA, 2Department of Biological Sciences, Northern Arizona many proteins bound to DNA (roadblocks). Conversely, RNA polymerase University, Flagstaff, AZ, USA, 3Center for Bioengineering Innovation, might displace a roadblocking transcription factor (TF) and compromise regu- Northern Arizona University, Flagstaff, AZ, USA. lation. The mechanism by which an RNA polymerase might elongate past Titin is a giant muscle protein that plays an important role in both sarcomere DNA-bound proteins without disturbing with their regulatory functions is assembly and passive muscle contraction. The titin gene contains 363 exons poorly understood. Many TFs from organisms spanning all kingdoms recog- in the human genome while the coding region of mouse titin has 347 exons. nize specific sites on DNA to shape the genome and regulate transcription The function of titin protein is tuned by the variation of exon composition. and, unlike nucleosomes, are not targets for combinatorial chemical modifica- This single gene is capable of generating hundreds of isoforms, but a limited tions that impact complex pathways. Instead, their regulatory effects stem number of isoforms have been shown to exist and the exon composition of these from the presence of multiple operators to which they bind with different af- many isoforms remains poorly characterized. Various splicing-related proteins finities and/or cooperatively. Such interactions often modify DNA topology, and small regulatory RNAs work together to produce muscle specific expres- such as DNA looping, but little is known about how topology affects tran- sion variations in titin. Muscular dystrophy with myositis (mdm) is caused by scriptional roadblocks either in vivo or in vitro. Recently we used scanning a homozygous deletion at the N2A/PEVK junction of titin, which leads to an force microscopy (SFM), to assay how the presence of a DNA loop affects 83 amino acid deletion. Using RNA-Seq and PCR techniques, we investigated elongation by RNAP through a lac repressor tetramer (LacI) roadblock. alterations in titin splicing in extensor digitorum longus (EDL) muscles from LacI oligomerizes to form a tetramer with two DNA binding domains and wild type and mdm mice. Among other differences, our results demonstrate is a strong roadblock to transcription elongation even when bound to just that expression of exons coding for the 4th to 6th Z-repeats is altered in EDL one binding site (operator). However, it can also mediate DNA looping be- muscles from mdm mice. While both wild type and mdm muscles express the tween two separate operator sites. We found that the strength of a roadblock same four splice variants, EDL muscles from mdm mice contain higher levels depends on the strength of the first operator encountered by RNAP and of the larger isoforms compared to wild type muscles. We are currently work- whether or that operator forms one end of a LacI-mediated DNA loop. To ing to elucidate the regulatory mechanisms that induce this shift in splicing be- assess how higher order TF oligomerization and operator multiplicity might tween wild-type and mdm muscles.

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Posters: Protein-Nucleic Acid Interactions II CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is a bacterial immune system, which has revolutionized life sciences through the 1259-Pos Board B168 introduction of a facile genome editing technology. In this system, the endonu- Investigating the Mechanism of DNA Recognition by a CRISPR-Cas12a clease Cas9 associates with guide RNAs to match and cleave complementary Nuclease DNA sequences, forming an RNA:DNA hybrid and a displaced non-target Wei Jiang. DNA strand. Thanks to a key DNA recognition element, a Protospacer Adja- Chemistry, University of Southern California, Los Angeles, CA, USA. cent Motif (PAM), Cas9 performs site-specific DNA cleavages, enabling to en- Clustered-Regularly-Interspersed-Short-Palindromic-Repeats (CRISPR) and gineer biological systems with unique efficiency and resulting in numerous CRISPR-associated (Cas) proteins provide an efficient means to target genomic applications in medicine and biotechnology. However, the mechanistic basis DNA sequences in a large variety of organisms, leading to a revolution in underlying the CRISPR-Cas9 function is unclear, preventing rational engineer- genome engineering. Both the class 2 type II Cas9, which has been widely ing of the system toward improved genome editing. We report on extensive mo- used in genome editing, and the recently discovered class 2 type V Cas12a lecular dynamics studies, including multi-microsecond and accelerated (Cpf1), use an effector complex comprising of a single protein activated by methods probing displacements over micro-to-milliseconds, characterizing CRISPR-encoded small RNA(s) (crRNA) to cleave double-stranded DNA at the mechanism of conformational activation of Cas9, from its apo form up to specific sites. However, Cas12a differs from Cas9 in key features of effector the nucleic acid binding. These simulations disclosed a mechanism for RNA protein, crRNA, and elements of recognition in target DNA, and has been re- recruitment in which the domain relocations cause the formation of a positively ported to show a higher degree of discrimination against mis-matches between charged cavity for nucleic acid binding. As well, we revealed the formation of a the guide RNA and the DNA. To elucidate the mechanism of DNA recognition catalytically competent Cas9, which is prone for the catalysis of DNA. We by Cas12a, which may underlie its enhanced substrate specificity, we are using showed how, upon DNA binding, the relocation of the catalytic HNH domain, a combination of cleavage, binding and competition assays to examine Cas12a assisted by conformational changes of the non-target DNA strand, activates recognition a variety of DNA constructs. The data showed for topologically Cas9 for catalysis. Finally, a mechanism of allosteric regulation, triggered by flexible DNA substrates, including those containing unpaired protospacer seg- PAM binding and activating the catalytic domains for concerted catalysis has ments, were bound at the cognate Cas12a active site even in the absence of pair- been discovered. Overall, our outcomes address the lack of mechanistic infor- ing between the RNA guide and the DNA target strand. However, these mation on CRISPR-Cas9, improving our understanding of the activation pro- substrates were not cleaved by the enzyme, suggesting that Cas12a employs cess. This information is critical for structure-based design of the CRISPR- proof-reading mechanisms subsequent to initial DNA binding to ensure proper Cas9 system, which could impact the development of improved genome editing cleavage. The results have implications on the mechanisms of Cas12a DNA tools. recognition, as well as in applications that rely on CRISPR binding to DNA, such as transcription regulation and base editing. 1262-Pos Board B171 Correlated Single Molecule Twist and Fluorescence Measurements on 1260-Pos Board B169 CRISPR/Cas Systems Duplex DNA Destabilization by Type V CRISPR-Cas Nucleases during Pierre Aldag1, Julene Madariaga Marcos2, Inga Songailiene3, Interrogation of DNA Felix Kemmerich1, Virginijus Siksnys3, Ralf Seidel1. Vladimir Mekler1, Leonid Minakhin1, Konstantin Kuznedelov1, 1Molecular Biophysics, University of Leipzig, Leipzig, Germany, 2Molecular Karthik Murugan2, Dipali Sashital2, Konstantin Severinov1,3. Biophysics, Centro Nacional de Biotecnologia, Madrid, Spain, 3Protein-DNA 1Waksman Institute, Rutgers University, Piscataway, NJ, USA, Interactions, Vilnius University, Vilnius, Lithuania. 2Department of Biochemistry, Biophysics & Molecular Biology, CRISPR/Cas systems are RNA-guided ribonucleoprotein (RNP) complexes Iowa State University, Ames, IA, USA, 3Institutes of Molecular Genetics with nuclease activity that provide prokaryotes with an adaptive defense mech- and Gene Biology, Russian Academy of Sciences, Moscow, Russian anism against foreign nucleic acids. The RNP complex recognizes complemen- Federation. tary target sites by base-pairing its RNA component with one of the strands of The prokaryotic CRISPR-Cas effector complexes locate their target sites by the target DNA while displacing the other one forming a so-called R-loop struc- scanning and interrogating the genomic DNA. The type II and type V ture. After completion of the R-loop formation, the target DNA is cleaved by a CRISPR-Cas nucleases cleave double-stranded DNA bearing sequences com- nuclease. However, CRISPR/Cas systems have shown to be highly promiscu- plementary to guide segments of CRISPR RNAs (crRNA) flanked by conserved ous during target selection allowing multiple mismatches to be tolerated be- PAM sequences; they have been successfully used for genome editing in various tween RNA and target DNA. Considering the vast potential of CRISPR/Cas organisms. Unpairing of a short PAM-proximal segment of DNA protospacer is systems in gene editing technology, it is crucial to fully understand the mech- a critical event that may determine the overall rate of target location. We sought anism behind the targeting process by these enzymes. Recently, we employed to elucidate mechanistic details of initial DNA interrogation steps by type V single molecule supercoiling measurements within magnetic tweezers to CRISPR-Cas nucleases Cas12a and Cas12b (also known as Cpf1 and C2c1, resolve the dynamics of R-loop formation by the Type I CRISPR/Cas system respectively). Using fluorometric and biochemical assays, we studied interac- Cascade in presence of mismatches in the target sequence. This revealed that tions of Cas12a and Cas12b effectors with a set of model DNA substrates that R-loop formation uses a sequential zipping process that initiates at the PAM mimic likely early intermediates on the pathway to the final R-loop complexes. motif upstream of the target. When encountering a mismatch, R-loop formation Consistent with reported data on the mechanism of DNA interrogation by type II stalls, followed by collapse or overcoming of the mismatch and eventually the Cas9 nuclease, we find that binding of type V effectors to PAM favors separation completion of the full R-loop. So far these experiments probed exclusively R- of several adjacent to PAM DNA protospacer base pairs even in the absence of loop formation without providing information on the actual binding of the pro- pairing between the crRNA guide and the protospacer. The separation is medi- tein and its conformational changes. Here we employ a combined magnetic ated in part by effector affinity for non-target DNA strand nucleotides in a tweezers and total internal reflection fluorescence microscopy setup to carry single-stranded conformation. In contrast, effector interactions with the nearest out correlated single-molecule force and fluorescence spectroscopy measure- to PAM target DNA strand nucleotides are energetically unfavorable in the type ments. Using fluorescently-labelled Cascade complexes we are able to follow V early intermediate complexes, presumably due to a steric clash between the association and dissociation events prior to the actual R-loop formation. These protein and DNA backbones. We suggest that these unfavorable interactions measurements reveal important details about the target recognition mecha- destabilize the DNA duplex and thus facilitate the DNA interrogation process. nisms by CRISR-Cas enzymes and allow to test emerging biophysical models We discuss the similarities and differences between the mechanisms of DNA of this process. interrogation by Cas9, Cas12a and Cas12b effectors. 1263-Pos Board B172 1261-Pos Board B170 Real-Time Observation of Target Search by the CRISPR Surveillance A PAM-Induced Signalling Activates the Communication between HNH Complex Cascade and RUVC in CRISPR-Cas9 Chaoyou Xue1, Yicheng Zhu1, Xiangmei Zhang2, Yeon-Kyun Shin1, Giulia Palermo1, Clarisse Ricci2, Yinglong Miao2, Martin Jinek3, Dipali G. Sashital1. J. Andrew McCammon2. 1 1 Department of Biochemistry, Biophysics and Molecular Biology, Iowa State Swiss National Science Foundation (SNSF) Advanced Post-Doctoral 2 University, Ames, IA, USA, Department of Statistics, Iowa State University, Fellow, University of California at San Diego, La Jolla, CA, USA, 2 3 Ames, IA, USA. University of California San Diego, La Jolla, CA, USA, University of CRISPR-Cas systems defend bacteria and archaea against infection by Zurich, Zurich, Switzerland. bacteriophage and other threats. The central component of these systems

BPJ 8621_8624 Monday, February 19, 2018 251a are surveillance complexes that use guide RNAs to bind specific regions through an induced-fit mechanism as the product of the equilibrium binding af- of foreign nucleic acids, marking them for destruction. Surveillance com- finity for DNA and the rate of R-loop formation. plexes must search a vast amount of DNA to rapidly locate targets and ensure timely immune response. A common strategy is to first search 1266-Pos Board B175 for short protospacer adjacent motif (PAM) recognition sequences located Exploring Mechanisms of Site-Specific DNA Cleavage with Single Mole- next to the target. It remains unclear how surveillance complexes can cule Sensitivity efficiently interrogate abundant potential PAM sites to find the lone Sadie Piatt, Allen Price, Stephen Parziale, Raquel Ferreira. target. Here, we used single-molecule FRET to visualize how the Type I- Emmanuel College, Boston, MA, USA. E surveillance complex Cascade searches DNA in real time. Using this sys- We use a variety of methods to study mechanisms of DNA cleavage using tem, we directly observe that Cascade rapidly and randomly samples DNA restriction endonucleases as a model system. In a single molecule technique, through nonspecific electrostatic contacts. Search kinetics are slowed signif- micron-sized beads are tethered with single DNAs in a microfluidic flow cell icantly in the presence of PAM sequences, and Cascade localizes at the and imaged using video microscopy. We have used this assay to study facil- PAM sites enabling interrogation of the adjacent sequence. We identify itated diffusion as well as the role of divalent cations. We have characterized several protein motifs within Cascade that are essential for either nonspe- the salt dependence of both NdeI and EcoRI and found that both enzymes cific sampling or positioning and readout of the PAM. Our findings exhibit a peak in cleavage rates near 80 mM, an observation consistent provide a comprehensive structural and kinetic model for the Cascade with the theory of facilitated diffusion. We are currently studying how target-search mechanism, revealing how CRISPR surveillance complexes DNA length affects the reaction rates and are studying both 1000 bp can rapidly search large amounts of genetic material en route to target DNAs and 200 bp DNAs. We are comparing our single molecule technique recognition. to ensemble assays which use quantitative analysis of gel electrophoresis. Preliminary data shows cleavage to be much slower in the gel assay as compared to the single molecule data, suggesting the two assays are limited 1264-Pos Board B173 by different steps. Our efforts are focused on improving this ensemble assay. Biophysical Analysis of Cas9 - DNA Interactions and Enzymatic Activity We are also developing single-molecule fluorescence methods to observe the with Electro-Switchable DNA Layers diffusion of EcoRI on DNA. In our technique, lambda phage DNA which Felix J. Kroener1,2, Ulrich Rant2. has been linearized and biotinylated on both ends is extended and tethered 1Technical University Dresden, Munchen,€ Germany, 2Dynamic Biosensors, to a surface. Using HiLo imaging, we have observed the diffusion and bind- Munchen,€ Germany. ing of catalytically inactive mutant EcoRI labeled with a quantum dot. We The analysis of the biophysical properties of Cas9 - DNA interactions is hope to use this method to directly observe DNA sliding by EcoRI. Our of fundamental importance for the development of new CRISPR/Cas9 work is currently focused on optimizing our method used to track enzyme applications. However, conventional technologies like SPR or BLI from search activity. inherent limitations and are prone to measurement artifacts.Here we present binding and enzymatic measurements with switchSENSE, a novel 1267-Pos Board B176 type of biosensor that utilizes electro-switchable DNA layers. The system Spatial Organization and Dynamics of RNA Processing in Caulobacter combines fluorescence with molecular dynamics measurements. For Crescentus the latter, DNA strands with a Cas9 target sequence are driven to oscillate Camille Bayas1, Jiarui Wang1, Marissa K. Lee1, Jared M. Schrader2, by alternating electric potentials on gold surface. Changes in DNA Lucy Shapiro2, W.E. Moerner1. motion upon Cas9 binding are monitored in real-time without the necessity 1Chemistry, Stanford University, Stanford, CA, USA, 2Developmental of Heparin to suppress unspecific binding.We present an assay to extract Biology, Stanford University, Stanford, CA, USA. binding kinetic rate constants of Cas9, as well as its cutting efficiency in RNA processing, specifically the regulation of RNA degradation and cleavage a single experiment on target and off-target DNA. We elucidate caveats and translation, is an integral part of gene expression. In bacteria, the lack of a > 1 1 in the measurements of very fast association rates kON 1E6 M s as membrane-enclosed nucleoid has suggested that these processes occur < 1 well as very slow dissociation rates kOFF 1E-5 s ,andshowexamples throughout the cytoplasm of the cell. However, due to the small size of bac- that expose the importance of controlling the ligand density on the biosensor teria and the diffraction limit, obtaining precise information on the location of surface in order to avoid measurement artifacts. Additionally, we present a biomolecules through the use of conventional fluorescence imaging is diffi- standardized workflow which allows efficient screening of sequence- cult. The conventional resolution of optical microscopes (200 nm) can be variations by hybridization of unlabeled target DNAs to a labeled standard circumvented by the time-sequential super-localization of single-molecule anchor-DNA. We compare different mismatch influences on binding of fluorescent labels, resulting in 5x resolution enhancement. We applied gRNA programmed Cas9 to immobilized DNA sequences and discuss ef- super-resolution (SR) and single particle tracking (SPT) techniques, in combi- fects on the association and dissociation rate constants, respectively.More- nation with a double-helix point spread function microscope, to study the over, we assessed the influence of Heparin on specific and unspecific three-dimensional (3D) spatial organization and dynamics of RNase E and ri- binding of Cas9 to its target sequence at different concentrations of Heparin bosomes in Caulobacter crescentus. We report the first 3D SR images and 3D and Cas9. We found that while Heparin suppresses unspecific binding of SPT of RNase E and ribosomes in Caulobacter. Our SR images show RNase m Cas9, at a concentration of 30 g/ml, the specific binding reduced to less E molecules strongly clustered in the cytoplasm of the cell, and ribosomes than 1%. weakly clustered throughout the nucleoid, while our SPT results show confinement of both RNase E and ribosomes. When transcription is inhibited, 1265-Pos Board B174 we find a more diffuse distribution, as well as a decrease in confinement, for DNA Unwinding is the Primary Determinant of CRISPR-Cas9 Specificity both RNase E and ribosomes. This suggests that active RNA processing and Shanzhong Gong, Helen H. Yu, Kenneth A. Johnson, David W. Taylor. activity, either RNA cleavage in the case of RNase E or translation in the case Molecular Bioscience, The University of Texas at Austin, Austin, TX, USA. of ribosomes, facilitates clustering and confinement. Together, our results sug- Bacterial adaptive immunity utilizes RNA-guided surveillance complexes gest that RNA processing is spatially organized within C. crescentus, and that composed of CRISPR (clustered regularly interspaced short palindromic this organization is related to the function of RNase E and ribosomes in RNA repeats)-associated (Cas) proteins together with CRISPR RNAs (crRNAs) to processing. target foreign nucleic acids for destruction. Cas9, a type II CRISPR-Cas effector complex, can be programed with a single guide RNA that base-pairs 1268-Pos Board B177 with the target strand of dsDNA, displacing the non-target strand to create an Global Landscapes of Protein-RNA Recognition Provide Quantitative R-loop, where the HNH and RuvC nuclease domains can cleave opposing Tools to Predict and Engineer Specificity in RNA Structured Elements strands. Cas9 has been repurposed for a variety of important genome engineer- Faruck Morcos, Qin Zhou, Zachary Campbell. ing applications. While many structural and biochemical studies have shed light Biological Sciences, University of Texas at Dallas, Richardson, TX, USA. on the mechanism of Cas9 cleavage, a clear unifying model has yet to emerge. The role of RNA structured elements is fundamental in biology. They help A detailed kinetic characterization of the enzyme reveals that DNA binding is build a network of regulatory interactions that has effects on the most important reversible, R-loop formation is rate-limiting, occurring in two steps, one for processes in the cell such as transcription, translation and mRNA decay. A rele- each of the nuclease domains. Although the HNH nuclease activity is stimu- vant biological phenomenon is the molecular interaction occurring between 2þ lated by Mg with a single measureable Kd, the RuvC activity requires two proteins and these RNA elements. Although such interactions have been exten- distinct Mg2þ binding events. The specificity constant for cleavage determined sively studied, the question of how proteins preferentially interact with

BPJ 8621_8624 252a Monday, February 19, 2018 different sequences but similar structures is still unresolved. Collections of in the RNA for modulating gene transcription. This work provides a basis for an known binding elements are insufficient to characterize the spectrum of poten- essential event for understanding transcription antitermination in pneumovi- tial mutations that contribute to functional RNA molecules. In this work, we ruses and is counterpart Ebola virus VP30. developed an integrated framework based on in vitro selection, high- throughput sequencing and global probabilistic modeling to quantify the land- 1271-Pos Board B180 scapes of protein-RNA recognition. This approach allowed us to characterize Solvent Accessibility of CRISPR-CAS9 Target DNA is Correlated with the way that sequence and structural elements confer RNA binding recognition Substrate Specificity to proteins P22N, 1N and BIV TAT. The parameters of our global model allow Travis H. Hand, Anuska Das, Emily Duboy, Mitchell Roth, us to discern the most important nucleotide sequence interactions that Chardasia Smith, Uriel Baptist, Hong Li. contribute to recognition. By creating a quantitative metric based on such Molecular Biophysics, Florida State University, Tallahassee, FL, USA. parameter inference, we are able to discriminate between regulated and non- Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)- asso- regulated elements in their genomic context as well as the design of functional ciated protein 9 (Cas9) is an RNA-guided DNA endonuclease that binds the variants that preserve or enhance specificity. We are able to verify such predic- substrate via base-pairing interactions between a guide RNA and a complemen- tions experimentally and use this framework to quantify pathways that reveal tary target sequence of DNA. Cas9 can be reprogrammed to target different permissive/disruptive evolutionary trajectories. Although we show our results sites with relative ease, making it a versatile tool in genome editing and ther- for a finite number of protein-RNA systems, our approach is broadly applicable apeutic applications. However, unwanted activities at different, but closely and easily transferable. Our framework provides a detailed characterization of related off-target DNA sequences remains a major barrier to the more wide- protein-RNA recognition landscapes with potential applications in unexplored spread application of Cas9 technologies. Innovative strategies are, therefore, systems. needed to improve the specificity of Cas9. We analyzed currently available Cas9 crystal structures and found a correlation between the buried solvent accessible area and tolerance to mismatches-as measured by Cas9 binding 1269-Pos Board B178 kinetics-in the target DNA. Positions with greater solvent exposure have a In Vitro Reconstruction and Analysis of Eukaryotic Catalytic Ribonucleo- greater sensitivity to mismatches, suggesting an impact of solvent in RNA- protein RNase P guided targeting specificity. Experimentally, our in vitro and in vivo tests Anna Perederina, Igor Berezin, Andrey S. Krasilnikov. with the previously characterized Acidothermus cellulolyticus Cas9 (AceCas9) Biochemistry and Molecular Biology, Penn State University, University on mismatched substrates showed a good agreement with this model. These re- Park, PA, USA. sults suggest that increasing solvent exposure of the bound target could be a RNase P is a ubiquitous ribonucleoprotein (RNP) complex primarily respon- general strategy for improving the specificity of Cas9. sible for the maturation of 5’-ends of tRNA. RNase P is an unusual enzyme as it relies on its RNA moiety for catalysis. In the course of evolution, RNase 1272-Pos Board B181 P retained the core elements of its catalytic RNA, but the protein part grew pro- Interaction between Capsid Coat Protein and MS2 Bacteriophage SSRNA gressively more complex. While bacterial RNase P is a two-component RNP with Different Loop Motif for Virus Assembly Process where the catalytic RNA is aided by a single small protein, in the eukaryotic Lokendra Poudel, Wai-Yim Ching. enzyme, a simplified version of this RNA is decorated by multiple (9-10) essen- Physics and Astronomy, UMKC, Kansas City, MO, USA. tial proteins. In MS2 bacteriophage single-stranded RNA virus, the genomic RNA plays vital The composition of eukaryotic RNase P has been established about two de- roles in the virus assembly process. Multiple dispersed RNA sequence/structure cades ago; however, the structural and functional characterization of this large motifs are central recognition motif to specific interact with capsid protein in an catalytic RNP proved to be very challenging. RNase P is a low-abundance assembly process referred to as packaging signal (PS) mediated assembly. The essential enzyme, and most of the progress in our understanding of the bacterial identification of these PSs was based on bioinformatics, geometric approaches and archaeal RNases P was achieved using enzymes reconstructed from their and sophisticated experimental protocols. We deal this problem by an alto- components in vitro. At the same time, previous attempts to reconstruct eukary- gether different approach, using a large-scale quantitative ab initio methodol- otic RNase P in vitro were not successful, mainly due to insolubility of its pro- ogy centered on critical aspects of the consensus PS recognition motif. DFT tein components. As the result, eukaryotic RNase P remained understudied and calculations are carried out on four nucleoprotein complexes (PDB IDs: poorly understood. 1ZDI, 1ZDH, 5MSF and 6MSF respectively) that are representative of the Here, we present the results of successful in vitro reconstruction of a catalyti- phage MS2 PSs. The calculated partial charge distribution of individual protein cally active S. cerevisiae RNase P RNP and its biochemical analysis. In our and RNA residues, as well as the calculated strength of the hydrogen bonding quest to reconstruct and analyze eukaryotic RNase P in vitro, we argued that (HB) configurations between them, enabled us to locate the exact binding sites the problematic, insoluble RNase P proteins may act as parts of larger, multi- of both nucleoprotein, corresponding to the sites exhibiting the strongest HBs. component protein subcomplexes. We have identified and isolated these pro- The formation of strong HBs can be traced to the change in the sequence of the tein subcomplexes, and used them to reconstruct and analyze eukaryotic ssRNA, leading to an information for PSs between the amino acids and nucle- RNase P RNP. obases. The specific interatomic HBs between atoms in the amino acids of the Our results eliminate the major obstacle to studies of eukaryotic RNase P, shed coat protein and the ssRNA are explored and quantified. Moreover, NAMD light on the RNA-protein interactions in this RNP, and can serve as a founda- simulation provided quantitative information on the strength of electrostatic in- tion for further analysis. teractions and the change in free energies between the four types of the capsid protein-RNA, exemplifying the role that modern computational techniques can 1270-Pos Board B179 play in further advancing the field of physical virology. Cooperative RNA Recognition by a Viral Transcription Antiterminator Gonzalo de Prat Gay. 1273-Pos Board B182 Fundacio´n Instituto Leloir, Ciudad Autonoma de Buenos Aires, Argentina. Selective Packaging of HIV-1 RNA by GAG Proteins RNA transcription of mononegavirales decreases gradually from the 3’ leader Ioulia F. Rouzina1, Robijn Bruinsma2. promoter towards the 5’ end of the genome, due to a decay in polymerase proc- 1Biochemistry, Ohio State University, Columbus, OH, USA, 2Department of essivity. In the respiratory syncytial virus (RSV) and metapneumovirus, the Physics and Astronomy, University of California, Los Angeles, Los Angeles, M2-1 protein ensures transcription anti-termination. Despite being a homote- CA, USA. tramer, RSV M2-1 binds two molecules of RNA of 13mer or longer per It is not understood how the HIV-1 retrovirus selects it genomic RNA (gRNA) tetramer, and temperature sensitive secondary structure in the RNA ligand is for packaging into virions from among the multitude of cellular RNA mole- unfolded by stoichiometric interaction with M2-1. Fine quantitative analysis cules in the cytoplasm of infected cells. Under in vitro conditions, the Gag pro- shows positive cooperativity, indicative of conformational asymmetry in the teins of HIV-1 have no specific binding affinity for the RNA J sequence, tetramer. RNA binds to M2-1 through a fast bimolecular association followed which has been shown to be the key recognition element of genomic RNA. by slow rearrangements corresponding to an induced-fit mechanism, providing We propose a search mechanism based on the two-state nature of the Gag pro- a sequential description of the time events of cooperativity. The first binding teins in solution. The minimum free energy state is the B (or ‘‘bent’’) state. In event of half of the RNA molecule to one of the sites increases the affinity this state, Gag is non-specifically bound to RNA molecules through the NC and of the second binding event on the adjacent contacting protomer by 15-fold, MA cationic subdomains and mobile. Next, in the E (or ‘‘extended’’) state, only product of increased effective concentration caused by the entropic link. This the NC domain of Gag binds to the RNA molecule that is being searched, while mechanism allows high affinity binding with an otherwise relaxed sequence the MA domain may bind to distant regions of RNA, small RNAs, small mol- specificity, and instead suggests a yet undefined structural recognition signature ecules such as PIP, or the plasma membrane. In the E state, Gag is immobile

BPJ 8621_8624 Monday, February 19, 2018 253a and has an enhanced binding affinity for the J sequence (1,2). In addition, un- 1276-Pos Board B185 like the B-state form E-state Gag has a binding interface for other E-state Gags, Inhibitory Effect of the DNA Tension on the CRISPR/Cas9 Activities thereby allowing stabilization of E state by multimerization and eventual capsid Suleyman Ucuncuoglu1, Kassidy Lundy1, Ozgur Sahin1,2. assembly. Alternation between B and E state Gag allows for an efficient search 1Biological Sciences, Columbia University, New York, NY, USA, 2Physics, mechanism that is similar to that of transcription factor proteins searching DNA Columbia University, New York, NY, USA. (3). (3) Slutsky, et.al. 2004. Kinetics of protein-DNA interaction: facilitated Invention of an adaptive immune system of bacteria and archaea opened a target location in sequence-dependent potential. Biophysical journal, 87(6), new venue for gene editing. Programmable nature of the Cas9 system offers pp.4021-4035. (1)Webb, et.al. Distinct binding interactions of HIV-1 Gag to an easy approach to edit any target DNA by replacing the complementary Psi and non-Psi RNAs: implications for viral genomic RNA packaging. guide RNA. CRISPR/Cas9 technology has already been implemented in a RNA. 2013 (8):pp.1078-88. (2) Comas-Garcia, et.al. 2017. Dissection of spe- wide variety of organisms that include many eukaryotic systems. However, cific binding of HIV-1 Gag to the ‘‘packaging signal’’ in viral RNA. eLife, eukaryotic DNA is usually much longer than the bacterial DNA and it is 6, p.27055. tightly packed inside the nucleus. Hence, DNA is under tension during the extraction for the genetic readout or the compaction for the storage. As an 1274-Pos Board B183 enzyme which mainly evolved in the bacterial systems, essentially nothing Characterizing Protein and RNA Interactions that Nucleate the HIV-1 is known how Cas9 copes with the DNA tension in the eukaryotic systems. Viral Assembly Here, we report that the Cas9 molecules failed to cleave the tensioned Ugonna Mbaekwe, Pengfei Ding, Michael Summers. DNA and the cleavage by Cas9 was restored when the tension was released. University of Maryland, Baltimore County, Baltimore, MD, USA. To observe hundreds of DNA molecules, we developed a single molecule Human immunodeficiency virus type 1 (HIV-1) has become a worldwide assay to apply tension to the target DNA while monitoring the cleavage activ- epidemic. During the HIV life cycle, the viral assembly is initiated by inter- ity. Our observation indicates that the DNA tension can be inhibitory for the action between the unspliced viral genomic RNA (vRNA) and its translated Cas9 targeting and cleavage. Combined with a recent report on the blockage product, the Gag polyprotein. Although there is a large excess of non-viral of Cas9 access by the nucleosomes, our results offer insight into why some RNA, Gag protein is able to efficiently package the vRNA. Gag contains Cas9 targeting attempts fail in eukaryotic cells. These findings might facilitate three domains: Matrix (MA), Capsid (CA), and Nucleocapsid (NC). The better Cas9 targeting strategies in eukaryotic cells and improve the therapeutic CA domain mediates Gag-Gag interaction resulting in the hexagonal Gag applications of CRISPR/Cas9 technology. shell that encapsulates the viral RNA while the NC domain of Gag recog- nizes exposed guanosines in the 50-leader (50-L) region of the vRNA genome 1277-Pos Board B186 for selective packaging. We seek to characterize the Gag-RNA interactions Single Molecule Study of hRap1 Regulated TRF2 Binding Motifs on essential to genome packaging using a truncated 50-L derivative and NC pro- Telomeric dsDNA tein constructs. We hypothesize that the hexameric structure of the CA Xiaodan Zhao1, Vinod Kumar Vogirala2, Meihan Liu1, Yu Zhou1, domain contributes to the RNA genome selection. The basic unit of the Sara Sandin2, Jie Yan3. immature viral shell is a hexamer of Gag; mutations at the hexameric inter- 1The Mechanobiology Institute, Singapore, Singapore, 2School of Biological face significantly reduce Gag’s selectivity towards dimeric viral RNA. Using Sciences, Nanyang Technological University, Singapore, Singapore, Nuclear Magnetic Resonance (NMR), Isothermal Titration Calorimetry 3Physics, National University of Singapore, Singapore, Singapore. (ITC) and electrophoretic mobility shift assays (EMSA) about sixteen bind- The homodimeric TRF2 subunits of shelterin protein complex bind to telo- ing sites for Gag proteins on the core encapsidation signal (CES) have been meric double-stranded DNA (dsDNA) and play an important role to maintain identified which promote the formation of a Gag hexamer. This protein-RNA the genomic integrity. Its function relies on how TRF2 modulates telomere initiation complex is suggested to function as the nucleation site to recruit structure and regulates associated proteins. In vitro studies have been shown more Gag proteins. Other techniques as cryo-electron microscopy are used that TRF2 unwinds dsDNA and promotes T-loop formation, which is sus- to study the structural information about this initiation complex. We aim pected to prevent recognition of chromosome ends as dsDNA breaks. TRF2 to solve the structure of this protein-RNA initiation complex which will pro- is able to recruit human Rap1 (hRap1) that in turn regulates TRF2 compacting vide the detailed molecular mechanism for selective genome packaging. dsDNA properties. Though TRF2 has been shown to induce higher order These studies will further our understanding of the mechanism of HIV structures in DNA, its compaction dependent end protection mechanism has genome selection, a stage in the life cycle that can be targeted with been challenged by latest study of fully assembled shelterin complexes in vi- therapeutics. tro. In order to gain more insights of how TRF2 and TRF2/hRap1 interact te- lomeric dsDNA, we performed single-DNA manipulation studies of TRF2 and 1275-Pos Board B184 TRF2/hRap1 binding to dsDNA under various DNA structural and topological A Flexible Region of PRP8 Interacts with U5 snRNA Loop I and is Func- constraints. We unveiled TRF2 binds to dsDNA with two binding motifs, tionally Linked to 5’ Splice Site Recognition which are dependent on experimental buffer conditions and stoichiometric ra- Andrew MacRae1, Megan Mayerle2, Robert Chalkley2, Melissa Jurica1. 1 2 tio of TRF2 to hRap1. TRF2 binds to torque constrained dsDNA with no UCSC, Santa Cruz, CA, USA, UCSF, San Francisco, CA, USA. chirality preference. The spliceosome is a macromolecular machine that catalyzes the removal of introns from pre-mRNA. Spliceosomes function through a series of 1277.1-Pos Board B186.1 orchestrated structural rearrangements involving dozens of proteins and Determining Nucleocapsid Protein Recognition of Non-canonical Sub- 5 uridine-rich small nuclear RNAs (U snRNAs). The U5 snRNA associated strate Hairpins protein Prp8 is an essential spliceosome scaffold proposed to exhibit mul- Henrietta Ehirim, Melanie Dillon, Catherine Volle. tiple conformations important for structuring the snRNA catalytic core and Biology, Cottey College, Nevada, MO, USA. the pre-mRNA itself. We devised a new lysine probing strategy to The nucleocapsid (NC) protein of HIV binds to and destabilizes the TAR compare human Prp8 lysine reactivity in multiple spliceosome intermedi- hairpin in the HIV genome. Studies have shown that G-containing base pairs ates from pre activation through post-catalysis. From these probing exper- next to a distortion in the duplex are prime sites for NC induced destabilization. iments we identified four conserved Prp8 lysine residues with differential Indeed, the NMR structure of the NC protein bound at the G/C base pair at the probe reactivity in pre-activated versus catalytic and post-catalytic human stem-loop interface, and flipping out the bases in the loop. While the NC protein spliceosomes. In spliceosome structures all four residues are modeled primarily binds to RNA, it can also recognize DNA hairpins. Here, we present within a region of Prp8 surrounding the U5 snRNA stem loop I, a structure basic characterization of NC protein binding to various DNA hairpins previ- important for 5’ exon positioning and splicing catalysis. In catalytic spli- ously shown to form in the eukaryotic genome. ceosomes two of the four lysines stabilize a highly negative groove in U5 snRNA stem loop I. Yeast expressing a Prp8 allele wherein these two ly- Posters: Chromatin and the Nucleoid I sines are mutated to alanine exhibit an intron retention defect and are less able to splice a reporter pre-mRNA with a non-canonical 5’ splice site 1278-Pos Board B187 consensus. Prp8 lysine to alanine mutant yeast genetically interact with al- Artificial Modification of Histones and the Effect on Epigenetics leles of splicing factors Isy1, and Snu114. Based on these genetic interac- Kyrsten M. Thibodeau. tions and catalytic spliceosome structures we propose that Prp8 regulates Bay Path University, Dalton, MA, USA. 5’ exon positioning through an interaction with U5 snRNA stem loop I In order for cells to package all of their genetic material, DNA is wrapped and that disruption of this interaction negatively affects the spliceosome’s around eight histone proteins (two copies each of H2A, H2B, H3, and H4) to ability to recognize the 5’ exon. make up the nucleosome. Nucleosomes are the fundamental building blocks

BPJ 8621_8624 254a Monday, February 19, 2018 of chromatin, which is the first step of compaction and the first level of gene 1281-Pos Board B190 regulation. The phrase ‘‘histone code’’ was coined in regard to those molec- Single Molecule Observation of Chromatin Compaction Reveals the Role ular processes that regulate the eukaryotic genome. Further study of the of Histone Tails and Their Epigenetic Modifications ‘‘histone code’’ and epigenetics has revolutionized the scientific commun- Sohn Byeong-Kwon. ity’s understanding of the regulation of gene expression. The Histone H4 Ulsan National Institute of Science and Technology, Ulsan, Republic of N-terminal tail has a region called the basic patch. It is thought that this re- Korea. gion is a hub for histone-modifying activity. Histone H2B lysine-123 The basic unit of DNA compaction in eukaryotic cells is the nucleosome, (H2BK123) is monoubiquitinated and this modification is required for the which is composed of DNA wrapped around histone octamer (H2A, H2B, proper regulation of transcription. In this study, H2BK123ub levels were H3, H4), and the control of its wrapping-unwrapping, the repositioning of measured and preparations were made to develop a novel amber codon sup- the histone octamer, and the compaction of the nucleosome array is central pression screen to study the histone H4 basic patch. The overarching goal of to the gene regulation mechanism. However, the mechanistic aspect of such the study was to artificially modify histones and their post-translational regulation mechanism is yet to be clearly understood. The nucleosomes modification processes in order to understand their mechanisms and there- dynamically form higher order structure of chromatin fibers to achieve fore better understand epigenetics. more compact state. Cells control the structure of chromatin fibers by intro- ducing isoforms of histone, modifications on histone tails, or change in 1279-Pos Board B188 nucleosome spacing, in order to regulate the accessibility of the DNA Mapping Combinatorial Epigenetic Modifications at Single Nucleosome to various nuclear proteins. Using magnetic tweezers combined with Resolution simultaneous single molecule fluorescence detection, we measured the Jen-Chien Chang1, Kazuhide Watanabe1, Takashi Umehara2, compaction-decompaction dynamics of nucleosome arrays. By controlling Yuichi Taniguchi3, Yuko Sato4, Hiroshi Kimura4, Akiko Minoda1. the DNA sequence composition and spacer length, making tail-deleted his- 1Division of Genomic Technologies, Riken Center for Life Science tone mutants, and also introducing site-specific epigenetic modification to Technologies, Yokohama, Japan, 2Division of Structural and Synthetic the tail of histone H3, we revealed their interplays to control the compaction Biology, Riken Center for Life Science Technologies, Yokohama, Japan, of chromatin and histone unwrapping. Here we present our preliminary re- 3Laboratory for Single Cell Gene Dynamics, Riken Quantitative Biology sults on these attempts. Center, Osaka, Japan, 4Department of Biological Sciences, Tokyo Institute of Technology, Tokyo, Japan. 1282-Pos Board B191 Post-translational modification of histone is one of the most well character- Chromatin Curtains: A Single-Molecule Method for Visualizing Histone ized epigenetic mechanisms that mediates many nuclear processes in the Marks on Chromatin Substrates In Vitro cell. Recent genome-wide techniques such as chromatin immunoprecipita- Liv E. Jensen, Bassem Al-Sady, Sy Redding. tion followed by the next-generation sequencing (ChIP-seq) combined University of California, San Francisco, San Francisco, CA, USA. with bioinformatics analysis provide evidences that specific combinations We have developed a single-molecule method for direct visualization of of histone modifications represent various chromatin states, and are associ- in vitro assembled chromatin. Using TIRF microscopy we observe fluores- ated with different regulatory functions. However, most of these methods cently labeled chromatin substrates of varying nucleosome densities in probe one histone modification at a time in a cell population; thus, the real time while maintaining spatially resolved individual fibers. Importantly, true combinatorial patterns at each histone or nucleosome obtained by inte- we are able to site-specifically introduce histone post-translational modifica- grative analysis of multiple experiments can be masked at highly dynamic tions to mimic naturally occurring heterochromatic patterns. In addition to genomic loci or by cell-to-cell heterogeneity. Here, we apply single- this framework, we have developed a set of fluorescently labeled reporter molecule fluorescent imaging in analyzing the coexisting histone modifica- proteins, based on histone reader domains, with which we can visualize co- tions at single-nucleosome resolution. First, we establish the assay by valent modification to histone tails. This system represents a valuable step detecting genome-wide level of combinatorial histone modifications using forward for studying the dynamics of heterochromatic marks at size and mononucleosomes from MCF10A cells. Next, we apply it to delineate the time scales that existing methods currently do not address. heterogeneous chromatin states during the Epithelial-Mesenchymal- Transition by comparing to time-course bulk ChIP-seq data. Further devel- 1283-Pos Board B192 opment of this technology includes enriching chromatin from genomic loci A Systematic Study of Nucleosome Core Particle and Nucleosome- of interest or integrating with a single-molecule sequencer. We expect this Nucleosome Stacking Structure 1 2 1 method to further elucidate the functions of chromatin in different states, Nikolay Korolev , Alexander P. Lyubartsev , Lars Nordenskio¨ld . 1School of Biological Sciences, Nanyang Technological University, with the ultimate goal in providing a more complete picture of how epige- 2 nome is modulated in various cellular processes. Singapore, Singapore, Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden. In most eukaryotes, DNA is packed into chromatin, consisting of linear ar- 1280-Pos Board B189 rays of the regular DNA–histone complexes, nucleosomes. Chromatin Investigating the Role of Histone Modification in Nucleosome Formation condensation is driven by the energetically favorable interaction between by CAG/CTG Repeats nucleosome core particles (NCPs). The close NCP-NCP contact, stacking, Whitli Thomas, Catherine Volle. is a primary structural element of all condensed states of chromatin in vitro Biology, Cottey College, Nevada, MO, USA. and in vivo. However, the molecular structure of stacked NCPs as well as the Huntington’s Disease is a neurodegenerative disorder caused by the expan- nature of the interactions involved in its formation have not yet been stud- sion of CAG/CTG trinucleotide repeats within exon 1 of the huntingtin gene. ied. We undertake an investigation of both the structural and physico- Previous research has demonstrated that the longer the repeat tract, the chemical features of the NCP and the NCP-NCP stacking. We introduce greater likelihood that CAG/CTG repeats will be incorporated into the an ‘‘NCP-centered’’ set of parameters (NCP-NCP distance, shift, rise, tilt, nucleosome, the most basic unit of DNA packaging in the genome. The his- and others) that allows numerical characterization of the mutual positions tone proteins that form those nucleosomes in the cell have many epigenetic of the NCPs in the stacking and in any other structures formed by the markers that can alter the packaging of the nucleosome. Additionally, these NCPs. The suggested approach is similar to the convention used for struc- modifications may change the affinity of the DNA for the histone core. Inter- tural classification of DNA and RNA double helices. NCP stacking in about estingly, while the interaction of CAG/CTG repeat DNA with histone pro- 150 published NCP crystal structures was analysed and statistics of the pa- teins has been extensively studied as a function of repeat length, there is rameters defining the stacking was determined. In addition, coarse grained yet to be a study that investigates the effect of the histones and their epige- (CG) Langevin molecular dynamics computer simulations modelling NCP netic marks on nucleosome formation by CAG/CTG repeats. Here, we ex- condensation was carried out. The CG model that was used to investigate press and purify histones from a recombinant source and use them to form NCP-NCP interaction takes into account details of the nucleosome structure nucleosomes in a nucleosome incorporation assay. We can then compare and adequately describes the long range electrostatic forces as well as the formation of nucleosomes with the recombinant histones to nucleosomes excluded volume effects acting in chromatin. CG simulations showed formed with histones isolated from chicken erythrocytes, thus elucidating good agreement with experimental data and reveal the importance of the the general effect of histone modification on nucleosome formation by H2A and H4 tail bridging and screening as well as tail-tail correlations CAG/CTG repeats. the stacked nucleosomes.

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1284-Pos Board B193 including compartments, topologically associating domains (TADs) and loops, Solution Ensemble of the macroH2A Nucleosome with Linker Sequence which are detected by chromosome conformation capture (Hi-C). To test Samuel Bowerman1, Srinivas Chakravarthy2, Jeff Wereszczynski1. whether cohesin shapes these chromatin features starting from the one-cell em- 1Physics, Illinois Institute of Technology, Chicago, IL, USA, 2Biology, bryo, we generated mouse knockout embryos deficient of Scc1-cohesin or the Illinois Institute of Technology, Chicago, IL, USA. protein Wapl, which regulates the residency time of cohesin on chromatin. Us- Eukaryotic organisms package their genetic material in a hierarchical scheme, ing single nucleus chromosome conformation capture (snHi-C), we discovered of which the nucleosome core particle is the fundamental unit. Each nucleo- that cohesin deletion pre-fertilization prevents the formation of TADs and chro- some binds approximately 147 basepairs of DNA to a protein octamer con- matin loops but not compartments in one-cell embryos. Moreover, we find that structed from two copies each of the H2A, H2B, H3, and H4 histones. Cells inactivating cohesin release by deleting Wapl results in differences in loop can epigenetically alter transcription by substituting canonical histones with strengths between the maternal and paternal genomes that may be related to a variant protein - one which has the same structure but a differing sequence. chromatin reprogramming. We further discovered that cohesin can influence The macroH2A variant is unique to the H2A family in that it possesses an extra inter-chromosomal interaction frequecies. We developed Langevin dynamics macro domain which is attached to the histone domain via a lysine-rich linker polymer simulations of a chromatin fiber undergoing active extrusion of chro- segment. While it has previously been suggested that this protein linker matin loops. We simulated cohesins actively juxtaposing flanking chromatin segment stabilizes the binding of extranucleosomal linker DNA basepairs, no from random loading sites, which resulted in good agreement with snHi-C structural studies exist to date. Here, we present small angle x-ray scattering data, reproducing loops, TADs, and genome-wide contact probability for the (SAXS) data on nucleosomes composed of an extended DNA sequence and various conditions. Our polymer simulations and snHi-C data are consistent three variations of H2A: the canonical histone, the histone domain of with a model where cohesin locally compacts chromatin forming loops and macroH2A, and the macroH2A histone domain plus linker sequence. We utilize TADs, and thus restricts inter-chromosomal interactions by active loop extru- a Monte Carlo algorithm to generate an ensemble of DNA breathing states, and sion, whose processivity is controlled by Wapl. Our simulations and experi- we reweight the population of states using the empirical SAXS data via an in- mental data provide evidence that cohesin-dependent loop extrusion house Bayesian protocol. We find that the canonical NCP and the truncated organizes mammalian genomes over multiple scales from the one-cell embryo macroH2A nucleosomes possess similar distributions of DNA-open breathing onwards. states, while the presence of the linker sequence biases toward larger popula- tions of a compact DNA-closed state. These findings support a mechanism 1287-Pos Board B196 for transcriptional repression in which the macroH2A linker segment reduces Chromatin Folding with DFRACT the accessibility of linker DNA basepairs to extranucleosomal factors. Alan Perez-Rathke, Jie Liang. Bioengineering, University of Illinois at Chicago, Chicago, IL, USA. 1285-Pos Board B194 Deciphering the detailed mechanisms governing gene regulation often requires High-Resolution Locus-Specific Mapping of Chromatin Contacts using 3-D reconstruction of the chromatin fold. Chromosome conformation capture Two-Photon Photoactivated DNA Cross-Linking techniques such as Hi-C provide insight into the chromatin folding state but Max Kushner1, Juan Wang1, Abdullah Ozer2, Judhajeet Ray2, John Lis2, only give averaged spatial information in the form of 2-D contact heat maps. Warren Zipfel3. Using these techniques alone, it is not possible to answer questions such as: 1Graduate Field of Biophysics, Cornell University, Ithaca, NY, USA, the existence of functional cellular sub-populations, the contact probabilities 2Department of Molecular Biology and Genetics, Cornell University, Ithaca, among multiple (>2) interacting loci, the causal folding relationships among NY, USA, 3Department of Biomedical Engineering, Cornell University, the spatially interacting genomic regions, or the identification of driver interac- Ithaca, NY, USA. tions governing the folding state. We present DFRACT (Dirichlet FRActal Spatial organization of the genome remains an interesting and not fully under- ChromaTin), a Bayesian method utilizing models of physical chromatin folding stood area of nuclear architecture and gene regulation. Recently, there have to offer probabilistic answers to these questions. We demonstrate our method been many technological and methodological developments towards this on a TAD locus within the K562 Hi-C data set of Rao et al. end from both imaging and genomic perspectives. Here we present a novel method to examine genomic contacts in vivo near a specific locus or nuclear 1288-Pos Board B197 region of interest. Using 3D localized two-photon excitation and a photoacti- Impact of the Linker Histone H1 on the Structure and Dynamics of Chro- vatable DNA crosslinker, the chromatin within a femtoliter volume around the matin Fibers: A Molecular Dynamics Study region of interest can be labeled with an affinity tag. By carrying out this pro- Francisco Rodriguez Ropero, Jeff Wereszczynski. cess on many cells using an automated microscope, we can then isolate the Illinois Institute of Technology, Chicago, IL, USA. genomic DNA and pulldown the crosslinked DNA. While several DNA Chromatin fibers consist of a biomacromolecular complex of DNA and pro- cross-linkers may be suitable for this method, we have initially focused on teins, which constitute the building block of eukaryotic chromosome. Nucleo- psoralen based compounds which are easily UV or two-photon photoactivated some core particles (NCPs) are the basic unit of chromatin fibers. They to form inter-strand DNA crosslinks preferentially at 5’-TpA dinucleotides in comprise 147 base pairs of DNA densely wrapped around eight core histone the genome. For photophysical optimization of the method, we use fluores- proteins (H2A, H2B, H3, and H4). NCPs within the chromatin fiber are inter- cently labeled genomic loci where 200 copies of a transgene have been inte- connected through a segment of linker DNA. NCPs and linker DNA often asso- grated at a single locus in U2OS cells. The DNA is extracted and purified ciate with a linker histone (H1 or H5), which leads to a higher compaction of using an affinity tag pull-down and the efficiency of crosslinking is quantified the overall chromatin fiber. via qPCR. After the optimal photoactivation parameters are determined, the In this communication we report results from 3 ms of fully atomistic Molecular genomic locus will be irradiated and the crosslinked DNA extracted and Dynamics (MD) simulations of 20 nm chromatin fibers formed by an octa- sequenced as a proof-of-principle experiment. The long-term goal is to create NCP structure, both with and without H1 linker histones bound in off-dyad po- a new method to bioinformatically map chromatin fragments at a nuclear re- sitions. To better understand the impact of linker H1 histone in chromatin struc- gion of interest, which can be performed before and after gene activation for ture and dynamics we discuss the differences in specific DNA-histone any gene of interest. interactions, inter-NCP interactions, chromatin flexibility, structure of linker DNA, as well as dynamic pathways between systems with and without linker 1286-Pos Board B195 H1 histone. A Mechanism of Cohesin-Dependent Loop Extrusion Organizes Mamma- lian Chromatin Structure in the Developing Embryo 1289-Pos Board B198 Hugo B. Branda˜o1, Johanna Gassler2, Maxim Imakaev3, Ilya M. Flyamer4, Characterizing Chromatin Geometry and Its Dependence on Local Nucle- Sabrina Ladst€atter2, Wendy A. Bickmore4, Jan-Michael Peters5, osome Environment Kikue¨ Tachibana-Konwalski2, Leonid A. Mirny3. Stefjord Todolli1, Ondrej Maxian2, Wilma K. Olson1. 1Harvard University, Cambridge, MA, USA, 2Institute of Molecular 1Center for Quantitative Biology, Rutgers University, Piscataway, NJ, USA, Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna, 2Department of Mathematics, Case Western Reserve University, Cleveland, Austria, 3MIT, Cambridge, MA, USA, 4MRC Human Genetics Unit, Institute OH, USA. of Genetics and Molecular Medicine (IGMM), Edinburgh, United Kingdom, Eukaryotic cell nuclei house their DNA in a highly compact, yet incredibly 5Institute of Molecular Pathology (IMP), Vienna, Austria. dynamic chromatin structure. This non-uniform and dynamic structure en- Higher order chromatin structure is established de-novo in the developing em- ables many processes of biological relevance, such as gene expression. bryo. It is long hypothesized that the protein cohesin plays an important role in The motions of chromatin facilitate the process of bringing together protein the establishment and maintenance of mammalian chromatin structures components, such as those required for transcription, that are situated far

BPJ 8621_8624 256a Monday, February 19, 2018 apart along the DNA sequence. A complete account of the connection be- 1292-Pos Board B201 tween the local features of DNA and protein and global motions of chro- DNA-Bending Non-histone Proteins Can Make Chromatin Irregular and matin is vital in understanding these processes. We have used a meso- More Compact scale representation of chromatin in Monte Carlo simulations of precisely Gaurav Bajpai1, Mandar Inamdar2, Dibyendu Das3, Ranjith Padinhateeri1. positioned nucleosomal arrays. Our simulations to investigate the effect of 1Biosciences and Bioengineering, IIT Bombay, Mumbai, India, 2Civil nucleosome spacing and histone tails on global chromatin properties feature Department, IIT Bombay, Mumbai, India, 3Physics department, IIT Bombay, a highly diverse chromatin fiber, with architectures beyond the widely refer- Mumbai, India. enced solenoid and zigzag/two-start models. In particular, our models with The function of a cell depends not just on the sequence of DNA in the cell, but integral and half-integral length linker DNA show very different geometries also on the Three dimensional(3D) organization of its chromatin. How chro- in terms of the nature of the stacking of nucleosomes with respect to one matin is organized in 3D inside the nucleus, in the length-scale of a gene, is another and the helical arrangements of the fiber. We employ a new method an open question. In vitro studies suggest that chromatin is organized into to efficiently characterize the nature of the inter-nucleosomal interactions. 30nm-wide fiber which was further packaged into the higher order chromatin This approach has enabled us to better interpret the mechanism by which structure. However, in vivo studies do not clearly observe the regular 30nm fi- the local nucleosome environment, including linker DNA, histone tails ber in living cells. In this work, we present a plausible explanation for the and the linker histone dictate global properties, including the large scale absence of 30nm fiber in vivo, and the presence of irregular chromatin seen bending and looping that affect long distance communication in chromatin in recent experiments. Using computer simulations, we study the organization fibers. of chromatin accounting for the presence of non-histone proteins(NHPs) that bend DNA. We find that DNA-bending NHPs can make chromatin irregular 1290-Pos Board B199 and more compact. Our work provides the density of NHP required for the Linker DNA Length Defines the Structure of Chromatin Fibers chromatin to be irregular. We calculate the packing ratios for different concen- Thomas B. Brouwer, Artur Kaczmarczyk, Nicolaas Hermans, trations of NHP. Margherita Botto, John van Noort. Lion, Leiden University, Leiden, Netherlands. 1293-Pos Board B202 In eukaryotes, DNA is compacted into chromatin and the mechanical and Single and Double Box HMGB Proteins Differentially Destabilize structural properties of chromatin fibers play an important role in gene Nucleosomes regulation. For well-defined, in vitro reconstituted nucleosomal arrays it Micah J. McCauley1, Ran Huo1, Nicole Becker2, Molly Nelson Holte2, has been shown that their higher order structure depends on the length Uma M. Muthurajan3, Ioulia Rouzina4, Karolin Luger3,5, of the linker DNA. Most studies however, focused on linker length consist- L. James Maher III,2, Nathan E. Israeloff1, Mark C. Williams1. ing of multiples of 10 base pairs and report zig-zag or a solenoidal struc- 1Physics, Northeastern University, Boston, MA, USA, 2Biochemistry and tures of stacked nucleosomes. In nature however, linker DNA lengths of Molecular Biology, Mayo Clinic College of Medicine and Science, 10nþ5 base pairs are most abundant. For short linkers fibers an alternative Rochester, MN, USA, 3Chemistry and Biochemistry, University of Colorado, anti-parallel zig-zag structure was recently proposed, but experimental ev- Boulder, CO, USA, 4Chemistry and Biochemistry, Ohio State University, idence is lacking. Here, we used multiplexed magnetic tweezers to mea- Columbus, OH, USA, 5Howard Hughes Medical Institute, Chevy Chase, MD, sure mechanical parameters of fibers with 20, 25, 50 and 55 bp linker USA. DNA. Based on the stiffness and the stacking energy, we conclude that Nucleosome disruption plays a key role in many nuclear processes including the 10nþ5 fibers fold differently than 10n fibers, indicating a dominant transcription, DNA repair and recombination. We combine atomic force micro- role of the linker DNA on chromatin folding and consequently epigenetic scopy (AFM) and optical tweezers (OT) experiments to show that high mobility regulation. These results suggest that nucleosome phasing should be group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new controlled with base pair accuracy in order to create specific higher order mechanism for regulation of chromatin accessibility. We find that both the dou- chromatin structures. ble box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins desta- 1291-Pos Board B200 bilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Transient Anomalous Subdiffusion Model of DNA-Binding Species in the Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This dif- Nucleus ference in nucleosome destabilization may explain why Nhp6A and Hmo1 Michael J. Saxton. function at different genomic sites. Hmo1 is enriched at highly transcribed ri- Dept Biochemistry & Molecular Medicine, University of California, Davis, bosomal genes, known to be depleted of histones. In contrast, Nhp6A is found CA, USA. across euchromatin, pointing to a significant difference in cellular function. Single-particle tracking experiments have measured the distribution of dwell times of DNA-binding species including CRISPR-Cas9, TetR, and LacI 1294-Pos Board B203 diffusing in living cells. The observed truncated power law distribution im- Dynamics of Histone H3 Tails in Mononucleosomes Studied by Single- plies transient anomalous subdiffusion. Monte Carlo simulations are used to Molecule FRET and MD Simulations characterize the time-dependent diffusion coefficient D(t) and to relate the Kathrin Lehmann1, Ruihan Zhang2, Suren Felekyan3, Ralf Kuhnemuth€ 3, time dependence to the dwell time distribution. The time dependence is Katalin Toth1. described in terms of the anomalous diffusion exponent, the limits D(0) 1Biophysics of Macromolecules, German Cancer Research Center, and D(N), and the crossover time between anomalous subdiffusion at short Heidelberg, Germany, 2Key Laboratory of Medicinal Chemistry for Natural times and normal diffusion at long times. We examine how the underlying Resources, Yunnan University, Kunming, China, 3Heinrich Heine Universit€at dwell time distribution is broadened by the statistics of escape to give the Dusseldorf,€ Dusseldorf,€ Germany. ideal observed distribution of dwell times. The actual observed distribution The nucleosome, consisting of two copies of each of the four histone proteins may be modified by experimental constraints. The simplest interpretation of H2A, H2B, H3, H4, and a 147bp long DNA, is the basic unit of chromatin the model is that the dwell times are actual binding times to DNA. One alter- compaction and key to gene regulation. The N-terminal tails of the four core native is that the dwell times are the periods of 1D diffusion on DNA in the histone proteins and the C-terminal tail of H2A are protruding from the core standard combination of 1D and 3D search. The initial anomalous regime particle in a disorder manner. Even though, the nucleosome has been widely represents the search of the DNA-binding species for its target DNA studied in the field of epigenetics, surprisingly little is known about the actual sequence, so this regime is of fundamental interest. Non-target DNA sites conformation of the tails and their dynamics. have a significant effect on search kinetics; false positives from bio- Here we focus on the conformation and dynamics of the H3 N-terminal tail at informatic studies are potentially rate-determining in vivo. Overexpression various NaCl concentrations and linker DNA length in experiments and MD of the DNA-binding species reduces anomalous subdiffusion because the simulations. Salt dependent conformational changes were measured by FRET deepest non-target binding sites are occupied and unavailable. Both binding with reconstitute Widom 601 mononucleosomes with a DNA length of 170 and obstruction affect diffusion. In the absence of a consensus model of bp, 190 bp or 210 bp. Nucleosomes were labeled on the H3 tail at position chromatin geometry, obstruction effects ought to be characterized by exper- K9 and at various positions on the intranucleosomal DNA or linker DNA. iment as well as by modeling. Suggested controls for obstruction are GFP as Both, bulk experiments using microplate scanning FRET spectroscopy a calibration standard among laboratories and cell types, and the DNA- (mpsFRET), and single molecule experiments using alternating laser excitation binding species with the binding site unobtrusively inactivated. (Supported (ALEX) or multidimensional confocal fluorescence spectroscopy with pulsed in part by NIH grant GM038133). interleaved excitation (MFD-PIE) were performed.

BPJ 8621_8624 Monday, February 19, 2018 257a

Our results confirm the previously described binding preference of the H3 tail rial chromosome and nucleoid structure. Here, we determined crystal structures towards the linker DNA at low salt concentrations as well as an influence of the of these chromosome-associated proteins in complex with native duplex DNA. linker DNA length on the H3 tail:DNA interaction. At higher salt concentra- Distinct DNA-binding modes of HUaa-and HUab elucidate fundamental fea- tions (> 600 mM), when nucleosome disassembly occurs, interaction of the tures of bacterial chromosome packing regulating gene transcription. By H3 tail with the linker DNA is disrupted and interaction with the inner DNA combining crystal structures with solution X-ray scattering results, we deter- gyre is favorized. The designed constructs enable us for the first time to our mined architectures of HU-DNA nucleoproteins in solution under near physio- knowledge to monitor conformational changes of the H3 tail, which underpins logical conditions. These macromolecular conformations and interactions the proposed dynamic nature of the H3 tail. result in contraction at the cellular level based upon in vivo imaging of native unlabeled nucleoid by soft X-ray tomography upon HUb and ectopic HUa38 1295-Pos Board B204 expression. Structural characterization of charge-altered HUaa - DNA com- Random Walk in the Realm of Chromatin plex reveals an HU molecular switch suitable to condense nucleoid and repro- Zhen Wah Tan, Enrico Guarnera, Igor N. Berezovsky. gram noninvasive Escherichia coli into an invasive form. Collective findings Bioinformatics Institute, Singapore, Singapore. suggest that shifts between networking, cooperative and non-cooperative Development of the chromosome conformation capture (3C) technique and its DNA-dependent HU multimerization control DNA compaction and supercoil- evolution from probing the interactions between a specific pair of loci to whole ing independently of cellular topoisomerase activity. By integrating X-ray crys- genome interactions (Hi-C) allowed us to enter an uncharted field of the chro- tal structures, X-ray scattering, mutational tests, and X-ray imaging that span matin structure, dynamics, and epigenetic regulation. Existing computational from protein-DNA complexes to the bacterial chromosome and nucleoid struc- approaches for the Hi-C data analysis can be roughly classified into two cate- ture, we show that defined dynamic HU interaction networks can promote gories, model-driven and data-driven, where the former is generally aimed at nucleoid reorganization and transcriptional regulation as efficient general mi- explaining features of the Hi-C data on the basis of proposed physical model crobial mechanisms to help synchronize genetic responses to cell cycle, chang- and the latter seeks to extract information about chromatin structure and inter- ing environments, and pathogenesis. Hammel, M., D. Amlanjyoti, F. E. Reyes, actions. Our goal is to explore chromatin using a minimum of simplifying as- J. H. Chen, R. Parpana, H. Y. Tang, C. A. Larabell, J. A. Tainer and S. Adhya sumptions on the structure and heuristic tuning parameters in the analysis. We (2016). SCIENCE Adv2(7): e1600650. represent the Hi-C data as an interaction network between the genomic loci and use Markov State Model for identifying the structural organization of chro- Posters: Membrane Physical Chemistry II matin. Specifically, assuming the diffusive character of the chromatin local contact formation, we construct a Markov jump process, mimicking a random 1298-Pos Board B207 walk in the chromatin media. The highly interacting loci form hubs in the chro- Electrostatics in Closed Systems matin interaction network, providing a foundation for the chromatin partition- Joel A. Cohen. ing and reconstruction of packing interactions in relation to their functional Physics, Univ of Massachusetts, Amherst, MA, USA. involvement. We show the existence of a structural hierarchy in chromatin, The solution to the Poisson-Boltzmann (PB) equation for the electrostatic describe a relationship between the intra/interchromosomal interactions and potential in an aqueous electrolyte depends on its boundary conditions different expression signal and epigenetic factor, and discuss future tasks in (b.c.). As a 2nd-order differential equation, PB requires 2 b.c. Common the study of chromatin structure in relation to gene expression and its b.c. are zero electrostatic potential and zero electric field in a reservoir regulation. with which the system is in equilibrium. The reservoir is usually assumed large and distant from charged surfaces. However, for charged colloids sus- 1296-Pos Board B205 pended in an electrolyte in a bottle, the reservoir might be neither large nor High-Resolution Mapping of Chromatin Dynamics during Transcription distant. The criterion for ‘‘large’’ is volume [ volume of all particles þ in Mammary Tumor Cells their screening clouds, or equivalently, linear dimensions of the reservoir Haitham Ahmed Shaban, Roman Barth, Kerstin Bystricky. [ sum of all particle radii þ their Debye screening lengths. Since Debye Laboratoire de Biologie Moleculaire Eucaryote (LBME) - Centre de Biologie length depends inversely on the square root of ionic strength, this criterion Integrative (CBI) - CNRS; University of Toulouse, Toulouse, France. can fail at low ionic strength. Such a system is considered isolated or Chromatin is defined to be highly dynamic for the regulation of gene expres- ‘‘closed,’’ since it is not in equilibrium with, and has no access to, a large sion. Control of gene expression mostly occurs at the transcription stage based reservoir. Without a reservoir, reference parameters such as a reservoir elec- on modifications of chromatin structure and transcription factors (estrogen-re- trostatic potential and bulk salt concentrations do not exist. New constants of ceptor alpha (ERa), in our case) along with RNA polymerase II dynamics. the system and new b.c. are required. In a closed system the conserved quan- However, it remains unclear how structural chromatin complexes respond tity is the number of ions N, rather than a reservoir ion concentration.We dynamically through coordinate movements to regulate genomic processes. show that N/V, the mean ion concentration in the system volume, plays a Also, the global and local motion correlation of chromatin with other transcrip- role analogous to the reservoir bulk ion concentration and provides a b.c. tion players is still debated. For this purpose, we developed a method to Zero electric field midway between 2 identical colloid surfaces, required estimate motion at sub-pixel resolution with high accuracy, which reveals by symmetry, provides the other b.c. Boltzmann statistics is still valid, but sub-diffraction vectorial information, although the intensity is diffraction- the electrostatic potential profile and ion distributions differ from those of limited, based on reconstructed dense global flow fields of fluorescent images. open systems due to the constraint on ion number, whereby populating This approach allows us to quantify the biophysical properties such as the diffu- higher energy states depopulates lower ones. Calculations for open and sion coefficient or the underlying types of motion of H2B tagged with GFP, and closed systems are compared to data for interacting charged liposomes in de- SiR-DNA as chromatin markers, and ERa tagged GFP in MCF-7 breast cancer ionized water. cells. We measure the correlated flows of chromatin markers, and ERa, based on direction and magnitude of motion at different time intervals. To follow the 1299-Pos Board B208 dynamic response during transcription, we studied the correlated motion of Isothermal Titration Calorimetry of Be2D and Ca2D with Phosphatidylser- chromatin in the absence and presence of 17beta-estradiol (E2) for (de) activa- ine Models Guides All-Atom Force Field Development for Lipid-Ion tion of transcription along with transcription inhibitors at initiation and inter- Interactions mediate transcriptional stages. Quantification of spatial smoothness shows Alison Leonard, Sergei Sukharev, Jeffery B. Klauda. that distinct motion boundaries over large time intervals, while for short time UMCP, College Park, MD, USA. intervals smooth transitions in a small neighborhood dominate. We observe Beryllium has multiple industrial applications, but its unsafe manufacturing is similar trends in chromatin dynamics upon pausing transcription at two associated with developing chronic inflammation in lungs known as berylliosis. different stages and long range correlations during transcription. Although high incidence of berylliosis was linked to a specific MHC allele that causes immune hypersensitivity to Be2þ, recent findings also indicate that Be2þ 1297-Pos Board B206 competes with Ca2þ for binding sites in phosphatidylserine (PS) and thus in- HU Multimerization Shift Controls Nucleoid Compcation hibits the recognition and phagocytosis of apoptotic cells, potentially causing Michal Hammel. necrotic inflammation. This study aims to develop additive force field param- Advanced Light Source, Lawrence Berkeley Lab, Berkeley, CA, USA. eters that would describe Be2þ competition for divalent ion binding sites on Molecular mechanisms controlling functional bacterial chromosome (nucleoid) anionic lipids critical for many signaling processes. We started with compaction and organization are surprisingly enigmatic, but depend partly Lennard-Jones parameters for Be2þ for the CHARMM additive force field upon conserved, histone-like proteins HUaa and Huab and their interactions based on interactions with water, including free energy of hydration and ion- that span the nano and mesoscales from protein-DNA complexes to the bacte- monohydrate interaction energy and separation distance. The results from

BPJ 8621_8624 258a Monday, February 19, 2018 isothermal titration calorimetry (ITC) for the binding affinities of Be2þ and Adsorption of indomethacin to gel-phase phospholipids was endothermic and Ca2þ to dimethylphosphate and acetate revealed that Be2þ has a stronger affin- entropically-driven while adsorption to fluid-phase phospholipids was ity than Ca2þ for binding with both component molecules. The existing exothermic and enthalpically-driven. Our studies suggest that indomethacin, CHARMM parameters for Ca2þ showed significant overbinding to acetate, like other arylacetic acid-containing drugs, preferentially interacts with ordered while the parameters recently developed for Be2þ resulted in overbinding to lipid domains. both component molecules. To adjust these affinities to fit the experimental free energies of binding, configuration probabilities and free energies of asso- 1303-Pos Board B212 ciation from molecular dynamics simulations were used to develop Lennard- Changes in the Biophysics of Lipid Memrbanes Mediated by Peptides and Jones interaction parameters between the ions and component molecules. Using Drugs the revised interaction parameters, binding energies of Be2þ and Ca2þ with Hannah M. Britt, Vian S. Ismail, Vanessa J. Lyne, Jackie A. Mosely, pure DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) monolayers were John M. Sanderson. calculated using free energy perturbation and compared with binding affinities Dept. of Chemistry, Durham University, Durham, United Kingdom. determined from Langmuir monolayer experiments, ITC measurements on sol- Peptides and drugs are subject to lipidation reactions in membranes, and in uble short-chain PS and DOPS liposomes. some cases appear to additionally facilitate lipid hydrolysis. Lipidation reac- tions involve acyl transfer from membrane lipids to reactive sites on a bound peptide or drug. For peptides, an amphiphilic structure appears to favor lipida- 1300-Pos Board B209 tion activity. For drugs, the depth of bilayer partitioning appears to be a crucial Association of Neurotransmitters with Lipid Bilayer Membranes factor. Partitioning studies, combined with rate profiles at varying temperature Brian Josey, Frank Heinrich, Mathias Lo¨sche. and pH reveal that, remarkably, the extent of lipidation of propranolol is signif- Physics, Carnegie Mellon University, Pittsburgh, PA, USA. icant even when the amounts of membrane-bound reactive species are Aimed to reproduce the results of electrophysiological studies of synaptic extremely low. In some cases, drugs also appear to promote lipid hydrolysis signal transduction, conventional models of neurotransmission are based on - to lysolipids, in competition with lipidation. Lipidation and hydrolysis the specific binding of neurotransmitters to ligand gated receptor ion channels. in vivo have been probed in order to demonstrate that these processes have sig- However, the complex kinetic behavior observed in synaptic transmission nificance for cell physiology. These experiments have also enabled membrane cannot be reproduced in standard kinetic model without the ad hoc postulation chemical reactivity to be correlated with adverse cellular phenomena such as of additional conformational channel states. On the other hand, if one invokes phospholipidosis. unspecific neurotransmitter adsorption to the bilayer—a process not considered in the standard models—the electrophysiological data can be rationalized with 1304-Pos Board B213 only the standard set of three conformational receptor states that also depend on Lipid Membranes as a Target for Reactive Aldehyde Action this indirect coupling to neurotransmitters via their membrane interaction. Un- Olga Jovanovic1, Mario Vazdar2, Elena E. Pohl1. like for anesthetics, experimental verification has been difficult because of the 1Institute of Physiology, Pathophysiology and Biophysics, University of low binding affinities of neurotransmitters to lipid bilayer. We quantify this Veterinary Medicine, Vienna, Austria, 2Laboratory for Chemistry in Model interaction with surface plasmon resonance (SPR) spectroscopy to measure Biological Systems, Rudjer Boskovic Institute, Zagreb, Croatia. the equilibrium dissociation constant of neurotransmitter membrane associa- Reactive aldehydes, such as 4-hydroxynonenal (4-HNE) and 4-oxononenal tion, as well as on and off rates under flow. Neutron reflectometry (NR) on arti- (4-ONE), are peroxidation products of polyunsaturated fatty acids formed in ficial membranes is used to characterize structural aspects of the association of cellular membranes during oxidative stress. Recently, we demonstrated that neurotransmitters with the membranes. Sparsely-tethered bilayer lipid mem- their interaction with phosphoethanolamine in the cell membrane alters the branes composed of zwitterionic (PC) and anionic (PS and PG) lipids were function of membrane carriers, such as mitochondrial uncoupling proteins, va- assembled and their interactions with serotonin, g-aminobutyric acid linomycin or CCCP (1,2). In this work, we observed that (i) 4-HNE increased (GABA), dopamine, and adenosine studied. SPR shows a wide range of binding the total membrane conductance, Gm, in mixed DOPE:DOPC bilayers in affinities for different neurotransmitters. NR shows that the ligand with the contrast to DOPC bilayers, (ii) the Gm increase depended on the cation concen- largest affinity, serotonin, penetrates the membrane deeply whereas GABA as- tration in buffer solution (iii) 4-ONE did not affect Gm at any lipid composition. sociates with the bilayer peripherally. We establish that some neurotransmitters Together with MD simulations the results offer an explanation as to why - interact non specifically with the lipid membrane matrix at physiologically 4-HNE and 4-ONE have a different effect on Gm and how a small, but impor- relevant concentration and that this interaction differs vastly for different tant difference in the chemical structure of 4-ONE compared to 4-HNE lead to neurotransmitters. different properties of biological membranes during oxidative stress. [1] O. Jovanovic, A. A. Pashkovskaya, A. Annibal, M. Vazdar, N. Burchardt, 1301-Pos Board B210 A. Sansone, L. Gille, M. Fedorova, C. Ferreri and E. E. Pohl, Free Radicals Membrane Composition Determines Mechanism of MIN Wave Cycle Biology and Medicine, 2015, 89, 1067-1076. Carsten So¨nnichsen, Weixiang Ye, Ruben Ahijado-Guzma´n, Sirin Celicsoy. [2] Malingriaux*, E.A., Rupprecht*, A., Gille, L., Jovanovic, O., Jezek P., Ja- Physical Chemistry, Johannes Gutenberg University, Mainz, Germany. burek, M., Pohl, E.E. (2013) Fatty acids are key in 4-hydroxy-2-nonenal-medi- The Min proteins bind and detach from lipid bilayers in the form of propagating ated activation of uncoupling proteins 1 and 2. PLOS ONE. waves which plays an important role in the cell division process. We use plas- monic nanoparticles covered by a supported lipid bilayer to study this process 1305-Pos Board B214 in detail. Using different sizes of nanorods, we are able to quantify the vertical Thermodynamic and Morphological Properties of Violacein Regulated by distance or z-position of the Min dynamic, i.e. where the Min proteins attach the Lipid Composition of Cellular Membrane Models at the Air-Water and detach. Surprisingly, we find the z-position depending on the lipid compo- Interface sition: in membranes containing cardiolipin CL, the Min dynamics is Karine D. Souza1, Katia R. Perez2, Nelson Duran3, Gisele Z. Justo1, happening at about 5nm vertical distance to the membrane, without CL, the Luciano Caseli1. 1 MinD/E protein waves oscillate directly on top of the lipid membrane. Our Ciencias Exatas e da Terra, Universidade Federal de Sao Paulo, Diadema- 2 finding is not consistent with all molecular models on Min dynamics and points SP, Brazil, Biophysics, Universidade Federal de Sao Paulo, Sao Paulo-SP, 3 towards a novel aspect of Min dynamics: the (local) lipid composition. Brazil, Universidade Estadual de Campinas, Campinas-SP, Brazil. Violacein is a naturally-occurring pigment with anti-bacterial, anti-fungal and 1302-Pos Board B211 anti-tumor properties. This substance is found in several species of bacteria and Thermodynamic Studies of Indomethacin Adsorption to Phospholipid accounts for their striking purple hues, showing recently uses for industrial ap- Membranes plications in cosmetics, medicines and fabrics. Some of this biological proper- Grace Yin Stokes. ties are therefore believed to be associated with their interactions with lipid Chemistry & Biochemistry, Santa Clara University, Santa Clara, CA, USA. surfaces, encouraging research on the identification of membrane sites capable Indomethacin is a non-steroidal anti-inflammatory drug (NSAID), and its inter- of its binding. With these ideas in mind, we investigated the interaction of vio- actions with supported lipid bilayers, may inform the molecular mechanism of lacein with models for cell membrane by using Langmuir monolayers of drug accumulation and retention in mammalian plasma membranes. We used selected lipids. Small amounts of the compound affected the surface the nonlinear optical spectroscopy second harmonic generation (SHG) to quan- pressure-area isotherms as well as the polarization-modulation reflection-ab- tify indomethacin adsorbed at the aqueous-bilayer interface and determine the sorption infrared spectra of the lipid monolayers, which pointed to a significant impact of lipid fluidity, cholesterol content, alkyl chain length and head group interaction. These effects depended on the electrical charge of the molecules charge. Experiments were conducted under physiologically-relevant drug con- that form the monolayer, and its activity was particularly distinctive for nega- centrations and at a range of pH, ionic strength and temperature conditions. tively charged phospholipids when compared to zwitterionic phospholipids.

BPJ 8621_8624 Monday, February 19, 2018 259a

Morphological analysis by using Brewster Angle Microscopy suggested that vi- the unexpected side-effect profile, which includes oddities such as hair growth. olacein at the air-water interface is homogenized when incorporated in both Previous studies have shown that cyclosporine is able to pass through cell mem- kind of lipids. Though the interaction of violacein with the lipids has affected branes, but the underlying mechanism has not been elucidated. It also has been the viscoelastic and structural properties of the floating monolayer, it is not able shown that cyclosporine A undergoes conformational shifts in response to to permeabilize lipid bilayers, as investigated using liposomes. These results changes in environmental factors. We used a gramicidin-based fluorescence consequently can help understand how violacein interacts on specific sites of assay, which is able to monitor changes in lipid bilayer elastic properties, to the membrane. Also, it was evidenced that the lipid composition of the film explore whether cyclosporine alters lipid bilayer properties. We found that: modulates the interaction with the lipophilic compound. first, cyclosporine is a bilayer-modifying molecule; and, second, that the ki- netics of bilayer modification is slow, much slower than the time courses 1306-Pos Board B215 observed with other compounds tested. This slow kinetics may reflect the Resveratrol Protects Membranes from PLA1 and PLA2 Hydrolytic Attack slow time courses of the conformational changes that have been observed in Yun Luo, Qinqin Fei, Wesley M. Botello-Smith, David Kent, other studies. This may imply that conformational shifts are required to convert Abdelaziz Alsamarah, Payal Chatterjee, Maria Lambros. cyclosporine from a water-soluble state to a more hydrophobic, bilayer- Pharmaceutical Sciences, Western University of Health Sciences, Pomona, modifying form that may be important for cyclosporine A’s movement across CA, USA. biological membranes. Background: Resveratrol is a polyphenol found in red wine. In this work we tested the hypothesis that resveratrol protects membranes from the hydrolytic 1309-Pos Board B218 attack of phospholipase A1, PLA1, and phospholipase A2, PLA2, which hydro- Sildenafil and Tadalafil Affect Ion Channels Formed by Antimicrobial lyze the sn-1 and sn-2 ester bonds of phospholipids, respectively, producing a Peptides via Membrane Dipole Potential fatty acid and a lysolipid. Anastasiia A. Zakharova, Svetlana S. Efimova, Daria A. Khaleneva, Methods: Molecular dynamics simulation studies were used to study the loca- Olga S. Ostroumova. tion of resveratrol within dipalmitoyl-phosphatidylcholine, DPPC, membranes Group of Ion Channel Modeling, Institute of Cytology of Russian Academy at 37C. Also liposomes were prepared with DPPC, and Distearoyl- of Sciences, St. Petersburg, Russian Federation. phosphatidylcholine, DSPC, having 0, 5, 9 and 33 mole % resveratrol and The aim of this report was to assess the possible effects of sildenafil and tada- were challenged for 10 minutes by the presence of PLA1, or PLA2. The result- lafil, the specific type-5 phosphodiesterase inhibitors, on the physicochemical ing free fatty acid, FFA, from the PLA1 and PLA2 challenge was measured us- properties of model lipid membranes. Changes in the steady-state membrane ing the ADIFAB and ADIFAB2 kits, respectively. conductance induced by Kþ-nonactin complexes and in the fluorescence inten- Results: Computational studies show that resveratrol decreases the bilayer sity of potential-sensitive lipid fluorescence probe, di-8-ANEPPS, were thickness and increases the membrane surface area. Also resveratrol has high measured to examine the changes in the boundary and dipole potential of lipid probability to form hydrogen bonds with sn-1 and sn-2 ester groups. When bilayers, respectively. We found that sildenafil and tadalafil decreased the DPPC and DSPC membranes were challenged with PLA2, there was signifi- dipole potential of membranes composed of DOPC, POPC, and multicompo- cantly less FFA in the presence of all of the concentrations of resveratrol nent bilayers, mimicking the membranes of vascular smooth muscle cells, compared to the control. This result signifies the fact that all resveratrol concen- and bathed in 0.1 M KCl (pH 7.4) on about 60 mV at 100 mM. Using differential trations were able to protect the DPPC and DSPC membranes from the PLA2 scanning microcalorimetry we showed that the tested PDE-5 inhibitors do not challenge. Furthermore, all the concentrations of resveratrol tested protected practically affect the temperature and cooperativity of the main transition of DSPC from PLA1 challenge, whereas 5 and 9 and not the 33 molar% resvera- DPPC at molar ratio to lipid of 1:10. The effects of sildenafil and tadalafil on trol protected DPPC from the PLA1 hydrolytic attack challenge. ion channels formed by gramicidin A and syringomycin E reconstituted into Conclusion: In conclusion, resveratrol assumes a protective position in the part lipid bilayers were also studied. Sildenafil increased the conductance of gram- of head group of the phospholipids bilayers proximal to fatty acyl chains and is icidin channels in DOPC-membranes bathed in 1 M KCl (pH 7.4) on 20%. Ta- able to protect the phospholipids from the hydrolytic attack of the PLAs. dalafil enhanced the steady-state number of open syringomycin channels in POPC-bilayers bathed in 0.1 M KCl (pH 7.4) by 6 times and diminished the 1307-Pos Board B216 single pore conductance on about 15%. We concluded that the alteration in 1,6-Hexanediol, Which Is Used to Disrupt Protein-Rich Liquid Droplets in ion channel activity was due to the changes in the dipole potential of mem- the Cell Cytoplasm, Does Not Disrupt Model Cell Membranes branes induced by the PDE-5 inhibitors. These data might provide some signif- Catherine Chang, Caitlin E. Cornell, Sarah L. Keller. icant implications for more complete understanding of action of these Chemistry, University of Washington, Seattle, WA, USA. pharmacological agents on cell membranes. The study was supported by the A surprising and exciting discovery in the past decade is that several proteins Russian Foundation of Science (# 14-14-00565). inside the cell cytoplasm can phase separate into liquid droplets (Brangwynne J. Cell Biol 2013 and Hyman et al. Annu. Rev. Cell Dev. Biol. 2014). Re- 1310-Pos Board B219 searchers often use a specific type of alcohol (1,6-hexanediol) to disrupt these Geometric Shape of Lipids versus Molecular Interactions in Membrane droplets. However, it was unknown whether hexanediol disrupts lipid mem- Pore Formation branes at the same concentration that is used to disrupt liquid droplets, which Neha Awasthi. would imply that hexanediol should not be used in cell studies. Here we per- Structural Molecular Biology, Georg August University, Goettingen, formed a vesicle leakage assay with DOPC vesicles loaded with carboxyfluor- Germany. escein dye at concentrations above which the dye self-quenches. In a positive Phosphoethanolamine (PE) lipids are considered crucial lipids for intracellular control, the detergent Triton X disrupts the vesicle membrane, causing an in- transport, viral entry via membrane fusion, antimicrobial peptide activity, and crease in fluorescence. In contrast, 1,6-hexanediol causes no increase in fluores- translocation of ions and cell-penetrating peptides. Transient membrane de- cence over the concentration ranges typically used in liquid droplet fects and pores appear across all these processes, and it is widely believed experiments. In conclusion, 1,6-hexanediol does not disrupt lipid model mem- that PE lipids favorably influence pore nucleation via their inverted cone mo- branes at experimentally-relevant concentrations, so it is suitable for in vivo lecular shape and intrinsic curvature. We demonstrate that the molecular shape studies of liquid droplets. and the intrinsic curvature of lipids do not determine the free energy of nucle- ating a membrane defect or pore. In fact, interactions between lipid head 1308-Pos Board B217 groups, and head-groups and solvent determine the energetics of pore nucle- Cyclosporine a Demonstrates Bilayer Modification with Slow Kinetics ation, and define whether pore nucleation is favored by enthalpy or entropy. Hanan A. Baker, Thasin Peyear, Olaf S. Andersen. Hence, contrary to belief, nucleating trans-membrane pores in a bilayer with Weill Cornell Graduate School of Medical Sciences, New York, NY, USA. PE lipids is energetically expensive due to extensive hydrogen bonding Cyclosporine A is a hydrophobic cyclic peptide that is widely used as an immu- network of PE lipids. In addition, we find qualitative differences between PE nosuppressant in the treatment of autoimmune diseases and prevention of organ and Phosphocholine (PC) lipids in their roles as zwitterionic components of rejection after transplantation. It has also been found to be cardioprotective and model bacterial membranes. Finally, we use potential of mean force (PMFs) is used as a topical treatment of keratoconjunctivitis sicca. Its cellular effect is to quantify the action of membrane active poly-arginines as model cell pene- to inhibit calcineurin, a serine phosphatase, which by dephosphorylating the nu- trating peptides. Our work highlights the critical nature of lipid composition clear factor of activated T cells leads to inhibition of transcription of the and arising interactions, and the limitations of shape based arguments, derived interleukin-2 gene in T-cells. This mechanism, however, does not account from geometric models in explaining the thermodynamics of membrane pore for many of the benefits conferred by cyclosporine A, nor does it account for nucleation.

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1311-Pos Board B220 During years literature indicated mitochondria as target-organelle for PDT, Ladderane Phospholipids Form Dense Membranes with Low Proton however more recent reports point out lysosomes as organelle-key to enhanced Permeability PDT efficiency. Photosensitizers (PS) that photodamage lysosomes impair the Frank R. Moss, Steven R. Shuken, Jaron A.M. Mercer, Carolyn M. Cohen, autophagic function pro-survival resulting in cell death associated to autophagy Noah Z. Burns, Steven G. Boxer. and may increase the PDT efficiency regardless the interaction level with mem- Chemistry, Stanford University, Stanford, CA, USA. branes. Therefore, once the photodamage in lysosomes has large potential to Anaerobic ammonium oxidizing (anammox) bacteria form a critical part of the oppose with paradigms in this field, we expect to unveil the lysosomal mem- nitrogen cycle by converting ammonium and nitrite to nitrogen gas. Anammox brane role in the cell death associated to the autophagy. The effects of photo- bacteria carry out their metabolism in the anammoxosome, a membrane-bound sensitization in lysosomal membranes were represented by giant unilamellar organelle that is comprised of the unique ladderane lipids. Ladderane lipids, vesicles (GUVs) made by bis-monoacylglycero phosphate (BMP) in this named for the ladder-like structure of fused cyclobutane rings in their fatty work. For photosensitization, we choose one photosensitizer that localizes in acid tails, have not been found anywhere else in nature, suggesting that they lysosomes: TPPS2a (porphyrin). The photooxidation of membranes with play a critical role in the anammox metabolism. It has been hypothesized TPPS2a increased generalized polarization (GP) values which may indicate that ladderane lipids prevent the diffusion of hydrazine (an anammox interme- that TPPS2a-photosensitization decreases membrane fluidity. Phase contrast diate), protons, or other species across the anammoxosome membrane. As microscopy was used to investigate the permeability of membrane to sucrose anammox bacteria have not been grown in axenic culture and grow extremely and glucose under GUVs photosensitization. Our results showed that slowly in enrichment cultures, researchers have not been able to isolate suffi- TPPS2a-photosensitization allows the permeability of sucrose and glucose cient quantities of pure ladderane lipids to determine the biophysical properties losing phase contrast of GUVs within 7 minutes of irradiation (1.6 mW). of ladderane membranes. Without knowledge of the physical properties of lad- This data is interesting to understand if TPPS2a causes pores that permit the derane lipids or genetic tools for studying the lipids in vivo, their biological leakage of enzymes and/or macromolecules, thus inducing a specific mecha- function remains unknown. We have developed efficient synthetic routes to nism of cell death (apoptosis and/or autophagy). We also found that TPPS2a- naturally occurring ladderane phospholipids and unnatural analogs. We show photosensitization leads to phase separation in GUVs made by that ladderane lipids have physical properties that are distinct from conven- BMP:DPPC:Cholesterol (1:1:1). It may have implications in cell death tional straight-chain lipids. Ladderane lipids form dense bilayers with slow signaling. lateral diffusion and dense monolayers with low compressibility. By varying Acknowledgements. Financial support by CNPq scholarship grant (150561/ the identities of the fatty acid tails, we establish structure-function relationships 2017-2) and FAPESP grant (2012/50680-5). for the different ladderane structures. These physical properties result in mem- branes with much slower rates of transbilayer diffusion of protons, which are 1314-Pos Board B223 pumped across the anammoxosome membrane and used to produce ATP. Interfacial Effects Dominate Ion Permeation through Membrane Channels These results suggest that ladderane lipids in the anammoxosome may prevent in Low Ionic Strength Solutions the dissipation of the proton gradient during the slow anammox metabolism. Antonio Alcaraz1, M. Lido´n Lo´pez1, Marı´a Queralt-Martı´n2, This role for ladderane lipids in the anammoxosome may partially explain Vicente M. Aguilella1. why anammox bacteria evolved such unique lipids. 1Dept. Physics (Lab. Molecular Biophysics), Universitat Jaume I, Castellon, Spain, 2Program in Physical Biology, Eunice Kennedy Shriver NICHD, 1312-Pos Board B221 National Institutes of Health, Bethesda, MD, USA. Energy Landscape of Membrane Deformations Predicts Mechanism of A number of protein channels and solid-state nanopores share the importance Pore Formation by Antimicrobial Peptides of electrostatic interactions between the permeating ions and the nanochannel. Sergey A. Akimov1,2, Oleg V. Kondrashov1,3, Timur R. Galimzyanov1,2, Ion transport at the nanoscale occurs under confinement conditions so that Irene Jimenez-Munguı´a2, Veronika V. Aleksandrova1, Peter I. Kuzmin1, interfacial effects such as access resistance (AR) become relevant. Here we Oleg V. Batishchev1. show that interpreting correctly electrophysiological measurements in terms 1Bioelectrochemistry, Frumkin Institute of Physical Chemistry and of channel ion selective properties requires the consideration of interfacial ef- Electrochemistry of RAS, Moscow, Russian Federation, 2Theoretical Physics fects, up to the point that they dominate protein channel conductance in and Quantum Technologies, National University of Science and Technology diluted solutions. We measure AR in a large ion channel, the bacterial porin ‘‘MISiS’’, Moscow, Russian Federation, 3Theoretical Physics, Moscow OmpF, by means of single-channel conductance measurements in salt solu- Institute of Physics and Technology (State University), Moscow, Russian tions with varying concentrations of high molecular weight PEG, sterically Federation. excluded from the pore. Comparison of experiments done in charged and Antimicrobial peptides are perspective agents for development of new gen- neutral planar bilayers shows that lipid surface charges modify the ion distri- eration of antibiotic drugs. They are thought to bind bacterial membranes via bution and determine the value of AR, indicating that lipid molecules are electrostatic and hydrophobic interactions. Partially embedded peptides more than passive scaffolds even in the case of large transmembrane proteins. cause elastic deformations of the membrane. We analyzed the energy land- We also found that AR might reach up to 80% of the total channel conduc- scape of the deformations arising around two peptide molecules accounting tance in diluted solutions, where current recordings reflect essentially the for deformations of splay, tilt, lateral compression/stretching under condition AR of the system and depend marginally on the pore ion concentrations of local volumetric incompressibility of membrane monolayers. Stationary and selective properties. These findings are of key importance for several configurations where the energy was minimal were obtained. The equilib- low aspect ratio biological channels that perform their physiological function rium states were shown to be almost independent on membrane tension, pep- in a low ionic strength and macromolecule crowded environment, just the two tide diameter and depth of its embedding as well as tilt angle. Locations of conditions enhancing the AR contribution. most probable formation of transversal pore were found by determining of membrane regions with maximal elastic stress. All such locations were at 1315-Pos Board B224 least several nanometers away from the peptides, thus, predicting that initial Water Permeability across the Droplet Bilayer Reveals Interaction pores should be purely lipidic. The density of the elastic energy was shown between Cholesterol and Polyunsaturated Lipids to be sufficient to cover about a half of the energy necessary to form the Sunghee Lee. pore, making formation of the pores possible at the expense of thermal fluc- Chemistry, Iona College, New Rochelle, NY, USA. tuations. The work was supported by Russian Science Foundation (project # Cholesterol can be the major component of total lipid in mammalian cellular 15-14-00060), Russian Foundation for Basic Research (project # 17-04- membranes, hence the knowledge of its association with lipid bilayer mem- 02070), and Ministry of Education and Science of the Russian Federation branes will be essential to understanding membrane structure and function. in the framework of Increase Competitiveness Program of NUST «MISiS» A Droplet Interface Bilayer (DIB) provides a convenient and reliable plat- (project # L4-2017-053). form through which permeability coefficient and activation energy for water transport across the membrane can be determined. In this paper, we present 1313-Pos Board B222 the effect of acyl chain structure in amphiphilic monoglycerides in the pres- Effects of TPPS2a-Photosensitization Lysosomal Membranes ence and absence of cholesterol on the permeability of water across DIB Tayana M. Tsubone, Rosangela Itri. membranes composed of these monoglycerides, where the acyl chain length, Institute of Physics, University of Sao Paulo, Sa˜o Paulo, Brazil. number of double bonds, and the position of double bond are varied system- Generating reactive oxygen species (ROS) in specific intracellular targets is the atically along the acyl chains, at two different cholesterol concentrations. best strategy to maximize the Photodynamic Therapy (PDT) efficiency, since Our systematic studies show dramatic sensitivity and selectivity of specific the short lifetime and high reactivity limite the ROS distribution inside cells. interaction of cholesterol with the monoglyceride bilayer having structural

BPJ 8621_8624 Monday, February 19, 2018 261a variations in acyl chain compositions. Our findings allow us to delineate the ON, Canada, 3BIOSS Centre for Biological Signalling Studies, Albert- exquisite interplay between membrane properties and structural components Ludwigs-Universit€at, Freiburg i.Br., Germany. and understand the balanced contribution of each. This successful demon- The action of membrane-active compounds involves permeabilizing the stration will provide important insights into the factors determinative of pas- lipid portion of membranes. For example, this applies to antimicrobial, sive membrane permeability in a wide variety of biologically relevant anticancer and cell penetrating peptides and their synthetic mimics, trans- systems. fection agents, and drug delivery vehicles. Membrane permeabilization is modelled by the release of an entrapped self-quenching fluorescent dye 1316-Pos Board B225 from lipid vesicles. This allows to determine the total leakage but also Solvatochromic Property in Lipid Bilayer Interphases Evaluated from the the distribution of leakage over the vesicle population. By a statistical Deconvolution of Time Resolved Emission Spectrum of Laurdan concept and kinetic studies, we characterize the strength of an individual Nozomi Watanabe1, Thomas K.M. Nyholm2, J. Peter Slotte2, Keishi Suga1, leakage event and discuss the molecular mechanisms such as asymmetry Hiroshi Umakoshi1. stress, curvature stabilization (toroidal pores, defects), large (oligomeric) 1Osaka University, Osaka, Japan, 2A˚ bo Akademi University, Turku, Finland. pores and combinations of these. We apply the concept to leakage induced Self-assembled lipid bilayer membranes are considered to have important roles by detergents, antimicrobial compounds and leakage upon phase transition in various bioprocesses. Raft domains, which are mainly composed of choles- of lipids. terol and sphingomyelin, form highly ordered microscale membrane domains Surprisingly, the strength of the individual leakage event can vary for a given with properties which are reported to contribute to functions such as membrane molecular mechanism. For example, the strength of leakage in asymmetric trafficking, virus infection, signal transduction and more. In order to understand binding and cracking-in can depend on the ratio of electrostatic to hydrophobi- the functions of raft domains, it is necessary to discuss the heterogeneity of cally driven interactions and varies with both, active agent and lipid composi- lipid diffusion because distributions of domains could determine that of pro- tion. The superposition or succession of different mechanisms (e.g. asymmetry teins and lead to control of cell functions. Evaluation of lateral diffusion stress and formation of defined pores at higher concentrations of active agent) regarding lipid mobility has been used as a major strategy to discuss the differ- can be assessed and may contribute to selectivity and activity. We better relate ences in fluidity. Besides, for the purpose of understanding the properties of results from model studies to biological cases and improve our understanding lipid bilayer interphases, the membrane polarity or hydration property of water of processes impossible to study directly, such as membrane-mediated antimi- molecules such as permeability and solvatochromic relaxation should be dis- crobial action. cussed. Exclusion of water molecules from the membrane surface can be considered to be a trigger for the interaction with external molecules. Despite 1319-Pos Board B228 the importance on this matter, the behavior of water molecules around lipid Oxysterol Translocation through Lipid Bilayers membrane surface is still unclear. The fluorescence from Laurdan (time Waldemar Kulig1,2, Heikki Mikkolainen2, Agnieszka Olzynska3, resolved emission spectra) was analyzed to detect how water molecules affect Piotr Jurkiewicz3, Lukasz Cwiklik3,4, Tomasz Rog1,2, Martin Hof3, the solvent relaxation in the hydrophilic region of lipid bilayer. Lifetime decon- Pavel Jungwirth2,4, Ilpo Vattulainen1,2. 1Department of Physics, University of Helsinki, Helsinki, Finland, volution could possibly help to understand the property or localization of the 2 fluorophore. Our results suggest that specific solvent relaxation states can be Department of Physics, Tampere University of Technology, Tampere, Finland, 3J. Heyrovsky´ Institute of Physical Chemistry, Academy of Sciences observed in sphingomyelin-containing bilayers. The hydration around lipid 4 head groups was evaluated with an aim for deeper insight into the formation of the Czech Republic, Prague, Czech Republic, Institute of Organic of heterogeneity in the lipid bilayer membrane. Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic. 1317-Pos Board B226 Cholesterol is a key molecule regulating various properties of animal cell Control of Ionic and Molecular Transport through Artificial Lipid Mem- membranes. Meanwhile, cholesterol is largely susceptible to oxidation, branes Containing Photopolymerizable Lipids with Applicability to Drug which results in oxysterols whose role in many regulatory processes can Delivery be significant, too. Oxysterols differ from cholesterol by the presence of Gamid Abatchev1, Caitlin Sall2, Daniel Prather2, Karsten Wake2, additional polar groups that are typically hydroxyl, keto, hydroperoxy, Daniel Fologea2. epoxy, or carboxyl moieties. Thus, the transmembrane distribution and 1Biomolecular Sciences, Boise State University, Boise, ID, USA, 2Physics, translocation dynamics of oxysterols can be substantially different from Boise State University, Boise, ID, USA. those of cholesterol, thereby influencing signalling across a membrane. The ability to precisely control delivery of a drug exclusively to a diseased Here, using atomistic molecular dynamics simulations, we present a novel site is crucial for any chemotherapy for which the therapeutic agent is char- mechanism for the passive translocation of oxysterols from one leaflet of acterized by a high toxicity to healthy cells and tissues. In this line, current a phospholipid membrane to the other. The new mechanism is viable for approaches include drug encapsulation into liposomes capable of evading tail-oxidized sterols, which are shown to translocate across a lipid bilayer the immune system, protecting their payload, and self-accumulation into tu- without rotation with respect to their principal molecular axis. Translocation mors. However, this approach suffers from the inability to control the of tail-oxidized sterols is demonstrated to be significantly faster than in the release of the drug, which in turn limits its efficacy. To alleviate this prob- commonly known translocation of cholesterol, where the molecule rotates in lem and to provide a more effective way for drug delivery, we propose using order to hydrate its OH-group both before and after translocation. We photopolymerizable lipids to control the permeability of artificial bilayer discuss how the observed mechanism may be associated with the biological lipid membranes upon UV-induced activation. Unlike previous reports, action of oxysterols as signalling molecules. our membrane composition was designed to promote the formation of lipid rafts, which facilitate local concentration of photopolymerizable compo- 1320-Pos Board B229 nents. In addition, the lipid rafts may be further stabilized by targeting pro- Theoretical and Computational Investigations into Lipid Bilayer Perme- teins, which further enhances membrane responsiveness to UV light. To ation of Drugs demonstrate this approach, we performed electrophysiology experiments Sunny Hwang, James C. Gumbart. to assess the changes in membrane permeability upon UV exposure. For Georgia Institute of Technology, Atlanta, GA, USA. improved relevancy, the transport of non-permeant, water-soluble dyes Transport phenomena across biomembranes are crucial processes in cellular through spherical bilayer membranes (liposomes) was investigated using biology, as well as in many medical, pharmaceutical, and environmental tech- fluorescence microscopy and spectroscopy experiments. In both cases, UV nologies. Therefore, determination of drug absorption is an important compo- exposure increased membrane permeability to ions and molecules, therefore nent of the drug discovery and development process in that it plays a key role in indicating achievement of controlled transport through artificial membrane the decision to promote drug candidates to clinical trials. The path of a drug systems. from the site of administration to its target cells or compartments implies the crossing of several semipermeable cell membranes, making it relevant to be 1318-Pos Board B227 able to predict whether and to what extent a molecule can pass through Membrane Leakage: Further Insight by Advanced Statistical Analysis of them. Lipid bilayers are an effective barrier to passive diffusion of ions and Vesicle Leakage some hydrophilic small molecules, but many others can permeate bilayers Stefan Braun1, Johannes Schnur1, Anja Stulz1, Heiko H. Heerklotz1,2, through passive diffusion at rates that depend on bilayer composition and prop- Maria Hoernke1,3. erties of the permeating solute. 1Pharmaceutical Sciences, Albert-Ludwigs-Universit€at, Freiburg i.Br., In this study, we consider the membrane deformation energy as the domi- Germany, 2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, nant factor in crossing the membrane into cells, as measured by in vitro

BPJ 8621_8624 262a Monday, February 19, 2018 cell-based experiments. We have investigated a new approach using the 1323-Pos Board B232 deformation free energy of a lipid bilayer based on the principle of a contin- 1/f Noise in the Anthrax Toxin Channel uum theory. To gain atomistic insight into the passive permeability process, Goli Yamini, Nnanya Kalu, Sanaz Momben Abolfath, we have used physics-based methods, namely molecular dynamics simula- Ekaterina M. Nestorovich. tions combined with the inhomogeneous solubility-diffusion model. The The Catholic University of America, Washington, DC, USA. estimated permeabilities from our method are compared with other popular ‘Pink’ or 1/f noise is a natural phenomenon widely observed in physics, eco- methods such as Quantitative Structure-Property Relationship (QSAR) nomics, astrophysics, biology, and even music and languages. In the anthrax analysis. toxin channel (PA63), it appears as fast but complete voltage-independent current interruptions with 1/f power spectral density. This complex kinetic Posters: Membrane Active Peptides and Toxins I behavior hampers the potential development of PA63 as an ion-channel biosensor. On the bright side of pink noise, the flickering in the anthrax toxin 1321-Pos Board B230 channel represents a mesmerizing phenomenon to investigate. Notably, iden- Screening of Medicines for Malaria Venture’s Pathogen Box for Cytotox- tical 1/f -type fluctuations are observed in the channel-forming components icity using a Gramicidin-Based Fluorescence Assay of clostridial binary C2 and iota toxins, which share functional and structural Thasin Peyear, Olaf S. Andersen. similarities with the anthrax toxin channel. Similar to PA63, they are evolved Physiology and Biophysics, Weill Cornell Medical College, New York, NY, to translocate the enzymatic components of the toxins into the cytosol. USA. However, the 1/f closures remained undetected in F427A PA63 mutant, where Neglected tropical diseases (NTDs) are a group of diseases that affect over the critical ‘hydrophobic belt’ of the PA63, conserved in C2 and iota toxin a billion people in the Developing World, and do not receive the attention channels and represented by the seven phenylalanine residues forming a nar- 4 they require. In order to contribute toward combatting NTDs, the Medicines row ring (so called ‘ -clamp’) in the channel lumen, is replaced by seven for Malaria Venture developed the Pathogen Box that contain drug-like mole- alanine residues. In this study we discuss the feasibility of several suggested cules with confirmed activity against malaria, tuberculosis, kinetoplastids and models for the 1/f noise. Our recent findings, including PEG partitioning ex- other pathogenic organisms that have been prioritized by the World Heath Or- periments, suggest that the 1/f noise of the PA63 channel occurs as a result of 4 ganization (WHO). To determine compounds in the Pathogen Box that have ‘hydrophobic gating’atthe -clamp region; the phenomenon earlier bilayer-modifying effects, which have been associated with cytotoxicity, we observed in many water-filled channels ‘fastened’ inside by hydrophobic are characterizing how these compounds affect lipid bilayer properties using belts. This finding can elucidate the functional purpose of 1/f noise and its a gramicidin-based fluorescence assay to probe for drug-induced changes in possible role in the translocation of the enzymatic components of binary the gramicidin monomer-dimer equilibrium. This assay exploits that drug- toxins. induced changes in lipid bilayer elastic properties alter the energetics of the 1324-Pos Board B233 monomer-dimer equilibrium, which is observed as a change in the number of Cryo-EM Structure of the Anthrax Toxin Protective Antigen Channel conducting channels (and the rate of channel-mediated ion movement across Bound to Lethal Factor lipid bilayers). Large unilamellar vesicle (LUV’s) loaded with a water- Nathan J. Hardenbrook1,2, Shiheng Liu3,4, Kang Zhou3,4, Jiansen Jiang3,4, soluble fluorophore are mixed with gramicidin channel-permeant fluorescence 3,4 1 þ þ Z. Hong Zhou , Bryan Krantz . quencher (TI ), and the rate of Tl influx is characterized by the fluorescence 1Department of Microbial Pathogenesis, University of Maryland Baltimore, quench rate. Changes in bilayer properties thus would be detected as changes in Baltimore, MD, USA, 2Department of Biochemistry and Molecular Biology, the fluorescence quench that occur due to the partitioning of the drug into the University of Maryland Baltimore, Baltimore, MD, USA, 3Department of bilayer. Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA, 4California NanoSystems Institute, 1322-Pos Board B231 University of California, Los Angeles, CA, USA. Interaction of Toxins and Peptides with Lipid Membranes Studied on a Anthrax toxin consists of three individual protein factors: the translocase Microfluidic Device channel forming protective antigen (PA), and the enzymatic factors lethal 1 € 2 € 3 1 Simon Bachler , Patrick Drucker , Alex T. Muller ,Celine Del Don , factor (LF) and edema factor (EF). Anthrax toxin is an excellent model sys- 2 3 2 Eduard B. Babiychuk , Gisbert Schneider , Annette Draeger , tem for studying the biophysical mechanisms of protein translocation. Bind- Petra S. Dittrich1. 1 ing of LF/EF to the PA pre-channel serves to stabilize their unfolding before Department of Biosystems Science and Engineering, ETH Zurich, Basel, translocation. There is limited high-resolution structural data of substrate Switzerland, 2Department of Cell Biology, University of Bern, Bern, 3 binding, specifically of the amino-terminal domain of LF (LFn), to the octa- Switzerland, Department of Chemistry and Applied Biosciences, ETH meric PA pre-channel. We were able to determine the high-resolution struc- Zurich, Zurich, Switzerland. ture of the heptameric PA in its pore conformation bound to LF at 4.6-A˚ . We developed a microfluidic device that allows for trapping, treatment, and Using lipid nanodisc, we were able to obtain random orientation of our analysis of up to 192 individual giant lipid vesicles to study the interaction PA toxin complex on a cryo-EM grid and therefore perform single-particle of molecules with lipid membranes. The vesicles were immobilized in hydro- analysis and structure determination. The PA7LF3 channel complex was in- dynamic traps where they could be isolated from the flow by actuating inte- serted into lipid nanodiscs. The structure contains the PA heptamer bound to grated round valves that are positioned around each trap. Fast exchanges of a single LF. Under these conditions, the C-Terminal domains of LF appears chemical solutions were accomplished by opening the valves without washing to be disordered, resulting in no electron density for this region of the pro- away the vesicles. tein. There appears to be no difference in the structure of the visible LFn We employed this device to monitor interactions of toxins and peptides with domain when bound to PA in the pore state vs. the pre-channel complex, lipid membranes and we observed various effects such as pore formation, with the first a-helix and b-sheet in LF unfolded and docked within the membranolysis, or permeation by means of fluorescence microscopy and a-clamp site. In addition, there appears to be a density present within the fluorescence correlation spectroscopy. (1) Exposure of pneumolysin to mem- phenylalanine clamp (F-clamp) site. Further studies are currently being per- branes with high cholesterol concentration within the lipid bilayer showed a formed in an effort to identify this density in an attempt to provide structural fast homogenous binding of pneumolysin to the membrane and pore forma- insight into the mechanisms by which proteins translocate through this PA tion within seconds. In contrast, phase-separated membranes composed of channel. ternary lipid mixtures resulted in a phase-selective binding of pneumolysin. (2) Binding of the antimicrobial peptide Lavracin to a membrane that resem- 1325-Pos Board B234 bles a bacterial cell led to the growth of membrane pores after the accumu- Elucidating the Unfolding Step in the Mechanism of Protein Translocation lation of peptide and rupture of the vesicles. (3) Permeation studies were using Anthrax Toxin done with the HIV-1 trans-acting activator of transcription domain and the Koyel J. Ghosal, Bryan A. Krantz. nona-arginine peptide. We found that the composition of the membrane Microbial Pathogenesis, University of Maryland Baltimore, Baltimore, MD, (lipids that induce a negative curvature or anionic lipids in combination USA. with cholesterol and neutral lipids) in-fluences its permeability for the tested Protein translocation across membranes is a critical step in many cellular pro- peptides. cesses, including bacterial pathogenesis. However, its molecular mechanism In conclusion, our microfluidic trap array constitutes a valuable platform for is poorly understood. The three-protein virulence factor, anthrax toxin that in- toxin or drug screening and can be used to visualize interactions of molecules toxicates cells via transmembrane protein translocation, is a well-defined with an artificial cell membrane. model for studying this process. Upon endocytosis of the anthrax toxin, the

BPJ 8621_8624 Monday, February 19, 2018 263a acidic endosomal pH triggers the protective antigen (PA) to form a transmem- were probed using FCS. Dye leakage studies on GUVs prepared with different brane channel, through which the two active enzyme components, the lethal lipid compositions indicated the preference of LLO towards the disordered re- factor (LF) and the edema factor (EF) are translocated to the host cell cytosol. gions of the membrane. Markedly different lipid diffusion dynamics were Protein translocation across the PA channel is catalyzed by two key non- observed in the leaked, unleaked and untreated vesicles indicating the connec- specific polypeptide binding sites – the a-clamp and F-clamp. The F-clamp tion between lipid mobility and its dependence on the membrane bound state being a very narrow opening, requires proteins to unfold in order for them to of the protein. Experiments with labelled LLO were carried out on GUV translocate. We hypothesize that small heterologous proteins such as Im7 can membranes and the diffusion of proteins on the GUV membrane is under be translocated through the PA channel and that they unfold through a rate- investigation. limiting transition state during translocation. To test our hypothesis, we ex- pressed and purified small proteins composed of the first 30 amino acids of 1328-Pos Board B237 LF followed by Im7 wild type (LF1-30.Im7 WT) and its mutants. Single Understanding the Pore-Forming Mechanism of Peptides Derived From channel translocation assays of the WT and its mutants were carried out using the N-Terminus of Sticholysin Haydee Mesa Galloso1, Uris Ros2,3, Pedro A. Valiente3, D. Peter Tieleman1. planar lipid bilayer electrophysiology. Results showed that the mutants trans- 1 located faster than the wild type, indicating that mutating the core residues Biological Sciences and Centre for Molecular Simulation, University of Calgary, Calgary, AB, Canada, 2Interfaculty Institute of Biochemistry, caused protein destabilization, which facilitated the unfolding process and 3 thus required less driving force for their translocation. Furthermore, denatur- University of Tuebingen, Tuebingen, Germany, Center for Protein Studies, ation studies using guanidine HCl will help determine the transition states, University of Havana, Havana, Cuba. from which, the critical step in translocation can be determined. Understand- Actinoporins are pore-forming toxins found in sea anemones that bind and oli- ing this mechanism can potentially facilitate in developing a new model for gomerize in membranes, leading to cell swelling, impairment of ionic gradi- targeted drug delivery. ents and, eventually, to cell death. These proteins when encapsulated into liposomes as an adjuvant, can induce a robust and specific CTL immune 1326-Pos Board B235 response. Therefore, they can be used in the design of vaccines capable of Cellular Entry of Diphtheria Toxin Does Not Require Formation of the forming pores and cross plasma and endosomal membranes to enhance im- Open-Channel State by its Translocation Domain mune response in anti-cancer treatments. Sticholysin I and II (StI and StII) Mykola V. Rodnin, Mauricio Vargas-Uribe, Alexey S. Ladokhin. are actinoporins produced by the Caribbean sea anemone Stichodactyla heli- Biochemistry and Molecular Biology, KUMC, Kansas City, KS, USA. anthus. Despite their high sequence identity (93%), these actinoporins exhibit Cellular entry of diphtheria toxin is a multistage process involving receptor differences in hemolytic activity that could be related to those found in their targeting, endocytosis and translocation of the catalytic domain across the en- N-termini. StII1-30 and StI1-31 are peptides spanning the first thirty (StII) or dosomal membrane into the cytosol. The latter is ensured by the translocation thirty-one (StI) N-terminal amino acid residues of StII and StI respectively. (T) domain of the toxin, capable of undergoing conformational refolding and Experimental evidence obtained by fluorescence and permeability assays sug- membrane insertion in response to the acidification of the endosome. Deci- gest that StII1-30 is more active than StI1-31 in cells. However, the molecular phering the molecular mechanism of this enigmatic system is especially determinants that explain this difference in functional activity are still un- important in light of the potential use for the T-domain for targeted drug de- known. Here, we used molecular dynamics (MD) simulations to characterize livery. While numerous now classical studies have demonstrated the forma- the mechanism of pore formation by StII1-30 and the effect of membrane cur- tion of an ion-conducting conformation, the Open-Channel State (OCS), as vature on its permeabilizing activity. The effect of StII1-30 aggregation and the final step of the refolding pathway, it remains unclear whether this channel binding to membranes was compared with its StI1-31 counterpart. Our data constitutes an in vivo translocation pathway or is a byproduct of the transloca- shows that, the higher hydrophobicity of StII1-30 facilitates not only its parti- tion. To address this question, we measure functional activity of known OCS- tion into the membrane hydrophobic core but also peptide oligomerization. blocking mutants with H-to-Q replacements of C-terminal histidines. We also Based on our MD simulations, we proposed that StII1-30 aggregation proper- test the ability of these mutants to translocate their own N-terminus across ties and its ability to induce lipid reorganization are key determinants in its lipid bilayers of model vesicles. The results of both experiments indicate lytic activity. This was tested by circular dichroism and atomic force micro- that translocation activity does not correlate with previously published OCS scopy experiments to confirm our outcome. Our results show an integrated activity. Finally, we determined the topology of TH5 helix in membrane- picture of cell membrane pore formation by these StII and StI derived inserted T-domain using W281 fluorescence and its depth-dependent quench- peptides. ing by brominated lipids. Our results indicate that while TH5 becomes a trans- bilayer helix in a wild-type protein, it fails to insert in the case of the OCS- 1329-Pos Board B238 blocking mutant H322Q. We conclude that the formation of the OCS is not Arginine Contributions to the Membrane-Active Properties of an Amphi- necessary for the functional translocation by the T-domain and that the OCS tropic Peptide from the CyaA Toxin Translocation Region Alexis Voegele1, Nicolas Sapay2, Daniel Ladant1, Alexandre Chenal1. is unlikely to constitute a translocation pathway for the cellular entry of diph- 1 2 theria toxin in vivo. A more likely scenario involves a transient passageway Institut Pasteur, Paris, France, Bioaster Technology Research Institute, due to the perturbation caused by the T-domain refolding on bilayer interface. Lyon, France. NIH:P30-GM110761. Bordetella pertussis, the whooping cough agent, secretes an adenylate cyclase toxin CyaA (1706 residue long) involved in the early stages of infection. CyaA 1327-Pos Board B236 intoxicates target cells by a unique process: it translocates its N-terminal cata- Studying Lipid Dynamics due to Listeriolysin O Binding and Pore Forma- lytic domain (residues 1 to 370) directly across the plasma membrane and pro- tion on Artificial Phospholipid Membrane Systems duces supraphysiological amount of cAMP, leading to cell death. However, the Ilanila Ilangumaran Ponmalar1, Ganapathy K. Ayappa2, Jaydeep K. Basu3. molecular process is largely unknown. Recently, we have shown that deletion 1Center for Biosystems Science and Engineering, Indian Institute of Science, of residues 375 to 485 of CyaA abrogates the translocation of the catalytic Bangalore, India, 2Department of Chemical Engineering, Indian Institute of domain into cells. We further identified a peptide, called P454 (residues 454 Science, Bangalore, India, 3Department of Physics, Indian Institute of to 484 of CyaA, translocation region), able to bind membrane, to adopt an a-he- Science, Bangalore, India. lical structure and to permeabilize lipids vesicles. Here, we have investigated Listeriolysin O (LLO), a pore forming toxin secreted by Listeria monocyto- the contributions of both arginines from P454 (R461 and R474) in membrane genes, is a cholesterol dependent cytolysin which binds to membrane choles- interaction, folding and membrane permeabilization steps. R461 and R474 terol and undergoes oligomerization to form pores ranging in size from 30 to were substituted by A, K, Q and E amino acids to characterize their contribu- 50 nm. Unregulated pore formation leads to membrane rupture and ensuing tions to the P454 peptide interaction with membrane. We found that peptide bacterial virulence in host cell. In this study, we use fluorescence correlation membrane insertion is dependent on lipid composition, attractive electrostatic spectroscopy (FCS) as well as super-resolution stimulated emission and deple- interactions and hydrophobic forces between P454 and membrane. Both argi- tion (STED) spectroscopy to correlate the changes in lipid dynamics upon nines are involved in the membrane partitioning via binding to the phosphate LLO binding and pore formation on phospholipid bilayers. Different sup- groups of the polar head of phospholipids, whatever the membrane composition ported lipid bilayer systems were used initially to understand the effect of investigated. While R474 is involved in peptide helical folding, R461 LLO on lipid dynamics. Studies using STED-FCS on supported lipid bilayer contribute to membrane permeabilization efficiency. Altogether, these data platforms reveal the changes in lipid diffusivities and deviation from Brow- indicate that both arginines are involved in the membrane-active behavior of nian diffusion in the presence of LLO. Additionally, the interaction of LLO P454 and are suggested to locally destabilize membrane to facilitate CyaA with giant unilamellar vesicles (GUVs) ranging in size from 5 to 50 micron translocation.

BPJ 8621_8624 264a Monday, February 19, 2018

1330-Pos Board B239 better understand this purinergic control of the biological activity of lysenin Membrane Activity of the Fungal Peptide Toxin Candidalysin channels, we analyzed the changes in voltage-induced gating induced upon Christian Nehls1, Julia Wernecke1,2, Laura Paulowski1, Mareike Lewke1, addition of increased concentrations of adenosine phosphates (ATP, and Helena S. Fabritz1, Julian R. Naglik3, Bernhard Hube4, Thomas Gutsmann1. AMP, respectively). Lysenin channels were inserted into planar bilayer lipid 1Division of Biophysics, Research Center Borstel, Borstel, Germany, membranes composed of asolectin, cholesterol, and sphingomyelin, and 2Deutsches Elektronen-Synchrotron, Hamburg, Germany, 3Dental Institute, their functionality with regards to voltage-induced gating was assessed Kings College London, London, United Kingdom, 4Department of Microbial from I-V plots recorded in response to linearly-variable transmembrane Pathogenicity Mechanisms, Hans Kno¨ll Institute, Jena, Germany. voltages. The I-V data has been examined within the frame of a two-state For decades, researchers have been trying to elucidate critical determi- gating model, which led to the quantification of the changes in the free en- nants of the pathogenicity of human fungi. Only recently, has an interna- ergy landscape elicited by the addition of adenosine phosphates. The com- tional consortium of scientists succeeded in identifying such a factor - a parison with similar data recorded upon addition of small amounts of peptide toxin secreted by the clinically important fungus Candida albi- multivalent cations (which act as ligands and induce gating) points towards cans. This peptide Candidalysin plays a crucial role during fungal infec- fundamentally different mechanisms of interaction. Multivalent cations bind tions of human mucosae. The peptide’s virulence manifests in the direct to the channel and elicit ligand-induced gating, which diminish the macro- damage of epithelial membranes, in the stimulation of a danger response scopic conductance, while anionic adenosine phosphates bind at a different signaling pathway and in the activation of epithelial immunity. We pro- site and reduce the macroscopic conductance most likely by a simple occlu- vided the first insights into the direct interaction between Candidalysin sion mechanism. and lipid membranes [1]. The peptide’s amphiphilic alpha-helical structure is described as a prereq- 1333-Pos Board B242 uisite for its binding to lipid membranes. In consequence of the initialized Biophysical Properties of Lysenin Channels binding, the peptide inserts between the lipid head groups and aligns par- Daniel Fologea. allel to the bilayer surface. Upon increasing surface accumulation, howev- Physics, Boise State University, Boise, ID, USA. er, the helix starts to penetrate the bilayer with oblique inclination. As a Lysenin, a pore-forming protein extracted from red earthworms, self- result, the bilayer is destabilized and transient local collapses occur. This assembles as a nonameric pore in artificial and natural lipid membranes con- carpet-like disintegration eventually leads to the disruption of the entire taining sphingomyelin. Although the cytolytic activity suggests a toxin-like membrane. A comparison of host, bacterial and fungal model membranes behavior, lysenin channels present notable features that are commonly shared will be used as an approach for understanding the selectivity of Candida- by ion channels including high transport rate and regulation by voltage and lysin for non-self membranes. The influence of peptide aggregation and ligands. In this work, we describe several salient features that set lysenin apart of C-terminal truncation on the mode of action of Candidalysin will be from other pore-forming toxins. The channel undergoes reversible voltage- discussed. induced gating at positive transmembrane potentials, which is satisfactorily [1] Moyes et al., Nature 532, 64-68 (2016). described as a Boltzmann distribution of the open and closed states. The lipid composition substantially modulates the voltage regulation, which is 1331-Pos Board B240 completely abrogated when neutral lipids are used to produce the supporting Lysenin Channel Reconstitution into Unsupported Droplet Interface lipid membrane. The channels present a large conductance hysteresis in Bilayers response to external voltages, and the history-dependence of the state distri- Christopher A. Thomas1, Devon Richtsmeier1, Aaron Smith2, bution indicates memory. Investigations of the temperature influence on the Peter Mullner3,2, Daniel Fologea1. voltage regulation reveal a strong rightward shift of the open probability dur- 1Department of Physics, Boise State University, Boise, ID, USA, 2Shaw ing inactivation, which manifests as the temperature increases. In contrast, the Mountain Technology LLC, Nampa, ID, USA, 3Micron School of Materials reactivation pathway is less sensitive to temperature changes, indicative of Science and Engineering, Boise State University, Boise, ID, USA. dynamic changes in the energy landscape stemming from conformational Droplet Interface Bilayers (DIBs) created at water/organic solvent interfaces changes of the channel. A similar behavior is shown when lysenin channels are becoming more and more popular as tools for investigating the biophysical are exposed to increased amounts of monovalent ions in the bulk electrolyte, properties of membranes and transmembrane transporters. The current ap- or to multivalent metal cations at amounts sufficient to yield electrostatic proaches for DIB preparation include the formation of a small spherical screening without inducing substantial ligand-induced gating. Our results droplet around a hydrogel support. While the stability of the formed mem- support the hypothesis that the conformational changes leading to channel brane is substantially increased by the support addition, the same support im- closure employ the movement of a specific protein domain deeper into the pedes any further changes in electrolyte composition, droplet size, later membrane. addition of molecules targeting the membrane, or a complete buffer exchange. In this work, we eliminated these shortcomings by creating DIBs without us- 1334-Pos Board B243 ing a solid support. The formation of the water droplet at the fine tip of a glass Inhibition of Bacterial Toxin Activity using Receptor-Based Peptides pipette enabled production of very stable bilayer membranes at the water/oil Eric Krueger, Shannon Hayes, Shailagne Yutuc, Angela C. Brown. interface. The solution composition was easily adjusted with a microfluidic Department of Chemical and Biomolecular Engineering, Lehigh University, injection system, which also allowed further addition of the pore-forming pro- Bethlehem, PA, USA. tein lysenin for full reconstitution in the membrane. The functionality of in- The leukotoxin (LtxA), secreted by the bacterium Aggregatibacter actino- serted lysenin channels was assessed from their response to regulatory mycetemcomitans, is a member of the RTX (repeats-in-toxin) family of stimuli such as voltage and multivalent metal ions used as ligands. The system toxins and targets leukocytes through several mechanisms. The toxin’s spec- was completed with a nanofluidic magnetic pump which allowed precise con- ificity for human white blood cells is driven by its recognition of the trol of the droplet size and membrane surface area, as inferred from micro- lymphocyte function-associated antigen-1 (LFA-1) integrin. This project in- scopy imaging and electrophysiology experiments. In conclusion, vestigates inhibition of LtxA-LFA-1 binding on living cells as an anti- unsupported DIBs may constitute a powerful tool for investigating the biolog- virulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we ical activity of transporters, along with fundamental physical features of are investigating using small peptides to block LtxA-binding sites on a b bilayer lipid membranes. LFA-1. Several binding sites on LFA-1, including a region on the L -pro- peller, have been proposed. Therefore, we have synthesized peptides corre- 1332-Pos Board B241 sponding to these domains and characterized their capability to inhibit LtxA ATP and AMP Reshape the Energy Landscape of Voltage-Gated Lysenin binding to LFA-1 and subsequent cytotoxicity in human immune cells. We Channels in a Charge and Concentration Dependent Manner found that four of the five peptides, specifically those corresponding to Daniel Prather1, Sheenah Lynn Bryant2, Nisha Shrestha2, sequential b-strands in the b-propeller domain, inhibited LtxA activity, K. Summer Ware1, Andy Bogard1, Philip Belzeski1, Daniel Fologea1. demonstrating the effectiveness of this approach. Further investigations 1Department of Physics, Boise State University, Boise, ID, USA, into the mechanism by which these peptides inhibit LtxA binding to 2Biomolecular Sciences Graduate Program, Boise State University, Boise, LFA-1 reveal a correlation between toxin-peptide affinity and cytotoxicity, ID, USA. leading to a diminished association between LtxA and LFA-1 on living cells. The interactions between lysenin channels inserted into artificial bilayer Our results demonstrate the possibility of using target-based peptides to lipid membranes and adenosine phosphates manifest as both a strong reduc- inhibit LtxA activity, and we expect that a similar approach could be used tion in conductance and a rightward shift of the voltage-induced gating. To to hinder the activity of other RTX toxins.

BPJ 8621_8624 Monday, February 19, 2018 265a

1335-Pos Board B244 1337-Pos Board B246 A Novel Membrane Peptide that Inhibits Cell Migration by Activation of Pyroglutaminated Ab-(3-42) Enhances Aggregation of Ab-Peptide on the Receptor Tyrosine Kinase EphA2 Neuronal Membranes at Physiological Concentrations: A FCS Analysis Justin M. Westerfield1, Daiane S. Alves1, Xiaojun Shi2, Yoshiaki Yano, An Takeno, Katsumi Matsuzaki. Vanessa P. Nguyen1, Robert H. Pullen III,3, Katherine M. Stefanski4, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Kristen Booth1,5, Jennifer Morrell-Falvey1,6, Bing-Cheng Wang7,8, Japan. Steven M. Abel3,9, Adam W. Smith2, Francisco N. Barrera1. The abnormal aggregation of amyloid b-peptide (Ab) into toxic oligomers and 1Biochemistry & Cellular and Molecular Biology, University of Tennessee, amyloid fibrils is central to the pathogenesis of Alzheimer’s disease. Nanomo- Knoxville, TN, USA, 2Department of Chemistry, University of Akron, lar concentrations of Ab-(1–40) and Ab-(1–42) are present at an approximate Akron, OH, USA, 3Department of Chemical and Biomolecular Engineering, ratio of 9:1 in the brain, and an increased level of more amyloidogenic and University of Tennessee, Knoxville, TN, USA, 4Genome Science & neurotoxic Ab-(1–42) is related to the early onset of the disease. Furthermore, Technology, University of Tennessee, Knoxville, TN, USA, 5Health Science interaction with cell membranes and more hydrophobic minor species, such as Center, University of Tennessee, Memphis, TN, USA, 6Biosciences Division, Ab-(1–43) and pyroglutaminated Ab-(3–42) (Ab-(3pE–42)) have been sug- Oak Ridge National Laboratory, Oak Ridge, TN, USA, 7Departments of gested to be involved in the initiation of the aggregation process. Physiology and Biophysics, Case Western Reserve University, Cleveland, In this study, we investigated the aggregation of major Abs on neuronal SH- OH, USA, 8Rammelkamp Center for Research, MetroHealth Medical Center, SY5Y cells by FCS. HiLyte Fluor (HL)-labeled Abs (HL-Ab-(1–40) or HL- Cleveland, OH, USA, 9National Institute for Mathematical and Biological Ab-(1–42)), 10 nM) and unlabeled Abs (10 nM) were mixed (total 20 nM) Synthesis, University of Tennessee, Knoxville, TN, USA. to examine the effects of various Ab species. HL-Abs were observed on cell Misregulation of the signaling axis formed by EphA2 and its ligand, ephrinA1, surface after incubation for 1 h, although they were monomeric because no sig- causes aberrant cell-cell contacts and leads to cellular transformation and ma- nificant FRET was observed between HL488-Ab and HL647-Ab. After longer lignancy in cancer. However, the activation mechanism of EphA2 is poorly un- incubation (48 h), both Ab-(1–40) and Ab-(1–42) formed 4-mers, estimated derstood. Also, solid tumors exhibit the Warburg effect, which results in an from decrease in diffusion coefficient on the cells. Addition of 10% Ab-(1–42) acidic extracellular microenvironment. Taking advantage of this property, we did not enhance the aggregation of Ab-(1–40). We found that trace amounts have used a novel approach to design TYPE7 (transmembrane tyrosine kinase (5%) of Ab-(3pE–42) enhanced the aggregation of Ab-(1–42) to 6-mers, peptide for Eph), a membrane peptide that targets EphA2. TYPE7 is pH- while Ab-(1–43) did not. Higher amounts (50%) of Ab-(3pE–42) did not responsive, a characteristic which provides solubility at neutral pH, but triggers enhance the aggregation of Ab-(1–42). In contrast to the case of Ab-(1–42), membrane insertion in acidic conditions. Decreasing the pH leads to increased the aggregation of Ab-(1–40) was not influenced by the addition of 5% Ab- partitioning and insertion of the peptide into the membrane. TYPE7 has a mem- (3pE–42). brane insertion pH50 of 6.2 (the pH point for 50% insertion). However, in the Thus, specific combination and mixing ratio of Ab species are important for the presence of the TM/JM domains of EphA2, the pH50 of TYPE7 increases to initial aggregation of Ab on cell membranes. 6.9. This effect does not occur in the presence of an unrelated TM domain, indi- cating specific interaction between TYPE7 and EphA2. Using FRET, we have 1338-Pos Board B247 determined the free energy of dimerization (DG) of the TM/JM domains of Towards a Nanoscale Description of the Interactions between Amyloid EphA2; we assess how the presence of TYPE7 alters this value. The activation Peptide Ab1-42 and Mutants with Membranes of EphA2 by TYPE7 shows important differences compared with activation by Mehdi Azouz1,2, Christophe Cullin2, Michel Lafleur1, Sophie Lecomte2. 1 a cross-linked version of the ephrinA1 (EA1) ligand. Interestingly, TYPE7 acti- Chemistry Department, Universite de Montreal, Montreal, QC, Canada, 2 vation does not involve phosphorylation of juxtamembrane residues Y588 and Chimie et Biologie des Membranes et Nanoobjets, Institut Polytechnique de Y594, suggesting a novel mechanism to release juxtamembrane inhibition of Bordeaux, Universite de Bordeaux, Bordeaux, France. the EphA2 kinase domain. Furthermore, while TYPE7 and EA1 inhibit cell Alzheimer’s disease is considered as the major form of dementia, with an esti- migration with similar efficiency, EA1 induces formation of larger EphA2 olig- mated number of 130 million of people living with the pathology by 2050, omers than TYPE7. This suggests formation of smaller oligomers might be suf- worldwide. In the project, we aim at determining the interactions between Alz- b ficient to fully activate EphA2. These results shed new light on the activation heimer’s neurotoxic peptide A 1-42 with some lipid components of neuron mechanism of EphA2. membranes. We focus our study on the effects of cholesterol and GM1 on the interaction with the peptide. Polarized Attenuated Total Reflection Infrared 1336-Pos Board B245 Spectroscopy (pATR-FTIR) allowed us to observe the impact of the membrane Ebola Virus Delta-Peptide Acts as an Enterotoxic Viroporin In Vivo composition on the interaction while high speed AFM in aqueous condition was Shantanu Guha1, Lilia Melnik2, Robert F. Garry2, William C. Wimley1. used to define the evolution of the lipid bilayer morphology upon the addition 1 Department of Biochemistry and Molecular Biology, Tulane University of the peptide. Key information about the kinetics of the bilayer fragmentation, 2 School of Medicine, New Orleans, LA, USA, Department of Microbiology peptide auto-assembly, and domain formation were collected. Lipid extraction and Immunology, Tulane University School of Medicine, New Orleans, LA, from bilayers by the peptide was quantified using the decrease of the nband in- USA. tensity observed by IR spectroscopy. Small amounts (10 mol%) of choles- Ebola virus (EBOV), part of the filovirus family, has a genome that encodes for terol, or GM1 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phospatidylcholine a partly conserved, 40-residue polypeptide, called the delta peptide, which is (POPC) membranes led to a significant lipid extraction unlike pure POPC bi- produced during Ebola virus disease pathogenesis. Sequence-structure analysis layers where the bilayers remained intact. In a complementary way, we could and a wealth of in vitro data suggest that the delta peptide is a viroporin, a observe membrane disruptions of lipid bilayers using AFM. The formation of diverse family of pore-forming peptides and proteins involved in replication peptide aggregates and of holes in bilayers were seen when membranes and pathogenesis of numerous viruses. Specifically, many filovirus delta pep- included cholesterol or GM1. These results highlight the pivotal role of mem- tides have a high abundance of cationic and aromatic residues while maintain- brane composition in the effect of Ab1-42. ing amphipathicity; these are properties that are common in membrane- Similar studies were carried out with a mutant form of the peptide forming sta- permeabilizing peptides. Full-length and conserved C-terminal EBOV delta ble oligomers with an anti-parallel b-sheet structure. ATR-IR spectroscopy and peptide fragments were shown to permeabilize synthetic lipid bilayers and mul- AFM demonstrated that this peptide had enhanced disrupting effects on mem- tiple cell types in vitro. Here, we follow up those results with an established branes. These findings highlight the crucial aspect of the aggregation state of mouse model of diarrheal pathology focusing on the small intestine. We hy- Ab1-42 and the potential greater toxicity of intermediates species generated dur- pothesized that the lytic activity observed against numerous cell types in vitro ing the fibrillation process. would also be apparent when testing the delta peptide in vivo. We found that the delta peptide exerts potent, dose-dependent enterotoxic activity against intesti- 1339-Pos Board B248 nal cells upon introduction to a closed intestinal loop. The peak activity of the Insertion Mechanism into the Lipid Bilayer of the pH Sensitive Atram Pep- peptide, which results in a diarrheal syndrome in the mice, occurs 9-12 hours tide and its Therapeutic Prospects after introduction to the small intestine. This conclusion is confirmed with his- Vanessa P. Nguyen1, Stephen J. Kennel2, Jonathan S. Wall2, tology studies in which we examined intestinal architecture and found that it Francisco N. Barrera1. was severely damaged by the delta peptide. Further, the density of mucin- 1BCMB, University of Tennessee - Knoxville, Knoxville, TN, USA, producing goblet cells in villi was diminished by the peptide. These results 2Departments of Medicine & Radiology, University of Tennessee Medical show that the EBOV delta peptide is a potential novel therapeutic target, and Center, Knoxville, TN, USA. neutralizing it could ameliorate some of the severe enterotoxic burden charac- The acidity-triggered rational membrane (ATRAM) peptide was designed to teristic of EBOV disease pathogenesis. have pH-responsive properties so that it can have the duality of being a

BPJ 8621_8624 266a Monday, February 19, 2018 peripheral or transmembrane peptide. ATRAM is a highly soluble peptide that 1342-Pos Board B251 partitions on the lipid membrane surface at neutral pH and will fold into the Using pH Sensitive Peptides for the Endosomal Release of Antibodies membrane as a transmembrane alpha helix at acidic conditions. Using biophys- Eric Wu1, Yilin Wang1, Kalina Hristova2, William Wimley1. ical techniques and fluorescence microscopy, we have furthered the under- 1Biochemistry, Tulane University SOM, New Orleans, LA, USA, 2Johns standing of the insertion pathway of ATRAM into lipid vesicles. Kinetic Hopkins University, Baltimore, MD, USA. studies show that peptide insertion occurs in at least three separate steps, Currently, the vast majority of cancer therapeutic antibodies target cell sur- including the oligomerization, helix formation, and membrane insertion. We face epitopes. However, there are many appealing targets that reside in the determined the membrane topology by evaluating the cellular uptake of fluores- cytosol that are either overexpressed or uniquely expressed in cancer cells. cently labeled ATRAM coupled to PEGylated liposomes. In addition, by per- These targets have not been fully explored, largely due to the difficulty in forming binding studies we have gained insight of binding cooperativity. delivering antibodies into the cell without significant degradation by the Finally, mice studies demonstrated that ATRAM has an extended circulation endosome/lysosome pathway. Development of a method that allows anti- half-life. Our data highlight the potential of ATRAM to be further developed bodies to escape this hydrolytic fate and be released into the cytosol would as a specific therapeutic agent for diseases that lead to acidic tissues, including open up an entirely new set of cancer therapeutic targets for exploration. cancer. The pH sensitive, pore forming peptide pHD108 is a 26 residue peptide that is a gain-of-function variant of melittin, a naturally found bee venom 1340-Pos Board B249 peptide known to form pores on membranes. Multiple acidic glutamate res- Use of Spontaneous Membrane Translocating Peptide for Cytosolic Deliv- idues in pHD108 are thought to confer the pH triggered membrane perme- ery of Biologically Active Polar Peptide abilization activity. In synthetic unilamellar vesicles, pHD108 has no Jenisha Ghimire, Taylor Fuselier, William C. Wimley. poration activity at pH 7 but at pH 5 it releases macromolecules at very Department of Biochemistry and Molecular Biology, Tulane University low peptide concentration. Thus pHD108 may enable release of uptaken School of Medicine, New Orleans, LA, USA. antibodies from endosomes. Little work has been done with pHD108 in The hydrophobic nature of cell membranes prevents the cytoplasmic entry of living cells or in animal models. The aims of this research include 1) modi- potentially useful polar cargoes. To evade this issue, the spontaneous mem- fying pHD108 via lipidation, conjugation, etc. to find the most effective brane translocating peptides (SMTPs) were previously selected via orthog- form of the peptide in terms of cellular membrane activity and endocytosis onal high-throughput screening for their ability to spontaneously rate, 2) determine the efficiency of endosomal release of macromolecules, translocate through synthetic lipid bilayers. These SMTPs are also able to including antibodies, 3) determine the mechanism behind endosomal translocate through the plasma membranes of cells, carrying polar dyes as release, and 4) use this model to screen different antibodies for cancer cargo. Based on this preliminary data, we hypothesize that SMTPs can be cell specific cytotoxicity. Success in this research would result in identi- used for cellular delivery of some biologically active, membrane- fying a potential cancer therapeutic as well as providing a novel means impermeant polar cargoes. To test this hypothesis, we use a bicyclic polar to study the blocking/neutralizing effects of antibodies against intracellular peptide phalloidin, a toxin isolated from the deadly Amanita phalloides targets. ‘‘death cap’’ mushroom, as a cargo and linked it to one of the SMTPs, TP2. Phalloidin inhibits actin polymerization, therefore we perform assays 1343-Pos Board B252 of cell mobility and viability to determine the ability of TP2 to translocate Synthetic Molecular Evolution of Hybrid Cell Penetrating Peptides that phalloidin through cell membrane into the cytoplasm. Our results verify Efficiently Deliver Peptide and Peptide Nucleic Acid Cargoes to Cells the cytoplasmic entry of this peptide-cargo conjugate, showing that SMTPs William Kauffman, William Wimley. can deliver useful cargoes to living cells. Department of Biochemistry and Molecular Biology, Tulane University, New Orleans, LA, USA. 1341-Pos Board B250 Peptides and peptide nucleic acids (PNAs) have long been recognized as Liposome Delivery System of Antimicrobial Peptides against Huanglongb- promising tools and potential therapeutics. Yet the cell membrane remains ing (HLB) Citrus Disease a significant barrier to their intracellular targets. Conjugation to cell pene- Jeanette C. Velasquez Guzman, Supratim Basu, Roel Rabara, trating peptides (CPPs) like pTat48-60 (tat) and pAntp43-68 (penetratin) fa- Loan K. Huynh, Gargi C. Basu, Hau B. Nguyen, Goutam Gupta. cilitates delivery, however delivery efficiencies remain low. Improving the New Mexico Consortium, Los Alamos, NM, USA. performance of known CPPs by rational design is hindered by the lack of Huanglongbing (HLB) is a vector-borne disease in citrus caused by Liberi- explicit design principles. Instead, here we use synthetic molecular evolu- bacter - a gram negative bacterium. Following its first detection in 2005, tion (SME) to generate and screen a CPP library containing 8,192 tat/pen- HLB has been widespread in Florida, Texas and recently appearing in Califor- etratin hybrid peptides to identify sequences with improved ability to nia. This fatal disease has serious damage on US citrus production but there is deliver PNA, using the Hela pTRE-LucIVS2 (Hela705) system. The parent no cure currently available yet. In this project, we focus on Liberibacter killing sequences poorly deliver PNA705 to cells. However, at 5mM peptide-PNA, in already infected citrus by anti-microbial peptides or therapeutic proteins via the top performing Delivery Peptide (PDEP)-PNA705 daughter sequence targeted liposome vesicle delivery. showed an 80-fold increase over PNA705 only treated cells in properly We have designed and synthesized eleven amino acid long membrane- spliced luciferase mRNA (qRT-PCR) and 33-fold higher standardized lumi- targeting small peptides with antimicrobial activity against Gram negative nescence values than the top performing parent sequence penetratin. The bacteria. Since Liberibacter is not culturable, our studies have been focusing PDEPs identified in this study are effective in multiple cell types, and on characterizing the peptide’s antimicrobial activity on several gram- deliver a peptide cargo to cells. The capabilities of the PDEPs make them negative bacteria surrogates such as Escherichia coli, Salmonella typhimu- a valuable research tool for delivery of membrane impermeable PNA or rium, Agrobacterium tumefaciens,andSinorhizobium meliottii.Thelast peptide sequences. This dramatic improvement in performance following two are Liberibacter surrogates. Our minimum inhibitory concentration a single iteration of SME is an indication of the power of this approach (MIC) experimental results indicated that P11-1 peptide is the most active to peptide sequence optimization. one. We have been developing a liposome vesicle delivery system for antimicro- 1344-Pos Board B253 bial peptides, therapeutic protein chimeras and non-toxic compounds. These Dissecting Drug Physico-Chemical Profiles as They Relate to their Bilayer capsules are similar to the virus-based delivery systems that have been used Modifying Potency for in planta drug delivery. Our ability to encapsulate therapeutic proteins Radda Rusinova1, Roger E. Koeppe II,2, Olaf S. Andersen1. and compounds has already been demonstrated using Green Fluorescent Pro- 1Weill Cornell Medicine, New York, NY, USA, 2Chemistry and tein (GFP), antimicrobial peptides and small molecules. We have success- Biochemistry, University of Arkansas, Fayeteville, AR, USA. fully encapsulated P11-1 antimicrobial peptide. In addition, we have been Amphiphilic compounds across many diverse drug classes alter lipid bilayer able to express and purify Citrus Tristeza Virus (CTV) capsid protein in properties. This is important because the hydrophobic coupling between both tobacco BY-2 cell cultures and in tobacco plant. Coating liposomes membrane proteins’ hydrophobic domains and the bilayer core results in en- with CTV capsid protein is expected to facilitate in planta transport and ergetic coupling between changes in lipid bilayer properties and membrane phloem targeting of the liposome vesicles. Additional effort on encapsu- proteins’ conformational preference, such changes in bilayer properties alter lating antimicrobial peptides and coating the liposome vesicles with CTV membrane protein function by a mechanism that does not involve drug bind- capsid protein to deliver the peptides to the phloem of citrus plants will ing to the protein. It thus becomes important to understand how drugs alter be demonstrated. bilayer properties at clinically relevant concentrations. Our previous work

BPJ 8621_8624 Monday, February 19, 2018 267a has shown that the drugs’ physico-chemical profile, rather than hydrophobic- decreased by rotating peptide to adopt perpendicular orientation to the mem- ity alone, dictate their bilayer-modifying potency. As drugs traverse varying brane plane. Low passive permeability of polar peptides across the planar environments, i.e. pH differences in segments of the gastro intestinal system, lipid bilayer may be improved upon thinning or other significant deforma- the ensuing changes in drug charge may alter bilayer partitioning, such that a tions of the bilayer. The computational method can also be used for cyclic drug’s pKa and the local pH (which determine the net drug charge) may peptides and small proteins with known structure. The method was imple- affect its interactions with cell membranes (and proteins). We explore this mented into a web server that is available through the CellPM website. question using gramicidin channels as reporters to quantify amphiphile ef- This web server uses as an input the amino acid sequence or 3D structure fects on lipid bilayer properties. We previously reported that amiodarone of a peptide in PDB format. As an output, the server produces the following and troglitazone alter bilayer elasticity at their clinical concentrations. To data: (1) peptide structure and parameters of its association with the mem- understand how pH affects drug interactions with the lipid bilayer, we brane, including energy of membrane binding, penetration depth and tilt examine changes in bilayer-modifying potency of a group of drugs with angle; (2) energy profile of a peptide along the membrane normal; (3) the varying pKa, including amiodarone and troglitazone. As we vary their log of the permeability coefficient that defines the ability of a peptide to charge by changing pH to below and above their respective pKa, we find passively cross the lipid bilayer; (4) downloadable coordinate file with opti- that increasing the charged population results in increased bilayer- mized peptide structures along the lowest energy translocation pathway; and modifying potency even though the bilayer partition coefficients of charged (5) the visualization of a peptide translocation pathway across the membrane compounds tend to be less than their neutral counterparts. The increased with GLmol. bilayer-modifying potency of charged compounds may be due to altered localization of charged compounds at the interface. We conclude that drug 1347-Pos Board B256 interactions with lipid bilayer, and their effect on bilayer properties, are Cell-Penetrating Peptide for Transcellular Transport: The Effect of highly dependent on its environment. Physico-Chemical Properties on Permeability Alexander Komin1, Ran Lin2, Honggang Cui2, Peter C. Searson1, 1345-Pos Board B254 Kalina Hristova1. Control of Cell-Selective Activity of Membrane-Active Polyleucine-Based 1Materials Science and Engineering, Johns Hopkins University, Baltimore, Peptides using Database-Guided High-throughput Screening MD, USA, 2Chemical and Biomolecular Engineering, Johns Hopkins Charles H. Chen1,2, Charles G. Starr3, Shantanu Guha3, Jochen Burck€ 4, University, Baltimore, MD, USA. Anne S. Ulrich4, William C. Wimley3, Martin B. Ulmschneider1,2. Many central nervous system (CNS) diseases (e.g. brain cancer, Alzheimer dis- 1Chemistry, King’s College London, London, United Kingdom, 2Materials ease) do not have an effective drug therapy. The major challenge lies in Science and Engineering, Johns Hopkins University, Baltimore, MD, USA, crossing the blood-brain barrier (BBB), which separates the circulating blood 3Department of Biochemistry and Molecular Biology, Tulane University from the brain. Cell-penetrating peptides (CPPs) have been proposed as a po- School of Medicine, New Orleans, LA, USA, 4Institute of Biological tential drug delivery vehicle across the BBB. However, while CPPs can enter Interfaces, Karlsruhe Institute of Technology, Karlsruhe, Germany. cells, many cannot efficiently translocate across to deliver drugs. We have iden- We have developed a new 14-residue antimicrobial peptide template sequence tified a CPP, called the CL peptide, that demonstrates an ability to deliver a containing four residues: L, D, K, and A. This template, called LDKA, which small-molecule cargo across an in vitro epithelial barrier at a moderately was designed using atomic detail structural information derived from unbiased high rate. In this study, we investigate the effect of secondary structure, charge, long-timescale equilibrium molecular dynamics folding, partitioning, and pore hydrophobicity, cargo, and other parameters on the barrier-penetrating property assembly simulations, shows low micro-molar antibacterial activity against of the CL peptide. several gram-positive and gram-negative bacteria. Furthermore, LDKA was shown to form pores in microbial and eukaryotic membrane models using fluo- 1348-Pos Board B257 rescent dye leakage assays. A library of 2,916 peptides was designed using this Cell-Penetrating Peptides with Antimicrobial, Transfection and Transduc- template sequence and high-throughput screened using an orthogonal vesicle tion Activities 1 1 1 1 leakage assay. Dyes of different sizes were embedded into vesicles with vary- Justine Wolf , Louic Vermeer , Arnaud Marquette , Morane Lointier , 1 1 1 2 ing lipid composition to simultaneously screen for both pore size and affinity to Jesus Raya , Philippe Bertani , Dennis Wilkens Juhl , Antoine Kichler , 3 3 € 3 4 5 charged and neutral membranes. From this screen, 9 different LDKA variants Martin Gotthardt , Max Wittmann , Regine Suss , Loic Hamon , Anne Galy , David Fenard5, Burkhard Bechinger1. were selected, sequenced, synthesized, and further characterized biophysically. 1 2 Remarkably, even though the selected sequences displayed only minor muta- Chemistry, University of Strasbourg, Strasbourg, France, Pharmacy, University of Strasbourg, Strasbourg, France, 3Pharmacy, University of tional changes, each of these peptides has unique functional properties, forming 4 5 either small or large pores and being selective for either neutral or anionic Freiburg, Freiburg, Germany, University of Evry, Evry, France, Genethon/ charged bilayers. Further screening of these peptides for bacterial and haemo- Inserm, Evry, France. lytic activity revealed good correlation between charged membrane model A family of histidine-rich peptides LAH4 was designed using linear cationic leakage and antibacterial activity, as well as neutral membrane leakage and peptides such as magainins as a template. These designed peptides have been haemolytic activity. Antimicrobial activity is different for each peptide and de- shown to exhibit considerable antimicrobial, nucleic acid transfection as well pends on the bacterial species. Analysis of the sequences reveals no clear motifs as cell penetrating activities. In contrast to their natural templates their mem- or explanation for these strong preferences, but suggests that hydrophobic brane interactions are strongly pH dependent. The delivery of cargo by these moment and sequence arrangement are more important for membrane selec- peptides is complex, involving many steps, which we investigated on a struc- tivity than net charge. The results reveal that simple sequence-function rela- tural and biophysical level. Recently, vectofusin-1, a member of the family tionships for peptides that can form a large repertoire of functional structures of LAH4 peptides has been shown to spontaneously self-assemble into helical may remain elusive and that it may be the peptide sequences propensity to coiled-coil structures, spherical aggregates, that further assemble into annular aggregate and assemble in a given environment that holds the key to functional and extended nanofibrils and hydrogels as a function of phosphate and in a prediction. pH-dependent manner. This bears considerable interest for the design of bioma- terials. Furthermore, the peptide has a strong capacity to enhance the gene 1346-Pos Board B255 transfer by lenti- and adeno associated viruses into the cell interior. Thereby, Modeling of Peptide Folding and Translocation across Membranes the fibers formed by this short peptide have gene therapeutic applications Andrei L. Lomize, Irina Pogozheva. ranging from monogenic and infectious diseases to cancer, by enhancing trans- College of Pharmacy, University of Michigan, Ann Arbor, MI, USA. duction levels of target cells and reducing the amount of lentivirus for greater A theoretical approach for fast computational modeling of three-dimensional safety and reduced costs. Vectofusin-1 promotes the entry of several retroviral (3D) structure, binding, folding, and passive diffusion of peptides across the pseudotypes into target cells when added to the culture medium, without cyto- lipid bilayer was developed. This method combines a thermodynamic model toxicity. These associate with viral particles allowing them to be easily pel- for a-helix and b-sheet formation, energy refinement of all-atom models, leted. These fibrils have a unique coiled-coil a-helical structure whereas positioning of the molecules in the bilayer using an anisotropic solvent most other viral transduction enhancers form b-amyloid fibrils. Our observa- model, and optimization of the rotational orientation of the molecule in tions define vectofusin-1 as a member of a new class of a-helical lentiviral every point of the transmembrane trajectory to find the lowest energy trans- transduction enhancers. Its coiled-coil fibril formation is reversible which bears location pathway. Calculation results are consistent with experimental considerable advantages in handling the peptide in conditions well-adapted to studies of more than 300 peptides in micelles and membranes, including scalable gene therapy protocols. References: J PepSci 21, 346 (2015), JPC B their secondary structure, membrane binding energies and tilt angles. 119, 9678 (2015), JBC 291, 2161 (2016), Acta Biomat (2017 in press), SciRep Modeling of translocation pathways shows that energy barriers can be 7:9585 (2017).

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Posters: Membrane Structure II Annu. Rev. Biophys. 46, 379-410. [4] M.T. Marty et al. (2016) Angew. Chem. 55, 550-554. 1349-Pos Board B258 1351-Pos Board B260 Solid-State 2H NMR Investigations of Viral M2 Ion Channel Drugs 1 1 2 1 Simulating GPCRS in Minimal Membrane Mixtures Soohyun Lee , Rami Musharrafieh , Xiaolin Xu , Andrey V. Struts , 1 1 2 3 3 1 1,2 Edward R. Lyman , Lewen Yang , Anne Robinson , Noah Malmstadt , Jun Wang , Trivikram R. Molugu , Michael F. Brown . 4 1 Ilya Levental . Department of Chemistry and Biochemistry, University of Arizona, Tucson, 1 2 Department of Physics and Astronomy, University of Delaware, Newark, AZ, USA, Department of Physics, University of Arizona, Tucson, AZ, USA, 2 3 DE, USA, Department of Chemical and Biomolecular Engineering, Tulane Department of Pharmacy and Toxicology, University of Arizona, Tucson, University, New Orleans, LA, USA, 3Department of Chemical Engineering, AZ, USA. Department of Materials Science and Engineering, University of Southern The influenza A M2 proton channel is pH-activated and essential for viral California, Los Angeles, CA, USA, 4Department of Integrative Biology and replication. Accordingly, M2 is an important target for antivirals such as Pharmacology, University Texas Medical School, Houston, TX, USA. adamantanes and spiro-piperidines that bind the channel pore to block pro- Many membrane proteins have been shown to be functionally dependent on ton flux [1]. NMR methods are powerful tools for measuring chemical cholesterol and other lipids. Both specific lipid binding sites and membrane structure and dynamics at atomic resolution. Therefore, we applied 2 curvature stress are thought to regulate function. In this work, a combina- solid-state H NMR spectroscopy to investigate the fundamental physical tion of coarse-grained ‘‘Martini’’ and all-atom simulations are used to properties for methyl-deuterated rimantadine-d3, amantadine-d-15,and 2 investigate these two mechanisms in the context of the A2A adenosine spiro-piperidine-d4 in the crystalline state [2, 3]. The H NMR longitudinal receptor. In unbiased simulations, the ‘‘cholesterol consensus motif’’ is Zeeman (R1Z) and quadrupolar-order (R1Q) relaxation rates and transverse QE observed to bind cholesterol in the active receptor state, but not when quadrupolar-echo decay R2 rates were measured as a function of temper- bound to antagonist. CG and Martini simulations together identify two ature to acquire molecular dynamics information related to the chemical new cholesterol binding sites on helices 5 and 6. One of the new sites is structure. Solid-state NMR spectra for rimantadine-d3 revealed a Pake also observed to bind cholesterol in several recent high resolution crystal powder pattern [2] with a residual quadrupolar splitting of 36 kHz, indi- structures of the protein, and in the simulations interacts with cholesterol cating fast anisotropic methyl rotation. Curiously, plots of R1Z and R1Q only when bound to the inverse agonist ZM241385. Metadynmics simula- relaxation rates as a function of temperature showed contrasting behavior tions are used to determine the affinity of these sites for cholesterol under versus a model assuming only methyl rotation contributing to relaxation. different conditions. Given that cholesterol content in plasma membranes Moreover, we observed multiple relaxation minima indicating complex varies with cell type and upon administration of widely prescribed pharma- molecular dynamics. Our data suggest a slower motion possibly due to mo- ceuticals, such as statins, understanding cholesterol-dependent function is lecular tumbling plus fast methyl rotation. Such composite phenomena are an important step towards exploiting membrane compositional variation generally expected in plastic crystals. Indeed, motional averaging of the re- for therapeutic ends. sidual quadrupolar couplings by slow molecular tumbling explains the experimental residual quadrupolar couplings. Detailed model-free spectral 1352-Pos Board B261 density analysis [3] provides the correlation times and activation energies Quantifying Nanoscale Morphological Features of the Primary Cilium of the respective motional processes. This work reveals new fundamental Membrane using Super-Resolution Fluorescence Microscopy chemical properties associated with a pharmaceutically important class Joshua Yoon, Lucien Weiss, Ljiljana Milenkovic, Tim Stearns, of drugs. Further studies will aim to elucidate the structural and dynamical W.E. Moerner. properties in membranes and proteins. [1] A.D. Balgi et al. (2013) PLOS Stanford University, Stanford, CA, USA. One 8, e55271. [2] T.R. Molugu et al. (2017) Chem. Rev. DOI:10.1021/ac- We present a method for capturing and quantifying the nanoscale morpho- s.chemrev.6b00619.[3] X. Xu et al. (2014) eMagRes 3, 275. logical features of the primary cilium in mammalian cells. This finger-like organelle protrudes outside the plasma membrane on the order of 2-5 mm and is specialized to help facilitate Hedgehog signaling pathway activation. 1350-Pos Board B259 To study the primary cilium structure, we fix cells where individual trans- Investigation of Photoinduced Oligomerization of Rhodopsin by Native membrane proteins, Smoothened, are tagged with single fluorescent labels. Mass Spectrometry Using three-dimensional super-resolution fluorescence microscopy, we 1 1 1 Steven D.E. Fried , William C. Resager , Suchithranga M.D.C. Perera , localize these proteins with 15-25 nm precision, providing us with a poin- 1,2 1 Michael F. Brown , Michael T. Marty . tillistic reconstruction of the ciliary membrane. We then apply an implicit 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, 2 surface fitting algorithm on our point cloud to generate a triangulated sur- AZ, USA, Department of Physics, University of Arizona, Tucson, AZ, USA. face mesh. This allows us to visualize and measure Mean Curvature (H)/ The photoreceptor rhodopsin is a canonical G-protein-coupled receptor Gaussian curvature (K) profiles along the shaft of the primary cilium. (GPCR) that is responsible for scotopic vision under low light conditions Not only do we measure the heterogeneity in shape from one cilium to [1,2]. Because of the dense packing of rhodopsin within the rod outer another, but we also show a shift in curvature toward negative K values segment membranes, the oligomeric state of the receptor in both when retrograde ciliary cargo transport is severely impaired. Our method inactive and activated conformations has been difficult to establish. Here, can be directly applied to studying membrane deformations present in we utilize native electrospray ionization mass spectrometry (ESI-MS) of various ciliopathies and cell features where curvature plays a role in deter- bovine rhodopsin in detergent micelles to probe the oligomeric state of mining the overall shape. the protein in response to photoactivation. The mass spectrum reveals that dark-state rhodopsin is a monomer with a single major peak corresponding 1353-Pos Board B262 to the predominant proteoform, plus several minor peaks indicating alterna- Methylene Volumes in Monoglyceride Bilayers are Larger than in Liquid tive post-translational modifications (PTMs). However, we discovered that Alkanes following activation by light, detergent-solubilized rhodopsin forms Brian C. Seper1, Anthony Ko2, Aaron Abma1, Andrew D. Folkerts1, higher-order oligomers. By comparing the time-dependent MS signal loss Stephanie Tristram-Nagle2, Paul E. Harper1. following online photobleaching of various proteoforms of rhodopsin, we 1Department of Physics and Astronomy, Calvin College, Grand Rapids, MI, observed the stabilizing effect of palmitoyl groups on the monomeric state USA, 2Department of Physics, Carnegie Mellon University, Pittsburgh, PA, of the receptor. The titration of POPC, POPE, and POPG lipids into the USA. detergent-protein complexes shows that specific lipids play differential The densities as a function of temperature of four fully hydrated saturated roles in stabilizing or destabilizing the monomeric state of the protein. monoglycerides with even chain lengths ranging from eight to fourteen Consistent with the known impacts of these lipids on rhodopsin stability were determined by vibrating tube densitometry and their phase transition [3], the POPE lipids favored receptor oligomerization, whereas POPC stabi- temperatures were determined by differential scanning calorimetry (DSC). lized the protein monomer. Future directions will further investigate this ef- We find the volume of a methylene group in a monoglyceride bilayer is 2 fect through the use of lipoprotein nanodiscs to yield new information on percent larger than in liquid alkanes, matching the methylene group volumes lipid-protein interactions [4]. Our results provide important new insights found in phosphatidycholine (PC) bilayers. Typically, the ratio of terminal into the mechanisms by which non-protein biological components regulate methyl volume (CH3) to methylene (CH2) volumes is assumed to be 2. By the oligomeric state of rhodopsin during the activation process. [1] analysis of alkane data, we find this ratio actually increases from 1.9 to 2.3 S.M.D.C. Perera et al. (2016) J. Phys. Chem. Lett. 7, 4239-4235. [2] U. with increasing the temperature from 0 C to 100 C. For a rough sense of scale, Chawla et al. (2016) Angew. Chem. 128, 598-602. [3] M.F. Brown (2017) we note that to effect a 2 percent reduction in volume requires of order 200

BPJ 8625_8627 Monday, February 19, 2018 269a atmospheres of pressure; pressures of this magnitude are biologically rele- 1356-Pos Board B265 vant. For instance, this amount of pressure is sufficient to reverse the effect Atomistic Insights into the Unique Roles of Lipopolysaccharide Modifica- of anesthesia. The component volumes obtained are an important parameter tions in Strengthening Bacterial Outer Membrane Defenses used for determining the structure of lipid bilayers and for molecular dy- Amy Rice, Jeff Wereszczynski. namics simulations. Illinois Institute of Technology, Chicago, IL, USA. Lipopolysaccharides (LPS) are a main constituent of Gram-negative bacterial 1354-Pos Board B263 outer membranes. Salmonella enterica, among other bacteria, are able to Molecular Dynamics Simulations of Stratum Corneum Model Membranes chemically modify the structure of their LPS in response to environmental Eric Wang, Jeffery Klauda. stimuli as a defense mechanism. Recent experiments have shown that these Department of Chemical and Biomolecular Engineering, University of modifications make the outer membrane more resistant to large lipophilic Maryland, College Park, MD, USA. drugs and antimicrobial peptides, and suggest that LPS modification prevents Recent studies in stratum corneum structure and permeability have focused the penetration of large molecules through a strengthening of lateral interac- tions between neighboring LPS molecules. We performed a series of nine 7- on the role of ceramides (CER). In this study, we use the all-atom m CHARMM36 (C36) force field to simulate bilayers using pure CER, binary s molecular dynamics simulations to study how each of three key S. enterica mixtures of phosphatidylcholine (PC) and CER, and ternary mixtures of LPS modifications affect important bilayer properties, with a focus on lateral CER, lignoceric acid (LA), and cholesterol (CHOL). For PC/CER bilayers, interactions and membrane structural characteristics. Our results highlight the conditions are replicated from experimental studies, and concentration unique effects each modification has on strengthening the bacterial outer membrane, and help improve our understanding of outer membrane chemical (XCER) is varied to probe the effect of CER on these systems. Based on the deuterium order parameters and bilayer thickness, C36 results are in good properties. agreement with experiment which supports the use of C36 for more diverse membranes. Additionally, XCER is shown to have a profound effect on nearly 1357-Pos Board B266 all membrane properties including surface area per lipid, chain order and tilt, Exploration of Lipid Composition in Circularized Nanodiscs area compressibility moduli, bilayer thickness, hydrogen bonding, and lipid Manuel Castro, James M. Hutchinson, Charles R. Sanders. clustering. Hydrogen bonding in particular can significantly affect other Biochemistry, Vanderbilt University, Nashville, TN, USA. membrane properties and can even encourage transition into the gel phase. Protein nanodiscs (NDs) are membrane mimetics which can simulate a lipid Despite CER’s tendency to condense the membrane, an expansion of CER bilayer environment by enveloping lipids with membrane scaffold protein lipids with increasing XCER is possible depending on how the balance be- (MSP). Recently, the lab of Gerhard Wagner advanced nanodisc technology tween various hydrogen bond pairs and lipid clustering is perturbed. Based by introducing covalent circularization of the MSP to increase disc homoge- on gel phase transitions, support is given for phytosphingosine’s role as a neity, resulting in circularized nanodiscs (c-NDs; ML Nasr et al, Nature hydrogen bond bridge between sphingosine ordered domains in the stratum Methods 2017). Here we report exploration of the range of lipid compositions corneum. Additionally, ternary CER/LA/CHOL mixtures are simulated at that can be accommodated in this new nanodisc format. We used electron mi- varying temperatures and unexpectedly reveal that the equilibrium state con- croscopy (EM) and dynamic light scattering (DLS) experiments to charac- tains inverse micelles in the center of the bilayer. The size of these inverse terize c-NDs of varying lipid composition under both protein-free and micelles is highly dependent on temperature with physiological systems con- membrane protein-integrated conditions. We also examined the quality of taining a majority of the ordered lamellar phase. Inverse micelles result from the NMR spectra of the cND-associated membrane proteins. Our results pro- lipid flipping of LA and CER which occurs over short (<50 ns) timescales. vide insight into optimization of sample conditions for structural biological Further study of these systems will involve varying CER type and probing investigations that employ circularized nanodiscs as model membranes. We the permeability of ethanol with umbrella sampling. thank ML Nasr for helpful discussion and the Wagner lab for making the re- agents needed for preparation of cNDs freely available. This work was sup- 1355-Pos Board B264 ported by US NMR grants R01 AG056147, R01 HL122010, and R01 095989. Molecular Dynamics Modeling of Pseudomonas Aeruginosa Biological MAC was supported by NSF graduate research fellowship program Membrane (ID: 2016222570). Ao Li, Xin Yong. Mechanical Engineering, Binghamton University, State University of New 1358-Pos Board B267 York, Binghamton, NY, USA. Nanoscale Structure of Lipid Bilayers Revealed By In-Silico and Experi- Pseudomonas aeruginosa, a common gram-negative bacterium and human mental Small Angle Neutron Scattering pathogen, could cause serious infections among the patients with burn wounds, Mitchell Dorrell1,2, Fred Heberle3, John Katsaras3,4, Ed Lyman2,5, cystic fibrosis, acute leukemia, organ transplants, and intravenous-drug addic- Alexander Sodt1. tion. The distinctive outer membrane (OM) structure of P. aeruginosa plays a 1Eunice Kennedy Shriver National Institute of Child Health and Human fundamental role in its virulence, colonization ability and inherent antibiotic Development, National Institutes of Health, Bethesda, MD, USA, resistance. Furthermore, outer membrane derived vesicles (OMVs) are para- 2Department of Physics and Astronomy, University of Delaware, Newark, mount in many disease-related processes. To provide critical insights into DE, USA, 3Biology and Soft Matter Division, Oak Ridge National OM functionality and OMV biogenesis, we conducted all-atomistic molecular Laboratory, Oak Ridge, TN, USA, 4Department of Physics and Astronomy, dynamic study of P. aeruginosa physiologically relevant asymmetric mem- The University of Tennessee, Knoxville, TN, USA, 5Department of brane. We hybridized a GLYCAM06-based force field with the Stockholm Chemistry and Biochemistry, University of Delaware, Newark, DE, USA. lipids force fields (Slipids) to model four asymmetrical membranes with Lipid Through the combination of simulations and numerical analysis with small angle A (LPA) as one leaflet and phospholipids 1,2-dipalmitoyl-sn-glycero-3-phos- neutron scattering, we probe the lateral organization of lipid bilayer mixtures. phoethanolamine (DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine Small Angle Neutron Scattering (SANS) is an excellent complement to optical (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-dio- techniques, as it reveals molecular scale structural details without fluorescent leoyl-sn-glycero-3-phosphoglycerol (DOPG) as the other, respectively. The probes. On its own, however, SANS is not sufficient to determine the organiza- detailed structure of membranes was characterized by a series of analysis of tion of the membrane. We therefore take a two-pronged approach, comparing transmembrane density, area per lipid, angle distribution, radial distribution molecular dynamics simulations of ternary mixtures to experimental data ob- function (RDF), and deuterium order parameter (SCD). The discrepancies of tained for the same mixtures. In-silico neutron scattering experiments on simu- area per lipid and backbone tilt angle of LPA in different membranes are quite lated membranes are compared to the experimental data, simultaneously subtle. The angle distribution together with the RDF provides the hydration validating the simulation data and providing a molecular scale model of the profile of two phosphate groups of LPA, which agrees well with experimental experimental system. Our all-atom molecular dynamics simulations use pre- observation. The liquid-crystalline phase of the membranes were determined existing software packages (e.g., Gromacs) to produce atomic trajectories of by SCD parameter and the different fluidity of LPA highlights the impact of ten microseconds in length. These trajectories are processed and analyzed using the corresponding phospholipid leaflet. Overall, the relative ordered acyl chain custom software to generate scattering intensities for comparison to experiment, of LPA compared to cytoplasmic cell membrane contributes to the low perme- assuming laterally averaged scattering within nanoscopic domains. Simulated ability of the bacteria outer membrane. The findings of this computational scattering intensities are fit to experimental data to extract information about investigation of P. aeruginosa outer membrane will further the understanding the lateral structure. In this way, the transverse variation in scattering length den- of microbial pathogenesis and aid in the design of effective treatments for in- sity across the bilayer — which, under fully deuterated solvent, is much more sig- fectious diseases. nificant than lateral contributions — is used to infer lateral structure.

BPJ 8625_8627 270a Monday, February 19, 2018

1359-Pos Board B268 micron large), rendering these complex models computational taxing. Thus, Lipopolysaccharide, Structure and Assembly of Bacterial Outer Mem- raising the question - what model complexity is needed for realistic lipid branes domain behavior? Boyan Bonev. Here we constructed CG Martini plasma membranes models of varying School of Life Sciences, University of Nottingham, Nottingham, United lipid complexity (7 up to 63 different lipid types). Lipid tail and headgroup Kingdom. combinations were varied within an expected biological distribution at Lipopolysaccharides (LPS) comprise the outermost leaflet in the cellular en- different resolutions. The different models where simulated in multiple me- velope of Gram-negative bacteria. Multiple phosphates and pyrophosphates dium to large-scale simulations. For each model, we analyzed a variety of enhance outer membrane stability, whilst also serving as surface epitopes for membrane physicochemical properties, including: lipid-lipid interactions, recognition by antimicrobials. Folding of outer membrane proteins in bacte- bilayer bulk material properties, domain formation and coupling between ria is mediated by the beta-barrel assembly machinery (BAM) complex. We the bilayer leaflets, evaluating which properties can be retained at lower investigate structural features of LPS using solid state and DNP-enhanced complexity. MAS NMR, as well as the role of LPS as receptors for antimicrobials. This work performed under the auspices of the U.S. Department of Energy by Species-specific phosphorylation in the outer core suggests the key role Lawrence Livermore National Laboratory under Contract DE-AC52- this LPS region plays in outer membrane stability and highlights vulnerabil- 07NA27344. Release number: LLNL-ABS-739405. ities to membrane attack. Liposomes containing LPS, particularly from rough phenotypes, show enhanced susceptibility to nisin, while LPS from 1362-Pos Board B271 organisms sharing ecological niches with antimicrobial producers provides The Molecular Structure of Human Red Blood Cell Membranes From modest enhancement. Enhanced susceptibility is seen with LPS from eupa- Highly Oriented, Solid Supported Multi-Lamellar Membranes thogenic K. pneumoniae and B. abortus compared to LPS from enteropatho- Sebastian Himbert, Rick J. Alsop, Maikel C. Rheinst€adter. genic S. enterica and gut commensal E. coli. The presence of LPS from Department of Physics and Astronomy, McMaster University, Hamilton, ON, B. melitensis, which is adapted to invade macrophages by immunostealth, Canada. appears almost silent to nisin. The existence of molecular complexes be- The preparation of Red Blood Cell (RBC) Ghosts is a well-known protocol in tween nisin and LPS was confirmed by solid state MAS NMR in most organ- biological and medical research and describes the extraction of the membrane isms except B. melitensis. LSP/nisin complex formation was also confirmed from RBCs. Another well-known protocol is the preparation of highly ordered in outer membrane extracts from E. coli. stacks of artificial lipid bilayers on silicon wafers. There are various attempts to adapt this protocol to a native cell membrane. For 1360-Pos Board B269 the first time we were able to combine both described techniques and to prepare Magainins in a Gram Negative Bacterial Membrane Mimic: A Structural highly ordered stacks of RBC membranes on silicon wafers [1]. These systems Analysis at Sub Nanometer Resolution can now be used as inexpensive and safe platforms for testing the effect of Michael Pachler. drugs and bacteria on RBC membranes in-vitro using biophysical techniques, Biophysics, University of Graz, Graz, Austria. such as X-ray and neutron diffraction, optical spectroscopy and atomic force Mixtures of magainin 2 (MG2) and PGLa are well known for their microscopy. We present direct experimental evidence that these RBC mem- enhanced synergistic killing of Gram-negative bacteria through a branes consist of nanometer sized domains of integral coiled-coil peptides, as membrane-mediated process. To elucidate the structural response of bacte- well as liquid ordered (lo) and liquid disordered (ld) lipids. Lamellar spacings, rial membranes to the peptides’ attack we performed small-angle X-ray and membrane and hydration water layer thicknesses, areas per lipid tail and neutron scattering experiments in combination with contrast variation using domain sizes were determined. The common drug aspirin was added to the palmitoyl oleoyl phosphatidylethanolamine (POPE)/ palmitoyl oleoyl phos- RBC membranes and found to interact with RBC membranes and preferably phatidylglycerol (POPG) (3/1 mol/mol) mimics of the inner plasma mem- partition in the head group region of the lo domain leading to a fluidification brane of E. coli. These model systems were shown previously to of the membranes, i.e., a thinning of the bilayers and an increase in lipid tail realistically capture the biological activity of the peptides. Our study spacing. included effects induced by the individual peptides, as well as their mix- [1] Himbert S. et al., The Molecular Structure of Human Red Blood Cell Mem- tures and a chemically-linked MG2-PGLa heterodimer upon addition to branes from Highly Oriented, Solid Supported Multi-Lamellar Membranes, POPE/POPG large unilamellar vesicles. At concentrations above 1/200 Scientific Reports 7, Article number: 39661 (2017). (peptide/lipid) both, the MG2/PGLa equimolar mixture and the MG2- PGLa heterodimer induced lipid phase separation. One of the coexisting 1363-Pos Board B272 phases was characterized by dehydrated multibilayers, presumably enriched Assembly of Cellular Envelopes - A Step Toward Cell-Scale Simulations in POPG and peptide. The second phase did not show any positional cor- Eric Shinn1, Emad Tajkhorshid1, Joshua Vermaas2. relations in the case of MG2/PGLa mixtures, but formed a cubic (Im3m) 1University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2National structure upon addition of the heterodimers. At lower peptide concentra- Renewable Energy Laboratory, Urbana, CO, USA. tions no positional correlations between the lipid bilayers were observed While molecular dynamics (MD) simulations have been able to elucidate the entailing a detailed structural analysis in terms of the scattering density pro- intricate mechanisms involved in protein function by focusing on isolated, file model. We found distinct effects on membrane thickness and area per single protein systems, they are hampered in their ability to characterize pro- lipid changes induced by the peptides. teins within the greater context of the highly complex cell interactome. To truly understand the behavior and mechanics of biological systems, proteins 1361-Pos Board B270 need to be studied in the broader context of the cellular environment. Tremen- Complex Biological Membranes: Capturing Bilayer Properties In Silico at dous advances in structural biology now allow us to resolve complex cellular Different Compositional Complexity structures like sub-cellular membranes and macromolecular protein assem- Helgi I. Ingolfsson, Tim S. Carpenter, Felice C. Lightstone. blies, indicating a growing demand for tools that enable cellular scale Biochemical and Biophysical Systems Group, Lawrence Livermore National modeling and simulation. To meet this need, we have devised a protocol Laboratory, Livermore, CA, USA. for assembling protein-embedded all-atom cellular envelopes. Lipids are Cellular plasma membranes contain hundreds of different lipid species that are selected from a library of conformations and arranged to form a spherical actively regulated by the cell. The reason for this large lipid diversity remains membrane using the Fibonacci sphere algorithm. Next, desired proteins are rather elusive. Specific lipids have been shown to serve as signaling molecules reduced to a simplified, coarse-bead representation that mimics each of their and bind to membrane associated proteins, modulating their function. The unique geometry. Grid-steered MD simulation situates the coarsely repre- diverse lipid types contribute differently to overall bilayer properties, control- sented proteins on the intended membrane surface and appropriately orients ling e.g. morphology, thickness, elasticity, and fluidity; membrane protein their intra- and extracellular domains. The proteins, once reverted to all- function can then be affected. Additionally, the lipids are inhomogeneously atom form, are used to generate an occupancy map which guides lipid mixed and domains of local lipid enrichment/depletion can sort proteins and removal from the membrane to make space for the proteins. Additional provide optimal local environments. grid-steered MD ensures that any remaining lipids offending the intended Recent molecular dynamics efforts using biologically complex lipid models protein volume are rectified. Finally, the proteins and membrane are merged at the coarse-grained (CG) Martini resolution have provided glimpses into into one system and solvated, ready for simulation. The strength of this pro- detailed lipid organization of cellular membranes. Due to the high tocol is that it can be extended to embed proteins into membranes of diverse complexity, 60 lipid types, large patches and long simulation times are shapes, marking a milestone in the progress toward modeling cell-scale pro- needed for proper sampling (e.g. multi microsecond long and 0.1 square tein-membrane systems.

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1364-Pos Board B273 1367-Pos Board B276 Molecular Mechanism of Central Nervous System Myelinogenesis: In Gangliosides and Lysolipids Regulate the Size of Membrane Rafts Depend- Vitro Self-Assembly of Myelin Membrane Lipid and Protein Structures ing on the Membrane Composition Andrew V. Molina, Ka Yee C. Lee. Anna S. Lyushnyak1,2, Vladimir D. Krasnobaev1,2, Timur R. Galimzyanov1,3, Chemistry, University of Chicago, Chicago, IL, USA. Sergey A. Akimov1,3, Oleg V. Batishchev1,2. The myelin sheath is an insulating, compacted, multilamellar biological mem- 1A.N. Frumkin Institute of Physical Chemistry and Electrochemistry of RAS brane that facilitates efficient propagation of action potentials down neuronal (IPCE RAS), Moscow, Russian Federation, 2Moscow Institute of Physics and axons, and is critical for proper physiological function. Recently, EM imaging Technology, Dolgoprudniy, Russian Federation, 3National University of has provided compelling in vivo data that puts to question the long-established Science and Technology ‘‘MISiS’’, Moscow, Russian Federation. mechanism for the formation of central nervous system (CNS) myelin. The new Gangliosides are the group of glycolipids abundant in brain and thought to be data have led to the proposal of a new model involving (1) the preformation of involved in amyloid aggregation and development of Alzheimer’s disease. myelin membrane tubules that are trafficked to the neuronal axon, where they Nevertheless, in some cases it can play even neuroprotective role. Here we (2) undergo a transition from tubular to lamellar form and thus form the final show that these contradictive activities of gangliosides can be the consequence compact myelin sheath [Szuchet et al. J. Struct. Biol. 190, 56-72 (2015)]. To of their influence on membrane liquid-ordered lipid domains, also called rafts. investigate this mechanism, we designed in vitro experiments to probe the in- Despite the existence of rafts in vivo is still an open question, they are widely teractions of myelin lipids as they (1) self-assemble into tubules and (2) tran- observed in model systems and can be some dynamical structures in cell mem- sition into lamellar form. Using fluid-cell AFM, TEM, and DLS, we have branes. Rafts are assumed to be important players in membrane signal transduc- investigated the self-assembly of lipidic tubules, their transition into the multi- tion and trafficking. Recently, we demonstrated both experimentally and lamellar structure of mature myelin, and how this process is modulated by lipid theoretically that ganglioside GM1 regulates the raft size distribution in two composition and the presence of myelin basic protein. Our data support a gal- different ways depending on the membrane cholesterol content. Therefore, it actosylceramide (GalCer) concentration-dependent response in lipid can both increase and decrease the line tension of the lipid domain boundary. morphology that drives a transition from stable tubules to nonspecific aggre- The same observations for the effect of lysolipids produced in the cell mem- gates with decreasing GalCer concentration. Our in vitro findings at high branes by enzymes phospholipase pointed us to the possible common mecha- GalCer concentrations align with the in vivo tubules observed in ovine embry- nism of the lipid molecular geometry and overall membrane composition on onic oligodendrocytes, suggesting that these structures of major myelin lipids the structure of membrane rafts and their influence on membrane processes. can be stable precursors for myelination. This work was supported in part by the Russian Foundation for Basic Research (project # 18-54-74001). 1365-Pos Board B274 The Biophysical and Biochemical Polarization of the Epithelial Plasma 1368-Pos Board B277 Membrane Role of Membrane Cholesterol in the Apoptosis Induced by Ginsenoside Allison Skinkle1, Barbara Diaz-Rohrer2, Ilya Levental2. Rh2, A Steroid Saponin 1Rice University, Houston, TX, USA, 2University of Texas McGovern Sandrine L. Verstraeten. Medical School, Houston, TX, USA. Cellular and Molecular Pharmacology, Louvain Drug Research Institute, Epithelial cells are structurally and functionally polarized, particularly evident in Brusssels, Belgium. their compositionally distinct apical and basolateral plasma membrane domains. Some saponins (digitonin, alpha-hederin, ginsenoside) exhibit several biolog- While extensive research has focused on the distinct protein compositions of ical and pharmacological activities such as antibacterial and anticancer effect. these membrane domains, little is known about differences in their lipid compo- Many studies attributed the activity of saponins to their interactions with mem- sition and resulting membrane biophysical properties. This limitation has per- brane cholesterol. sisted due largely to the lack of an effective method for efficiently separating In this study, we focus on the ginsenoside Rh2, the main metabolite of ginseng. apical from basolateral plasma membranes. Here, we developed a novel tech- Our goal is to explore whether and how cholesterol could play a role in the mo- nique for the efficient and specific separation of apical and basolateral plasma lecular mechanisms involved in apoptosis induced by Rh2. membranes. By isolating Giant Plasma Membrane Vesicles (GPMVs) from We first characterized the Rh2 cytotoxicity using U937 cells depleted or not in polarized Madin-Darby Canine Kidney (MDCK) cells, we found that the apical cholesterol. We observed higher cellular uptake of Rh2 upon cholesterol deple- and basolateral plasma membranes have distinct biophysical properties. The api- tion explaining why the Rh2-induced apoptosis is observed earlier in cal membrane was more tightly packed than the basolateral membrane and had cholesterol-depleted cells compared to non-depleted cells. Mechanism includes significantly greater domain stability, suggesting robust differences in their lipid the inactivation of Akt/PI3K cell survival pathways, known to be associated compositions. We have extended the GPMV technique to quantify the compre- with cholesterol-enriched membrane domains. hensive, detailed lipidome of the apical and basolateral plasma membranes, al- We therefore developed biophysical approaches and produced artificial mem- lowing for comparison of differences in lipid head groups, but also in fatty branes composed of cholesterol or not to assess its potential role in the Rh2- acyl length and unsaturation. Furthermore, we are developing methods to define induced cytotoxicity. Using large unilamellar vesicles (LUVs), we investigated the compositional and biophysical asymmetry of these membranes. Comparison whether Rh2 could interact with membrane and examined its effect on different of the apical and basolateral lipidome reveals novel connections between lipid biophysical parameters: fluidity (fluorescence anisotropy of diphenylhexatriene composition and the biophysical properties of membranes, and more importantly and generalized polarization of laurdan), permeability (calcein release) and how these properties direct epithelial cell polarization. fusion (R18 dequenching). Rh2 increases membrane rigidity, induces the release of calcein and provokes hemifusion of membranes. These effects appear 1366-Pos Board B275 higher and/or earlier in liposomes without cholesterol as compared to lipo- Liquid Disordered and Liquid Ordered Nanodomains Present in the somes containing cholesterol. POPC-Sterol Model System Altogether, our data suggest that Rh2 insertion within the bilayer could be Fernando Favela-Rosales1, Iva´n Ortega-Blake2, Jorge Herna´ndez-Cobos2. enhanced upon low cholesterol content, thereby explaining earlier and greater 1Ciencias Ba´sicas, ITSZO, Sombrerete, Mexico, 2Biofı´sica, Instituto de Rh2-induced apoptosis in cholesterol-depleted cells as compared to non- Ciencias Fı´sicas, Cuernavaca, Mexico. depleted cells. Moreover, regarding earlier and/or higher changes of biophysi- Molecular dynamics simulations have shown to be a powerful tool for the un- cal liposomal membrane properties in the absence of cholesterol, cholesterol derstanding of biological processes at molecular level. The modelling of lipid could not be considered as the specific target of Rh2. On the contrary, choles- bilayers with this technique is a common practice nowadays, leading to infor- terol seems to depress the sensitivity of membrane to Rh2. mation on the behavior of such system inaccessible by other means. On the other hand, simple lipid bilayers have proved to be reliable for studying cell 1369-Pos Board B278 membrane phenomena, in particular, the POPC-sterol system where a proposed Structural Transitions in Ceramide Cubic Phases during Formation of the mixed phase region leads to polyene increased activity. In order to advance in Human Skin Barrier the understanding of this system, molecular dynamics simulations were per- Christian Wennberg1, Magnus Lundborg1, Ali Narangifard2, Lars Norlen2, formed along this phase diagram, the results support the existence of nanodo- Erik Lindahl3. mains. We suggest that the reason for increased activity could be due to 1Erco L€akemedel AB, Stockholm, Sweden, 2Dept. Cell & Molecular Biology, adsorption along the intermediate phases. In this work, we look into the differ- Karolinska Institutet, Solna, Sweden, 3Dept. Biochemistry & Biophysics, ence in nanodomain formation for the POPC-sterol system between cholesterol Stockholm University, Stockholm, Sweden. and ergosterol trying to fully understand the selectivity of polyenes for the latter The main barrier that protects our body from the environment is the outermost one. Funding: DGAPA-PAPIIT-IG100416. layer of our skin, the stratum corneum. The barrier function is maintained by

BPJ 8625_8627 272a Monday, February 19, 2018 stacked layers of saturated long-chain ceramides, free fatty acids and choles- These results reveal a mammalian mechanism for homeostatic membrane remod- terol, but we know surprisingly little of the detailed molecular structure. It is eling - analogous to homeoviscious adaptation in poikilotherms - wherein cells believed to be formed through a reorganization of glycosylceramide-based bi- remodel their membrane lipidomes in response to dietary lipid inputs in order layers with cubic-like symmetry into ceramide-based bilayers with stacked to maintain functional membrane phenotypes. lamellar symmetry. The process is accompanied by deglycosylation of glyco- sylceramides and by gradual dehydration of the lipid complex, which eventu- 1372-Pos Board B281 ally turns it into skin. We show that it is possible to model a simplified Predicting Spectral Properties of Polarity Sensitive Dyes with QM/MM version of this formation process in molecular dynamics simulations, and in Simulation particular study the effects of deglycosylation and dehydration on bilayers of Swapnil Baral1, Bjorn Baumeier2, Edward Lyman1. human skin glycosylceramides and ceramides, which fold in 3D space with cu- 1Physics and Astronomy, University of Delaware, Newark, DE, USA, bic (gyroid) symmetry. Deglycosylation of glycosylceramides destabilizes the 2Mathematics and Computer Science and Institute for Complex Molecular cubic lipid bilayer phase and triggers a cubic to lamellar transition. Further- Systems, Eindhoven University of Technology, Eindhoven, Netherlands. more, subsequent dehydration of the deglycosylated lamellar ceramide system Polarity sensitive, lipophilic dyes such as Laurdan report lipid packing in bio- closes remaining pores between adjacent lipid layers and locally induces a cer- membranes, as the emission spectrum is red shifted in more polar environments. amide chain transformation from hairpin-like to splayed conformation. By em- In simple membranes, the dye is more accessible to solvent in more disordered ploying electron microscopy simulation, it is possible to model expected cryo- membranes, and the spectral shift is well-explained by dipolar relaxation of the EM micrographs from the simulations, and these agree surprisingly well with solvent. However, in more complex systems other factors may contribute, espe- original cryo-EM imaging of stacked ceramide layers in the stratum corneum. cially hydrogen bonding between the environment and the chormophore. An This both provides insight into the formation of the skin barrier, and molecular approach has been developed in which the local environment is first sampled models that could help us better understand the permeability of skin. by classical molecular dynamics simulation of the dye in different environments, followed by prediction of the absorption spectrum by numerical quantum me- 1370-Pos Board B279 chanics. Simulation results are presented for an optimized model of Laurdan Plant Polyphenols Induced the Polymorphic Phase Transition of Mem- and C-Laurdan for use with the CHARMM family of forcefields in a variety brane Lipids of membrane environments. Several different quantum methods are compared, Svetlana S. Efimova, Olga S. Ostroumova. including time-dependent density functional theory and GW-BSE. Institute of Cytology of RAS, St-Petersburg, Russian Federation. Flavonoids are called ‘‘natural modifiers of biological reaction’’ due to their 1373-Pos Board B282 ability to change the response of the human body to allergens, viruses and onc- Humectants’ Influence on the Nanostructure and Thermotropic Behavior ogens. This is evidenced by their antioxidant, anti-inflammatory and antipara- of Fully Hydrated Phospholipids sitic and antibacterial properties. However, its use is limited by low relatively Ngai Ying Denise Li, Michael Rappolt. bioavailability. At the present time, the most perspective way to increase the School of Food Science and Nutrition, University of Leeds, Leeds, United bioavailability of polyphenols is used the complexes of modifiers with lipid Kingdom. vesicles. We had found that flavonoids (phloretin, butein, 4’-hydroxychalcone, Humectants are hygroscopic substances used to retain moisture in many personal naringenin, quercetin, myricetin, biochanin A, genistein, cardamonin, licochal- care products, particularly in phospholipid-based skin creams and haircare prod- cone A, liquiritigenin) are able to induce a polymorphic phase transition of ucts. Here we studied the effect of humectants on the thermotropic behavior and DOPC. Large unilamellar liposomes were prepared by extrusion methods. nanostructure of fully hydrated 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine, The lipid:polyphenol ration was equal to 3:1, 1:1, and 1:3. Visualization of DPPC, from 25 to 50 C. Three humectants have been investigated: betaine, sar- the polyphenol-induced non-bilayer lipid structures enriched with polyphenols cosine and acetamide monoethanolamine, AMEA. Small angle X-ray scattering, were processed by laser scanning confocal microscope, Olympus FV3000. SAXS, differential scanning calorimetry, DSC, and isothermal calorimetry, ITC, Phloretin, cardamonin, biochanin A, genistein, quercetin and myricetin caused have been applied. The general trend is that all humectants induce an increase in the appearance of needle-shaped spherical non-bilayer structures, while the both the pre-transition temperature (gel to ripple phase) and the main transition addition of butein and naringenin in the liposomal suspension leads to the temperature (ripple to fluid phase). At lower concentrations of humectant appearance of rod-like structures. The morphology of non-bilayer structures (0.05-0.4 M), however, we observed a lowering in the pre-transition temperature. induced by polyphenols does not depend on the ratio of lipid:polyphenol. Deduced electron density profiles of the gel phase of DPPC display for all three The addition of 4’-hydroxychalcone, liquiritigenin and licochalcone A to the humectants similar effects: the membrane thickness marginally increases with liposome suspension did not induced a polymorphic phase transition of lipids. increasing concentration of humectant, and concomitantly the interstitial water Our results indicated a dependence of the polyphenol ability to induce the for- layer decreases significantly. This suggests that the membrane becomes more mation of non-bilayer structures on the number of OH groups in the molecule. rigid upon addition of humectants leading to a reduced inter-membrane repulsion It is proposed to apply the methods of electron microscopy and small-angle X- (reduction of the Helfrich undulation force). Furthermore, DSC results reveal ray scattering to study the thin structure of the polyphenol-induced non-bilayer strong enthalpy variations compared to DPPC in pure water. Complementing lipid structures. The study was supported in part by RFS (#17-74-10137). ITC measurements on the solution/dissolution enthalpy of the humectants confirmed that changes in the lipid/water partition coefficient across the main 1371-Pos Board B280 transition are responsible for an overall increase of the enthalpy in the presence Homeostatic Remodeling of Mammalian Membranes in Response to Die- of AMEA and leads to a decrease with sarcosine. Betaine instead has no great tary Lipid Perturbations is Essential for Cellular Fitness influence on the main transition enthalpy. In conclusion, by combining SAXS, Kandice R. Levental, Eric Malmberg, Ilya Levental. DSC and ITC (i) nanostructural changes in the DPPC bilayer as well as (ii) Integrative Biology and Pharmacology, McGovern Medical School at the different lipid/water partitioning behavior of the humectants can be unraveled. University of Texas Health Science Center at Houston, Houston, TX, USA. A major fraction of cellular bioactivity occurs at membranes, with the lipidic ma- 1374-Pos Board B283 trix constituting a functional, dynamic interface that actively regulates protein ac- Dumbbell-Shaped Janus Dendrimersomes Exhibit Lamellar to Sponge tivity and cell physiology. Proper membrane functionality requires maintenance Phase Transitions of a narrow range of physical properties under challenge from external inputs. Samantha Wilner, Qi Xiao, Virgil Percec, Tobias Baumgart. The most prominent example of such maintenance is homeostatic adaptation of Chemistry, University of Pennsylvania, Philadelphia, PA, USA. membrane properties to temperature variation, a fundamental and ubiquitous Self-assembling amphiphilic Janus dendrimers, composed of hydrophobic and design feature in ectothermic organisms. However, such responsive membrane hydrophilic branches, are promising models for understanding the complexity adaptation has not been widely investigated in homeotherms. Here, we report of biological membranes. Modeling the transitions of membrane shapes from that challenging mammalian membrane homeostasis by dietary lipid inputs leads lamellar to non-lamellar is particularly desirable as these transitions play essen- to robust lipidomic remodeling to maintain membrane physical properties. Specif- tial roles in key cellular processes, such as membrane fusion and fission, and ically, supplementation with polyunsaturated fatty acids (PUFAs) leads to rapid occur in response to external stimuli, including drug treatment and heat. We and extensive incorporation of the exogenous fats into membrane lipids, inducing previously reported that mixtures of novel hydrogenated, fluorinated, and a reduction in membrane packing. These effects are rapidly compensated for by hybrid Janus dendrimers self-assemble into dumbbell-shaped structures. upregulation of saturated lipids and cholesterol, via activation of the mammalian Here, we show that these structures undergo a thermally induced lamellar-to- sterol regulatory machinery, specifically SREBP pathway, resulting in recovery of sponge transition. The sponge phase (L3) is characterized as an isotropic membrane fluidity. Inhibition of membrane remodeling results in decreased multi-connected 3D bilayer that forms when the surfactant monolayer of a cellular fitness when membrane homeostasis is challenged by dietary PUFAs. bilayer exhibits a spontaneous curvature toward the bulk solvent. We used

BPJ 8625_8627 Monday, February 19, 2018 273a confocal microscopy, fluorescence polarization, fluorescence recovery after protein interactions. The membrane composition of GPMVs closely resem- photobleaching, and cryo-electron microscopy to demonstrate that this transi- bles the plasma membrane lipidome and proteome, but lacks active remod- tion occurs not only for the dumbbell-shaped vesicles but also for single- eling and cytoskeletal support. In addition to the complex composition, component dendrimersomes made of either hydrogenated or fluorinated den- GPMVs are expected to retain the plasma membrane structural characteris- drimers. Consistent with the formation of a 3D bilayer network, we show tics, thus allowing access to material parameters otherwise concealed in the that dendrimersomes become more permeable to water soluble fluorophores af- active cellular system. Here, we report bending rigidity values for GPMVs ter transitioning to the sponge phase. These findings suggest that we have the at varying GPMV isolation conditions. Further, we correlate them with the potential to control sponge formation by tuning the spontaneous curvature of membrane viscosity and order assessed from Laurdan general polarization the membrane to generate a range of thermally inducible sponge-like structures. and fluorescent lifetimes of molecular rotors. When aspirated in micropi- These dendrimersomes may be useful in (1) drug delivery, since non-lamellar pettes, GPMVs show unexpected mechanical properties, which are not delivery vehicles can promote endosomal disruption and cargo release and (2) found in simple lipid bilayers. GPMVs exhibit droplet-like instabilities modeling isotropic membrane rearrangements of biological systems. upon increasing aspiration pressures. We find that the apparent elastic modulus matches closely reported values of cellular cortical tension. To un- 1375-Pos Board B284 derstand how the passive GPMV membrane gives rise to cell-like responses, Electrostatically Controlled Domain Size and Alignment in Phase Sepa- we employed fluorescent lipid analogs, nanotube pulling experiments and rated Lipid Multilayers electron microscopy. We detect membrane reservoirs exhibiting lipid sort- 1 2 2 1 1 Suho Lee , Yuno Lee , Ji Hyun Bak , Dae-Woong Jeong , Jae-heui Lee , ing and investigate their role in the observed soft elastic response. Addition- 3 2 1 Hyun Hwi Lee , Changbong Hyeon , Myung Chul Choi . ally, fluorescent lifetime imaging of a membrane order sensitive dye helps 1Department of Bio and Brain Engineering, KAIST, Daejeon, Republic of 2 3 to clarify the GPMV membrane structural response to mechanical tension. Korea, KIAS, Seoul, Republic of Korea, Pohang Accelerator Laboratory, The results contribute to the understanding of the plasma membrane Pohang, Republic of Korea. structure and enable synthetic reconstitution of plasma membrane like Ordered domains in cellular membranes, called lipid rafts, are involved in signal comportments. transduction, cellular transport, and disease development. They compartmen- talize membrane proteins, thus provide functional hotspots for those cellular pro- cesses. Especially, when two membranes communicate, domain size growth and 1378-Pos Board B287 positional proximity between these domains in distinct membranes can be neces- Structural and Mechanical Effects of Calcium on the Lipid Bilayer sary for efficient cell-cell interaction, synaptic vesicle fusion, and drug delivery Nicholas E. Charron, Pei Yin Yang, Huey W. Huang. through vesicles. Here, using synchrotron X-ray reflectivity and fluorescence mi- Physics and Astronomy, Rice University, Houston, TX, USA. croscopy, we describe our recent findings that domain size and alignment in Calcium is a ubiquitous divalent cation involved in fundamental biochem- phase separated lipid multilayers can be controlled by salt concentration in ical and biophysical processes. Its integral roles in cell signaling, protein- aqueous phase. We also describe the effect of depletion force as well. mediated fusion, and action potential propagation have been well studied with a variety of techniques. Calcium is also commonly maintained at strong 1376-Pos Board B285 asymmetric concentration gradients, which often span several orders of Effect of PEG, Lipid Composition and Formulation on Vesicle Lamellar- magnitude, across mammalian cell membranes in proper physiological con- ity: A Small Angle Neutron Scattering Study ditions. Yet, there has been a comparative lack of investigation of the effect Valeria Nele. of divalent cations on lipid bilayer structure and mechanical properties. Of Materials, Imperial College, London, United Kingdom. the few studies done, experimental methods are limited to undefined resolu- Liposomes are vesicles comprising a phospholipid bilayer and inner aqueous cav- tions and indirect spectroscopic or fluorescent techniques. This is in part due ity, and are well-established systems for drug delivery applications. They are able to the inherent difficulty in preparing well aligned samples with ions for to encapsulate both hydrophobic and hydrophilic compounds. Among the param- high resolution experiments; common multilayer preparation techniques eters to account for when designing a liposomal drug carrier lamellarity (number are subject to miscibility limits and sample segregation. Computationally, of consecutive lipid bilayers within one vesicle) is very important: it affects both molecular dynamics simulations have predicted a calcium ‘‘condensing’’ ef- encapsulation efficiency and efflux rate through the membrane of the encapsu- fect that is associated with bilayer thickening and increased hydrocarbon lated drug. Despite the relevance of liposomes as drug carriers, a comprehensive chain order. However, these methods are limited by still-developing ap- study on how lipid composition and formulation method affect vesicle lamellarity proaches to modeling divalent cation structure and interactions. Here, we is still lacking. Here, we combined small-angle neutron scattering (SANS) and report lamellar x-ray diffraction measurements of lipid bilayer thickness cryo-TEM to elucidate the membrane structure of vesicles prepared using three in partially charged multilayers containing calcium. Additionally, we report different formulation methods (film hydration followed by freeze-thawing (FT) an apparent calcium-induced mechanical softening effect in giant unilamel- or agitation on a shaker (AS), and reverse phase evaporation (REV)) and extruded lar vesicles (GUVs) as measured by micropipette aspiration. This effect is through a 100 nm membrane. We examined two phospholipids: 1-palmitoyl-2- marked by a measurable reduction in the elastic stretching coefficient of oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3- GUVs in calcium-rich environments. Both the thickening and mechanical phosphocholine (DPPC), both widely used in the liposome field. Our results softening effects may be important for proteins and molecules that couple show that POPC vesicles prepared via the FT or AS methods retain a substantial directly to the lipid bilayer in physiological calcium concentrations. Future amount of multilamellarity after extrusion through 100 nm membranes. This is investigations involving PG-containing membranes and bacterial protoplasts also true for DPPC vesicles, although the extent of residual multilamellarity is may help lead to a better understanding of how invading bacteria thrive in much lower. The introduction of a poly(ethylene)glycol (PEG)-modified lipid calcium-rich environments, or if calcium-rich membrane domains have in the vesicle composition results in the formation of unilamellar vesicles after some non-trivial influence on the mechanisms of antibiotics and/or host de- extrusion when the PEGylated lipid content is at least 0.5 mol% for POPC ves- fense antimicrobial peptides. icles and 0.1 mol% for DPPC vesicles. As for the REV method, unextruded DPPC vesicles are already unilamellar, while unextruded POPC vesicles still exhibit some bilamellarity. Extruded, REV-prepared POPC vesicles are unila- Posters: General Protein-Lipid Interactions I mellar instead. In conclusion, our results provide important insights into the effect of formulation method and lipid composition on producing liposomes with a 1379-Pos Board B288 defined membrane structure. Theoretical and Computational Modeling of the Rupture Force Distribu- tion in Peptide Lipid Interactions 1377-Pos Board B286 Milica Utjesanovic, Kanokporn Chattrakun, Krishna Sigdel, Gavin M. King, Cell-Like Mechanical Response in Passive Plasma Membrane Vesicles Ioan Kosztin. Jan Steinkuhler€ 1, Tripta Bhatia1, Iztok Urbancic2, Erdinc Sezgin2, Department of Physics & Astronomy, University of Missouri - Columbia, Martin Westermann3, Reinhard Lipowsky1, Rumiana Dimova1. Columbia, MO, USA. 1Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces Peptide-lipid interactions are essential for understanding various cellular pro- Science, Potsdam, Germany, 2MRC Human Immunology Unit, Weatherall cesses and their mechanisms. Both experimental and theoretical study of these Institute of Molecular Medicine, Oxford, United Kingdom, 3Electron interactions is notoriously difficult due to the complexity of the system and the Microscopy Center, Medical Faculty Friedrich-Schiller-University Jena, wide range of time and length scales involved. Atomic force microscopy Jena, Germany. (AFM) based force spectroscopy can be used to quantify the strength of Giant plasma membrane vesicles (GPMVs) which are isolated from adherent peptide-lipid interactions, at the single molecule level, by measuring the corre- cells are an emerging model system for the study of lipid-lipid and lipid- sponding detachment (last rupture) force F. Based on the experimental and

BPJ 8625_8627 274a Monday, February 19, 2018 all-atom molecular dynamics simulation results, we argue that the contact be- ronment. We now report that in the process of scrambling, lipid headgroups tween a peptide and lipid membrane right before detachment is localized at the traverse a dynamically revealed hydrophilic pathway in the region between level of one or two residues, in general, situated at the end of the peptide. Thus, transmembrane helices 6 and 7 of the protein while their hydrophobic tails we show that the experimentally determined rupture force distribution function, remain in the bilayer environment. We present quantitative kinetic models P(F), can be modeled as a weighted combination of several independent escape of the translocation process based on Markov State Model analysis of the processes of Brownian particles across free energy barriers. Each detachment simulation trajectories. As key residues on the lipid translocation pathway pathway is characterized by (i) an energy parameter (barrier height, U0); (ii) are conserved within the class of rhodopsin-like GPCRs, including the a geometric parameter (spatial extent of the potential barrier, x0); and (iii) a ki- beta1- and beta2-adrenergic receptors and the adenosine A2A receptor, our re- netic parameter (intrinsic escape rate, k0). While the parameters U0 and x0 are sults illuminate new aspects of GPCR structure and dynamics while providing specific to individual residues, k0 and the weight parameters need to be deter- a rigorous basis for the design of variants of these proteins with defined mined through the fitting process. The method has been successfully applied to scramblase activity. reproduce the experimentally measured P(F) for several short peptides interact- ing with model lipid bilayers. 1382-Pos Board B291 Work supported by the Burroughs Wellcome Fund (Career Award at the Sci- G-Protein-Coupled Receptor Activation through Membrane Deformation entific Interface), the NSF (CAREER Award #: 1054832), and the MU Nipuna Weerasinghe1, Steven D. Fried1, Suchithranga M.D.C. Perera1, Research Board. The computation for this work was performed on the HPC Anna R. Eitel1, Udeep Chawla1, Trivikram R. Molugu1, Andrey V. Struts1, infrastructure provided by RCSS at the University of Missouri, Columbia Michael F. Brown1,2. MO. The HPC equipment for the computational work is supported by NSF 1Chemsitry and Biochemsitry, University of Arizona, Tucson, AZ, USA, CNS-1429294. 2Department of Physics, University of Arizona, Tucson, AZ, USA. G-protein-coupled receptors (GPCRs) are the largest family of cell membrane- 1380-Pos Board B289 bound proteins that detect molecules outside the cell and transduce signals to Soft Matter Influences on G-Protein-Coupled-Receptor Activation Probed activate intracellular functions. Rhodopsin is a canonical GPCR, which upon by FTIR and UV-Visible Spectroscopy photoactivation undergoes a series of conformational changes, leading to a Michael F. Brown1,2, Blake Mertz3, Eglof Ritter4. chemical equilibrium between inactive Metarhodopsin-I (MI) and active 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, Metarhodopsin-II (MII) states. Membrane protein function directly depends AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA, on protein hydration, and indirectly on hydration-modulation of lipid-protein 3C. Eugene Bennett Department of Chemistry, West Virginia University, interactions, hydrophobic matching, and the membrane spontaneous curvature Morgantown, WV, USA, 4Institut fur€ Medizinische Physik und Biophysik, [1]. Here we investigate rhodopsin activation by monitoring membrane hydra- Charite Universit€atsmedizin Berlin, Berlin, Germany. tion by various osmolytes. Using UV-visible spectroscopy, we discovered that Rhodopsin is an essential prototype for studying G protein-coupled receptor the rhodopsin MI/MII equilibrium is very sensitive to osmolyte size. Large os- (GPCR) structure and function in lipid membranes. Here we address the molytes support stability of the preactive MI state. The effect of osmotic stress role of the membrane soft matter (lipids and water) in GPCR function at for rhodopsin in membranes is explained through a back shift of the MI/MII the membrane level. We postulate this process involves an energy landscape equilibrium due to protein dehydration, and a forward shift due to lipid mem- mechanism (ELM), wherein an ensemble of activated states is affected by brane deformation. Solid-state 2H NMR spectroscopy shows that dehydration membrane protein-lipid bilayer interactions as described by a flexible surface of the lipid head groups yields a reduction in area per lipid, and correspond- model [1]. Rhodopsin was incorporated into lipid vesicles (POPC, DOPC, ingly an increase of bilayer thickness [2]. Increasing either the hydrophobic mixtures with DOPE) and investigated by simultaneous Fourier transform bilayer thickness or monolayer spontaneous curvature leads to a greater frac- infrared (FTIR) and UV-visible spectral acquisition. Spectral reduction and tion of active MII rhodopsin [1]. We attribute the forward shift of the metarho- analysis via pH titration curves indicated that more than one activated state dopsin equilibrium by small osmolytes (e.g., sucrose, PEG200) to a membrane exists [2] supporting the ELM concept. Moreover, temperature changes lipid effect. The back shift by large osmolytes is ascribed to withdrawing water have a marked effect on rhodopsin activation, decoupling the two protonation from the protein. In the absence of membrane lipids, when the protein is sol- switches necessary for full activation. By manipulating the lipid environment ubilized in detergent, small osmolytes favor the preactive MI state as seen we validated the flexible surface model and ELM, whereby lipids with nega- experimentally. Further work entails the introduction of spectroscopic methods tive monolayer curvature facilitate rhodopsin activation [1]. Thermodynamic to test the effect of membrane deformation on rhodopsin activation, and the parameters show the pH-dependent activation of rhodopsin leads to a confor- sensitivity for membrane lipid composition. [1] M.F. Brown (2017) Annu. mational ensemble (Meta-II state) that corresponds to different tiers of an Rev. Biophys. 46, 379. [2] J.J. Kinnun et al. (2015) Biochim. Biophys. Acta enthalpy-entropy folding funnel. Activation of rhodopsin involves conforma- 1848, 246. tional states that are collectively designated as Meta-II (energy landscape mechanism). Our results challenge the paradigm of a linear reaction sequence, 1383-Pos Board B292 and point to a parallel mechanism of rhodopsin activation in a natural lipid State Dependent Interactions of Lipids with GPCR Revealed by MD Sim- membrane environment. Membrane lipids bias the ensemble of activated sub- ulations using In Vivo-Mimetic Membranes states, which depends on both the lipid head groups and the acyl chain unsa- Wanling Song, Mark S.P. Sansom. turation. Formation of Meta-II may thus involve a partial unfolding of the Oxford University, Oxford, United Kingdom. receptor that exposes recognition elements for the G-protein (transducin) (or G protein-coupled receptors (GPCRs) constitute the largest superfamily in the other effectors) that are buried in the dark state. [1] M.F. Brown (2017) mammalian genome and are targeted by over 30% of the marketed drugs. Annu. Rev. Biophys. 46, 379-410. [2] M. Mahalingam et al. (2008) PNAS Membranes are known to have modulatory effects on GPCRs. However, 105, 17795-17800. the mechanism of such effects in a complex cell membrane environment re- mains elusive. We carried out coarse-grained MD simulations on the Adeno- 1381-Pos Board B290 sine A2a receptor embedded in bilayers comprised of 10 biologically relevant The GPCR Opsin Translocates Lipids via a Dynamic Mechanism Specified lipid species. Three conformational states of the receptor, i.e. the inactive by Markov State Model Analysis of Molecular Dynamics Trajectories state, the active state, and the active state with a mini-Gs protein bound Giulia Morra, Asghar M. Razavi, Kalpana Pandey, Harel Weinstein, were simulated to study the influence of protein-lipid interactions on receptor Anant K. Menon, George Khelashvili. dynamics. The simulations revealed a clear division of annular lipids from Weill Cornell Medical College, Cornell University, New York, NY, USA. bulk lipids. The binding affinities of the former showed a degree of sensitivity Several heptahelical membrane proteins belonging to the class of rhodopsin- to the conformational state of the receptor, suggesting that these lipids may like G protein-coupled receptors (GPCRs) have been shown to act as consti- play a role in regulating receptor activity. In particular, we demonstrated tutive phospholipid scramblases: when reconstituted into synthetic vesicles that PIP2 is able to enhance the association of the A2aR with the mini-Gs pro- they catalyze the transbilayer translocation of >10,000 phospholipids per sec- tein. The tight interactions of PIP2 with conserved basic residues on the C ter- ond, which is at least 9 orders of magnitude faster than the rate of spontaneous minal helix suggested that PIP2 may facilitate the translational movement of lipid flip-flop. The molecular mechanism by which these proteins facilitate this helix required for G protein activation. Our results suggest the existence rapid lipid scrambling is not known. To address this problem, we carried of a ’mega-receptor’ the functional properties of which will be determined by out large-scale ensemble atomistic molecular dynamics (MD) simulations both the protein and the bound lipids. This may expand the potential drug- (>50 ms simulation time) of the opsin GPCR in an explicit membrane envi- gable space of GPCRs.

BPJ 8625_8627 Monday, February 19, 2018 275a

1384-Pos Board B293 any binding of PH domain to pure POPC membranes which suggests the asso- Cholesterol Interactions with the A2A Adenosine Receptor: All-Atom, ciation is electrostatically driven. We also show a novel and stable binding pose Coarse-Grained, and Metadynamics Simulations of PIP3 lipid to the PH domain which to best of our knowledge has been Lewen Yang. observed for the first time. Furthermore, by performing the additional well- University of Delaware, Newark, DE, USA. tempered metadynamics calculations we have studied the unbinding dynamics By mole, cholesterol is the most abundant component of animal cell plasma and energetics of WT and E345K GRP1 PH domain from the membrane which membranes. Many membrane proteins have been shown to be functionally suggests the stronger binding of PH domain with the membrane having PIP3 dependent on cholesterol, several of which have also been shown to bind lipids and role of sentry mutation (E345K) and its membrane binding dy- cholesterol at well-defined locations on their membrane-facing surface. In namics. Based on our simulations results we propose a binding mechanism this work, a combination of coarse-grained ‘‘Martini’’ and all-atom simulations of GRP1 PH domain with inositol lipids. are used to identify two new cholesterol binding sites on the A2A adenosine receptor, a G-protein coupled receptor that is a target for the treatment of Par- 1387-Pos Board B296 kinson’s disease. One of the sites is also observed to bind cholesterol in several Multivalent Membrane Lipid Targeting by the Calcium-Independent C2A recent high resolution crystal structures of the protein, and in the simulations Domain of Slp-4/Granuphilin interacts with cholesterol only when bound to the inverse agonist Abena Watson-Siriboe, Aml Alnaas, Jack Henderson, Sherleen Tran, ZM241385. The affinity of cholesterol at different sites and in different recep- J. Ryan Osterberg, Nara Lee Chon, Tatyana Lyakhova, Julianna Oviedo, tor states is obtained by metadynamics simulations. Given that cholesterol con- Hai Lin, Jefferson Knight. tent in plasma membranes varies with cell type and upon administration of Chemistry, University of Colorado Denver, Denver, CO, USA. widely prescribed pharmaceuticals, such as statins, understanding Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab cholesterol-dependent function is an important step towards exploiting mem- effector responsible for docking insulin secretory vesicles to the plasma mem- brane compositional variation for therapeutic ends. brane prior to exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, binds plasma membrane SNARE complex pro- 1385-Pos Board B294 teins via a central linker region, and contains tandem C-terminal C2 domains Investigating Cholesterol Dynamics and Interactions with the Dopamine (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate Transporter using a Membrane Mimetic Model (PIP2). Its C2A domain has previously been shown to bind PIP2 or its soluble Muyun Lihan1, Emad Tajkhorshid1,2. analogues with low micromolar affinity; however, the domain docks with low 1 Center for Biophysics and Quantitative Biology, University of Illinois at nanomolar apparent affinity to PIP2 in lipid vesicles that also contain back- UrbanaChampaign, Urbana, IL, USA, 2Department of Biochemistry, ground anionic lipids such as phosphatidylserine (PS). Here we show using a University of Illinois at UrbanaChampaign, Urbana, IL, USA. combination of computational and experimental approaches that this high- Cholesterol, an integral component of animal lipid membranes, is involved in affinity membrane interaction arises from concerted binding at multiple sites modulation of both membrane physical properties and a wide variety of mem- on the C2A domain. In addition to the previously identified, PIP-selective brane proteins. Conventional computational studies of cholesterol-protein in- lysine cluster, a second cationic region of the protein domain contributes sub- teractions have been either using coarse-grained methods or relying on > ms stantially to its affinity for physiologically relevant lipid compositions. Muta- all-atom simulations due to the slow dynamics of lipids, in order to provide use- tions at the two sites decrease affinity for PIP2 and background anionic ful information. The highly mobile membrane-mimetic (HMMM) model has lipids, respectively, while combined mutations at both sites are needed to block been previously reported to study interactions between peripheral membrane binding to physiologically relevant membranes. Docking and molecular dy- proteins and phospholipids with expedited lipid reorganization. Here, we namics simulations indicate that binding at the nonspecific site varies with pro- have extended the use of the HMMM model to investigate interactions between tein conformation, suggest a possible allosteric mechanism of cooperativity integral membrane protein and cholesterol. Cholesterol dynamics were first between the two sites. Overall, multivalent lipid binding by the Slp-4 C2A closely examined in the HMMM model with extensive all-atom molecular dy- domain provides selective recognition and high affinity docking to the plasma namics simulations. Both lateral diffusion and flip-flop motion were shown to membrane. be significantly accelerated at cholesterol concentrations ranging from 5% to 50%, while atomic density profiles and internal dynamics of cholesterols 1388-Pos Board B297 with proper restraints in the HMMM membranes were found similar to those The Membrane Tether of the RAS Signaling Protein Drives Nanocluster- in full-length lipid membranes. The HMMM model was then applied to study ing by Fly-Casting for Anionic Lipids cholesterol interactions with an integral membrane protein, dopamine trans- Chris Neale, Angel Garcı´a. porter. The cholesterol binding sites and detailed interactions with the dopa- Los Alamos National Lab, Los Alamos, NM, USA. mine transporter observed in the HMMM membrane were compared with Cancer is a devastating disease that results from pathological modifications to those from microsecond all-atom molecular dynamics simulations and experi- cellular decision making processes. In healthy cells, the protein-mediated mental data. Our results show great potential and advantages of using the signaling networks that regulate growth and movement are tightly regulated. HMMM model to identify cholesterol binding sites with enhanced efficiency However, mutations that disrupt or over-activate signaling proteins can drive and clarify cholesterol-protein interactions without losing atomic details. uncontrolled cell growth. Ras, a peripheral membrane signaling protein, har- bors activating mutations in 20% of all cancers, especially those of the 1386-Pos Board B295 pancreas, colon, thyroid, and lung. Understanding GRP1 PH Domain-Lipid Interaction using an Accelerated In theory, the signaling output from overactive Ras mutants can be decreased Membrane Modelunderstanding GRP1 PH Domain-Lipid Interaction by small molecules that bind and stabilize inactive states. However, crystallo- Using an Accelerated Membrane Model graphic structures of Ras reveal a relatively smooth surface lacking substantial Shashank Pant1,2, Emad Tajkhorshid1,2. hydrophobic crevices for drug binding. Therefore, we use molecular dynamics 1Center for Biophysics and Quantitative Biology, University of Illinois, simulations to explore the topography of Ras proteins and the dynamic acces- Urbana-Champaign, Urbana, IL, USA, 2Beckman Institute of Advanced sibility of their effector-binding surfaces. Our long-term goal is the identifica- Science and Technology, Urbana, IL, USA. tion of potentially druggable cavities at intermolecular interfaces, including Phosphatidylinositol (3,4,5)-triphosphate (PIP3), behaves as a second those between Ras and the cell membrane. messenger by recruiting a broad spectrum of Pleckstin Homology (PH) do- Our molecular dynamics simulations indicate that, in Ras isoform K-Ras4B, the mains to the membrane surface. The specific interaction of PIP3 lipids with prenylated 19-residue C-terminal highly variable region (HVR) membrane an- the GRP1 protein is essential for the key processes like cellular growth, chor remains disordered as it pulls Ras’ globular G domain closer to mem- DNA synthesis and apoptosis. This study reports all-atom molecular dynamics branes enriched in negatively charged (anionic) lipids. Furthermore, we show simulations using Highly Mobile Membrane Mimetic accelerated model of the that although Ras’ globular G domain interacts promiscuously with its own association of GRP1 PH domain with PIP3, PIP2 containing lipid bilayers. The HVR and membrane lipids, there are preferential and potentially targetable in- PH domain was placed in different orientations at around 20 A˚ away from teractions. Finally, simulations of multiple HVR peptides indicate that intermo- the phosphate plane of the membrane producing 10 different membrane lecular contacts predominantly involve the G domain-proximal (N-terminal) models, each of which was simulated for 150ns. In order to show the im- region of the HVR, consistent with a fly-casting mechanism that augments portance of surrounding lipids in the association of GRP1 PH domain and ion- the self-association of Ras proteins in the context of disordered membrane ositol lipids we have constructed different lipid bilayers namely pure patches enriched in anionic lipids. This targeted adsorption of the K-Ras4B POPC, 80:20 POPC:POPS, 80:19:1 POPC:POPS:PIP3, with PIP2, 73:20:5:2 HVR to anionic membrane surfaces may play a role in regulating the multime- POPC:POPS:PIP2:PIP3 with WT and E345K PH Domain. We didn’t observe rization ability of downstream effector proteins such as Raf kinase.

BPJ 8625_8627 276a Monday, February 19, 2018

1389-Pos Board B298 primes Vt for dimerization. Since these questions are difficult to address with Specific Coating of Cellular Lipid Droplets by a Giant and Repetitive experiments, we tackle the problem with a combination of coarse-grained Amphipathic Helix and atomistic molecular dynamics simulations. By systematically screening Manuel Gimenez Andres1, Sandra Antoine-Bally2, Marco M. Manni3, with coarse-grained simulations, we determined the main binding orientations Cathy Jackson2, Bruno Antonny3, Alenka Copic2. of monomeric Vt on a PIP2 containing model membrane. This allowed us to 1Institut Jacques Monod, CNRS, Universite Paris Sud, Paris, France, 2Institut characterize PIP2 interactions at unprecedented detail. On the one hand, our Jacques Monod, CNRS, Universite Paris Diderot, Paris, France, 3IPMC, findings confirm the central role of residues that have been previously identified CNRS, UniversiteCoˆte d’Azur, Valbonne, France. in crystal structures and in mutational studies. On the other hand, we discovered Lipid droplets (LDs) are dynamic organelles that play an essential role in additional, stable and transient, binding sites. Moreover, the simulations sug- cellular lipid homeostasis; however, the mechanisms of selective protein target- gested a plausible mechanism how PIP2 binding can lead to the release of an ing to LDs to mediate their function are poorly understood. Many LD proteins unstructured region of Vt, which exposes a surface, that mediates dimer inter- interact with LDs via amphipathic helices (AHs), which can mediate direct and actions. Since large conformational changes are not accessible to coarse- reversible binding to lipid surfaces. We use a uniquely long and monotonous grained simulations we back-transformed coarse-grained simulation snapshots AH, found in the mammalian LD protein perilipin 4, to probe the physico- to atomistic systems to study the process more closely. Finally, we combined chemical properties of the LD surface. We show that this AH is completely un- constraints from experiments with our data to generate possible conformations structured in solution but can adopt a highly helical conformation, over a length of a Vt-dimer bound to a membrane that can be tested by future experiments. of 660 amino acids, when in contact with lipids. By the use of fluorescently- tagged AH variants, we show that LDs are relatively permissive for AH bind- 1392-Pos Board B301 ing, suggesting a surface with abundant lipid packing defects, in agreement Cooperativity in Membrane Binding By C2AB Tandem Domains of with predicted behavior of a phospholipid monolayer on a neutral lipid core Synaptotagmin-7 and Synaptotagmin-1: A Comparative Study (Bacle et al., 2017, Biophys J, 112:1417). Whereas a slight increase in the hy- Hai Tran, Lauren Anderson, Jefferson Knight. drophobicity of perilipin 4-derived AHs makes them more promiscuous for University of Colorado Denver, Denver, CO, USA. binding to other cellular compartments, perturbing the distribution of charged Synaptotagmin 1 (Syt1) and synaptotagmin 7 (Syt7) contain analogous tandem residues in the polar face causes a striking loss of LD targeting. These results C2 domains, C2A and C2B, which serve as Ca2þ sensors to trigger fusion of suggest that the physico-chemistry of the perilipin 4 AH is exquisitely tuned to secretory vesicles during exocytosis. Functionally, Syt1 triggers fast release ensure LD specificity. In vitro, we find that the purified wild-type AH binds of neurotransmitters, while Syt7 is involved in slower processes such as hor- poorly to bilayer membranes. In contrast, it can interact efficiently with neutral mone secretion. It has been previously shown that Syt1 C2 domains bind mem- lipids and is capable of forming small uniformly-coated oil droplets. Accord- branes cooperatively, penetrating deeper into membranes as the C2AB tandem ingly, overexpression of this AH in cells overcomes a decrease in LD stability than as individual C2 domains. In contrast, our previous study suggested that associated with phospholipid depletion. We propose that by substituting for the two C2 domains of Syt7 bind membranes independently, based on their the phospholipid monolayer, perilipin 4 may be important for stabilization of dissociation kinetics from liposomes. Here, we further investigate the interdo- LDs when phospholipids are limiting, for example during periods of LD main interaction of Syt1 and Syt7 C2 domains by measuring Ca2þ sensitivities, growth. dissociation kinetics, and insertion depth of individual and tandem domains during binding to synthetic liposomes with physiologically relevant lipid com- 1390-Pos Board B299 positions. The Syt7 C2AB tandem was found to have greater Ca2þ sensitivity Single Molecule Parallax Fluorescence Quenching Measurements Reveal than either single domain from equilibrium Ca2þ titration data. Stopped-flow C2 Domain Cooperativity in the Membrane Penetration Activity of fluorescence spectroscopic measurements show that Syt1 C2AB dissociates Otoferlin much slower than either of its isolated C2 domains while for Syt7, the largest Shauna C. Otto1, Nicole J. Hams1, Weihong Qiu2, Colin P. Johnson1. population of the C2AB tandems has a comparable dissociation rate to the C2A 1Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA, domain and a subpopulation dissociates at a much slower rate. Furthermore, 2Physics, Oregon State University, Corvallis, OR, USA. like Syt1, the C2B domain of Syt7 penetrates membranes more deeply when Otoferlin is a vesicle-associated transmembrane protein consisting of six C2 present in the C2AB tandem than it does as individual domain, suggesting domains that is proposed to act as a calcium sensor for exocytosis in inner cooperative insertion. Based on these findings, we propose that the C2 domains hair cells in the ear. Otoferlin has been shown to be essential for vesicle fusion of Syt7 coinsert into membranes as they do in Syt1, but interdomain coopera- but the mechanism of action is poorly understood. It is thought that otoferlin tivity in Syt7 contributes less to the energetics of membrane binding than brings the vesicle and plasma membrane into close apposition by the dipping in Syt1. action of its many C2 domains into the plasma membrane, thus facilitating fusion. Previous studies have demonstrated that the innermost C2 domain, 1393-Pos Board B302 C2F, interacts with the plasma membrane lipid PIP2, but in a calcium indepen- Toward Understanding the Mechanism of Calcium-Inhibited Membrane dent manner. Here we have used TIRF microscopy to measure single molecule Binding of the SLP-2 C2A Domain parallax quenching of the noncanonical amino acid acridone incorporated into Timothy Spotts1, Sam Willstead2, Abena Watson-Siriboe1,3, the C2F domain to determine the extent of membrane penetration as a function Jefferson Knight1. of adjacent C2 domains. Our results indicate that the C2F domain in the context 1Chemistry, University of Colorado Denver, Denver, CO, USA, 2Chemistry, of the C-terminal doublet (C2EF) construct inserts in a calcium sensitive University of Southampton, Southampton, United Kingdom, 3Integrative manner and in the context of the C-terminal otoferlin triplicate (C2DEF) Biology, University of Colorado Denver, Denver, CO, USA. C2F inserts itself more deeply into the membrane. Based on these results, we Synaptotagmin-like proteins (Slps) are membrane-binding proteins that play propose that the C2F domain targets otoferlin-containing vesicles to the plasma key roles in trafficking of large dense-core vesicles for exocytosis. In particular, membrane, while the C2E domain confers calcium-sensitivity to the dipping Slp-2 is responsible for docking hormone secretory vesicles to sites of exocy- process. Studies conducted with the soluble protein, domains C2ABCDEF sup- tosis in pancreatic alpha cells and other cell types. Like all Slp family members, port the hypothesis that the remaining domains may act to stabilize membrane Slp-2 contains an N-terminal Slp-homology domain, a linker region of unde- dipping at the carbonyl-tail interface of the membrane. fined structure, and two C-terminal membrane-binding C2 domains (C2A and C2B). The C2A domain of Slp-2 has been shown to have a unique response 1391-Pos Board B300 to Ca2þ ions, in that it binds anionic liposome membranes in the absence of Membrane Binding and Dimerization of Vinculin Tail Ca2þ but dissociates upon addition of sufficient Ca2þ. This Ca2þ dependence Lukas Braun1, Ingmar Scho¨n2, Viola Vogel1. contrasts with most other C2 domains, which bind lipid membranes in either 1HEST, ETH Zurich, Zurich, Switzerland, 2Molecular and Cellular aCa2þ-dependent or Ca2þ-independent fashion. To investigate the mechanism Therapeutics Royal College of Surgeons in Ireland, Dublin, Ireland. of this unusual Ca2þ inhibition, a series of biophysical and spectroscopic mea- Vinculin is an essential scaffold protein that reinforces the link between the surements has been completed. First, the affinity of the well-purified C2A cell-adhesion machinery and the actin cytoskeleton. Binding to the phospho- domain for liposomes containing different compositions of key anionic lipids lipid Phosphatidylinositol 4,5-bisphosphate (PIP2) triggers the membrane asso- was measured using steady-state and stopped-flow fluorescence spectroscopy. ciation and subsequent dimerization of Vinculin tail domains (Vt). Structural Next, the effect of Ca2þ on C2A domain binding to each of the liposome com- and biochemical data have identified residues that are involved in lipid binding, positions was measured. It was found that Ca2þ does inhibit membrane binding, as well as the conformational transitions that are necessary for dimer formation. albeit at significantly higher concentrations than previously reported. Although However, the binding orientations of both the monomer and the dimer on the the protein-liposome affinity varies by >10-fold among the different lipid com- membrane remain controversial. Moreover, it is unclear how PIP2 binding positions tested, the Ca2þ concentration required for inhibition varies relatively

BPJ 8625_8627 Monday, February 19, 2018 277a little, suggesting a nonspecific mechanism. Current efforts are directed toward in many cases unclear. Molecular Dynamics (MD) simulations allow details of quantifying Ca2þ and other metal binding affinities using a luminescence-based biomolecular interactions to be examined on an atomistic or near-atomistic scale. Tb3þ displacement assay, and measuring effects of Ca2þ and lipids on protein For protein-membrane interactions, a multiscale approach may be employed, us- structure using EPR and NMR. ing a coarse-grained representation to simulate binding followed by refinement of the binding pose with full atomistic representation. Previously, a multiscale 1394-Pos Board B303 approach was used to investigate PTEN-membrane interactions; however this Exploring Ferrocyanide-Mediated Photoreduction Mechanics of Cyto- study used the truncated PTEN crystal structure, containing the catalytic phos- chrome C in the Presence and Absence of Cardiolipin phatase domain (PD) and lipid binding C2 domain but missing several disordered Dmitry Malyshka, Reinhard Schweitzer-Stenner. regions. Here, we have modelled these missing regions and utilized umbrella Drexel University, Philadelphia, PA, USA. sampling and multiscale simulations to investigate the behaviour of full-length Upon interacting with cardiolipin (CL), ferricytochrome c has been shown to lose wild type and mutant PTEN binding to PIP-containing membranes at both an en- its native M80 ligation and gain peroxidase activity in what is known as a pivotal ergetic and structural level. In particular, we have demonstrated a role of the step in the protein’s role in initiating apoptosis. The system can be difficult to disordered regions in directing productive membrane binding and the potential study spectroscopically due to the co-existence of multiple conformational states, importance of residues at the PD-C2 interface - a hotspot for disease- and yet this co-existence of native and partially-unfolded conformations is an associated mutations - in maintaining productive domain orientations. This important factor to quantify due to its clear biological significance. We have pre- work shows how MD can be used to increase our understanding of interactions viously shown that ferrocyanide-mediated photoreduction as probed by Soret between proteins and membranes in both health and disease. band resonance Raman (RR) spectroscopy can be used to quantify the amount of non-native conformers of cytochrome c during binding to CL-containing 1397-Pos Board B306 membranes when analyzed using the model of Pandiscia and Schweitzer- The Role of Lipid Chemistry in Alpha-Synuclein Membrane Binding and Stenner. However, the question of whether the photoreduction itself perturbs Aggregation the system by pumping the protein into the reduced state - or whether the Sandra Rocha, Pernilla Wittung-Stafshede. oxygen-mediated oxidation of ferricytochrome c as shown by Serpas et al. re- Biology and Biological Engineering, Chalmers University of Technology, stores the equilibrium - has still remained unanswered. RR spectra measured un- Gothenburg, Sweden. der anaerobic conditions show that the equilibrium is indeed shifted towards the Alpha-synuclein (a-Syn) is a small protein protein whose aggregation is the hall- ferro- state even in the presence of CL after extended periods of time. Further- mark of Parkinson’s disease. The protein is intrinsically disordered in solution more, we explored the mechanics of photoreduction itself by measuring RR but it forms an a-helix structure when bound to membranes. The interaction spectra at different 442 nm laser wattages and ferrocyanide ion / cytochrome c of a-Syn with lipids is related to its proposed function, i.e., regulation of synaptic concentrations. This method was extended to different laser lines in order to plasticity and neurotransmitter release, but it can also modulate aggregation into determine whether the mechanism of photoreduction is excitation wavelength amyloids. Recently, we studied the interaction of a-Syn with vesicles of DOPS dependent. This idea was further explored using different redox couples. (1,2-dioleoyl-sn-glycero-3-phospho-L-serine) and DOPG (1,2-dioleoyl-sn-glyc- 0 1395-Pos Board B304 ero-3- phospho-(1 -rac-glycerol)) and found that the lipid head-group chemistry a Lipid Binding Preferences of the Alternatively Translated Region of modulates lipid-protein interactions and affects the amyloid fiber formation. - PTEN-Long Syn has higher affinity for DOPG membranes and the protein aggregates at Anne-Marie Bryant1, Karin Plante1, Alonzo Ross2, Gericke Arne1. faster rates in the presence of DOPG vesicles as compared to DOPS vesicles. 1Worcester Polytechnic Institute, Worcester, MA, USA, 2UMass Medical Current efforts focus on revealing the underlying mechanisms for the interaction a School, Worcester, MA, USA. and the consequences for the lipid vesicles upon -Syn binding. Preliminary re- a PTEN-Long is a 576-amino acid translational variant of phosphatase and tension sults, using flow linear dichroism spectroscopy, for membrane bound -Syn a homolog on chromosome ten (PTEN), a tumor suppressor gene and antagonist to show that the long axis of the -Syn N-terminal helix is initially perpendicular phosphoinositide-3-kinase (PI3K) signaling. PTEN-Long (PTEN-L) exhibits an to the membrane normal for both DOPS and DOPG lipid bilayers. However, alternative translation region (ATR) with an additional 173 N-terminal amino over time, the protein orientation changes and the DOPG membranes become a acids at the N-terminal end to the normal PTEN open reading frame. PTEN-L more oriented, possibly due to -Syn inserting deeper into the membrane. In a is secreted from cells, can exist outside the cell and shows activity towards addition to bulk experiments, we have successfully probed -Syn binding to in- dividual vesicles in real time by monitoring changes in their scattering intensity PI(3,4,5)P3 after cell entry. Therefore, PTEN-L may have therapeutic uses by restoring a functional tumor suppressor protein to tumor cells. The mechanism with single-particle resolution imaging. by which PTEN-L enters cells is currently not understood. The ATR region of PTEN-L is evolutionary conserved and contains a polyarginine stretch with ho- 1398-Pos Board B307 mology to cell permeable peptides (such as HIV TAT). This suggests that Membrane-Mediated Gramicidin Interactions Determine Peptide Clus- PTEN-L may be able to traverse the plasma membrane passively, but it hasn’t tering and Enhance Channel Formation 1,2 2,3 2,3 been proven. The N-terminal ATR part of PTEN-L is intrinsically disordered, Oleg V. Kondrashov , Timur R. Galimzyanov , Sergey A. Akimov , 4 which we confirmed using CD and FTIR spectroscopy. Our FTIR studies did Yuri N. Antonenko . 1Moscow Institute of Physics and Technology, Dolgoprudny, Russian not reveal any observable structural change of PTEN-L ATR upon lipid binding 2 to PC, PS, PI and PI(4,5)P containing vesicles. However, we found that the Federation, A.N. Frumkin Institute of Physical Chemistry and 2 Electrochemistry RAS, Moscow, Russian Federation, 3National University of integrity of model lipid bilayers composed of PS are compromised upon inter- 4 action with PTEN-L’s ATR and we are further studying this with lipid leakage Science and Technology "MISiS", Moscow, Russian Federation, Belozersky experiments. The lipid binding preferences of the ATR part of PTEN-L, as well Institute of Physico-Chemical Biology, Lomonosov Moscow State as the full-length protein are being investigated using surface plasmon resonance University, Moscow, Russian Federation. (SPR). This study aims to gain insight on understanding PTEN-L’s ATR mem- Gramicidin is a membrane channel-forming antibiotic that is thoroughly inves- brane interactions at the molecular level. tigated. The peptide monomer is embedded into a single monolayer of a bilayer membrane. The through channel is formed by transmembrane dimerization of 1396-Pos Board B305 two peptide molecules. The details of the monomer interaction remain un- Structural and Energetic Details of the Binding of PTEN to Phosphatidy- known. Using continuous elastic model we studied the interaction of two gram- linositol Phosphate-Containing Membranes through Molecular Simula- icidin monomers embedded into a lipid bilayer. The calculated dependencies of tions dimer formation frequency on membrane tension and channel lifetime on mem- Fiona B. Naughton1, Antreas C. Kalli2, Mark S.P. Sansom1. brane thickness proved to be in perfect agreement with available experimental 1Department of Biochemistry, University of Oxford, Oxford, United data. The energy profile of the interaction was calculated for peptides located in Kingdom, 2Leeds Institute of Cancer and Pathology, University of Leeds, the same monolayer and in the opposite monolayers of lipid membrane. We Leeds, United Kingdom. found out that the energy of the system had a minimum when two gramicidin PTEN (Phosphatase and Tensin Homologue) is among the proteins most peptides were separated by a few nanometers for both cases. The calculated commonly found mutated in cancer, and has been associated with numerous other interaction energy profile was used to analyze the gramicidin molecules diseases. This is due to its role as a negative regulator of cell growth and signal- ensemble behavior. The simplest model of 2D Van der Waals gas was applied ling pathways, catalysing the dephosphorylation of the second messenger to calculate the critical temperature of the ensemble condensation. The calcu- PI(3,4,5)P3 to PI(4,5)P2 at the inner leaflet of the plasma membrane. However, lated temperature was found to be approximately 3 times higher than the phys- the exact details of the interaction of PTEN with PIPs and the membrane in gen- iological temperature, which indicates that gramicidin peptides in membrane eral, particularly in relation to the effect of clinically observed mutations, remain could form clusters, in agreement with recent experimental data. The designed

BPJ 8625_8627 278a Monday, February 19, 2018 elastic theory is general and may be used for the analysis of membrane- TheT-cellImmunoglobulinandMucincontaining(TIM)proteinfamily mediated interactions of the inclusions of different nature: transmembrane mol- consists of three membrane proteins with immunoglobulin domains ecules, amphipathic peptides, etc. The work was supported by the Russian that have been shown to bind phosphatidylserine (PS) in vitro and PS- Foundation for Basic Research (project # 17-04-02070). containing membranes in vivo. TIM1, TIM3, and TIM4 are each associated with distinct physiological roles yet they share the same conserved calcium- 1399-Pos Board B308 coordinating PS binding pocket. The TIM proteins are expressed on CY3/CY5 Fluorophore-Lipid Interactions and their Effects on Membrane different cell types and respond to different conditions despite recognizing Protein Dynamics the same ligand. We show that in addition to the presence of PS in a mem- Kin Lam1,2, Emad Tajkhorshid2,3. 1 brane, the bulk properties of the membrane modulate the binding of the TIM Department of Physics, U of I Urbana-Champaign, Urbana, IL, USA, proteins. Moreover, the three members differ in their sensitivities to these 2Beckman Institute for Advanced Science and Technology, Urbana, IL, USA, 3 bulk properties, allowing us to characterize the membrane conditions that Department of Biochemistry, Center for Biophysics and Computational optimize binding for each TIM protein. Characterization of their membrane Biology, U of I Urbana-Champaign, Urbana, IL, USA. contextual binding helps inform how the different TIM proteins fulfill their Organic fluorophores, such as Cy3 and Cy5, have been widely used as chemical distinct roles despite their conserved PS binding pocket. In order to investi- labels to probe dynamics and functions of membrane proteins. While several gate TIM proteins’ sensitivity to bulk membrane properties, we combine water-soluble fluorophores have been reported interacting strongly with mem- thermodynamic binding measurements with Molecular Dynamics (MD) brane lipid, detailed fluorophore-lipid interactions and the effect of fluorescent informed structural analysis. X-ray reflectivity experiments analyzed by labeling on the natural dynamics of target proteins has not been revealed by MD-refined models of the protein-membrane system reveal candidate resi- experiment. We employed the highly mobile membrane mimetic (HMMM) dues which may contribute to membrane-binding in addition to the PS bind- model in a large set of all-atom molecular dynamics simulations to unravel ing pocket. In particular, our analysis revealed, in each of the TIM proteins, the spontaneous partitioning of sulfo-Cy3/Cy5 fluorophores into lipid bilayers a single tryptophan that buries into the membrane upon binding. Fluores- with different lipid compositions. Spontaneous membrane partitioning of Cy3/ cence spectroscopy of this tryptophan is thus used as an indicator for bind- Cy5 captured in these simulations reveals two steps. Electrostatic interaction ing since the tryptophan’s emission spectrum shifts upon burial. Binding between Cy3/Cy5’s sulfo-group and lipid headgroups facilitates the fast mem- experiments under a variety of membrane conditions allow us to measure brane association of fluorophores, followed by slow insertion into the lipid the affinity as a function of bulk membrane properties. bilayer core. Using biased-exchange umbrella sampling, free energy calcula- tions with full-tailed lipids revealed a higher energy barrier for partitioning 1402-Pos Board B311 into negatively charged (PS) membrane than uncharged (PC/PE) membrane. EPS15 Forms Membrane Bound Networks that Promote Localized Assem- Furthermore, the effect of fluorophore-lipid interactions on membrane protein bly of the Clathrin Coat has been examined by covalently linking Cy5 to the transmembrane helices of Grace Kago1,2, Justin Houser1, Wilton T. Snead1, Wade F. Zeno1, glycophorin A. Equilibrium simulations show non-trivial effect of Cy5-tagging Carl C. Hayden1, Eileen M. Lafer3, Jeanne C. Stachowiak1,2. on the helix-tilting angle and lateral diffusion inside a membrane, depending on 1Biomedical Engineering, University of Texas at Austin, Austin, TX, USA, the position of tagging site relative to the membrane. Our results suggest that 2Cell and Molecular Biology, University of Texas at Austin, Austin, TX, fluorophore-lipid interactions can affect the structure and natural dynamics of USA, 3UTHSCSA Center for Macromolecular Interactions, University of membrane proteins. Texas at San Antonio, San Antonio, TX, USA. 1400-Pos Board B309 Assembly of clathrin-coated vesicles, an essential cellular process, requires The Interaction of Amphipathic a-Helix Bundle Proteins with Neutral remodeling the membrane surface into a highly curved vesicle, loaded with Lipid Droplets specific cargo proteins and receptors. The clathrin-coat is recruited to the Mona Mirheydari1, Elizabeth K. Mann2, Edgar E. Kooijman2. membrane surface by a synchronized assembly of protein adaptors that 1Rutgers University, New Brunswick, NJ, USA, 2Kent State University, are localized at the nascent vesiculation site. The recruitment of these pro- Kent, OH, USA. teins depends on i) binding to the plasma membrane lipid phosphatidylino- Lipid droplets (LDs) are dynamic cell organelles with structure similar to sitol 4,5-biphosphate (PtdIns (4,5) P2) and ii) binding with each other. One that of lipoproteins: a core dominated by triglycerides and other neutral of the earliest proteins to arrive at nascent CME sites is epidermal growth lipids surrounded by a phospholipid-protein monolayer. The phospholipid factor receptor pathway substrate clone 15 (Eps15). Interestingly, the monolayer covering the neutral lipid core makes for a unique assembly recruitment of Eps15 is independent of both clathrin and cargo proteins. for lipid-protein interaction. We study these interactions via liquid droplet Structural studies using electron microscopy have reported that Eps15 as- tensiometry, using droplet shape to measure the surface tension of a triolein sembles into homo-dimers and tetramers. These findings led us to inquire droplet in an aqueous medium. For the first time, we use lipid mixtures to whether Eps15 assembly could promote initiation of clathrin coated pits. form the phospholipid monolayer. We report two distinct types of experi- Here we show that the ability of Eps15 to oligomerize enables the assembly ments: 1) Dynamic measurements in which we oscillate the surface area of stable protein clusters at membrane surfaces. These dynamic clusters of the lipid droplet, analogous to the area changes naturally occurring in serve as hotspots for recruitment of other adaptor proteins including epsin1 LDs. These experiments allow us to deduce surface pressure vs. area iso- and AP180, which ultimately facilitate preferential recruitment and assem- therms for different lipid mixtures. They also allow us to test the stability bly of the clathrin coat. Taken together, these results are significant because of the phospholipid and protein layer on the surface of the triolein droplet. they show that an Eps15-derived protein network can provide a stable, yet 2) Insertion measurements, which allow us to characterize the protein affin- flexible platform for local assembly and growth of clathrin coated mem- ity and insertion for lipids with different negative curvatures and charges. brane invaginations. We used two amphipathic a-helix bundle proteins namely apoLp-III (full length protein) and apoE-3 (N-terminus). We show that bundle unfolding Posters: Exocytosis and Endocytosis is critical for protein interaction with the triolein-phospholipid interface. Important differences between apoLp-III and apoE-3 are discussed in terms 1403-Pos Board B312 of the ability of the helix bundle to unfold. Drug Delivery Platform Based on Amphiphilic Poly-N-Vinyl-2- Pyrrolidone: The Role of Size Distribution in Cellular Uptake 1401-Pos Board B310 Anna L. Luss1, Camilla L. Andersen2, Irene G. Benito2, Rafael C. Marzo2, The Membrane Matters: Sensitivity of TIM Proteins to Bulk Membrane Zaida H. Medina2, Martin B. Rosenlund2, Sven B. Romme2, Properties in Binding Phosphatidylserine Pavel P. Kulikov1, Cristian P. Pennisi3, Mikhail I. Shtilman1, Daniel Kerr1, Zhiliang Gong2, Gregory T. Tietjen3, Adrienne Luoma4, Leonid Gurevich2. Charles L. Dulberger5, Erin J. Adams4, Ka Yee C. Lee6. 1D. Mendeleyev University of Chemical Technology of Russia, Moscow, 1Program in Biophysics, University of Chicago, Chicago, IL, USA, Russian Federation, 2Materials and Production, Aalborg University, Aalborg, 2Department of Chemistry, University of Chicago, Chicago, IL, USA, Denmark, 3Health Science and Technology, Aalborg University, Aalborg, 3Surgery: Transplant & Immunology, Yale School of Medicine, New Haven, Denmark. CT, USA, 4Committee on Immunology and Department of Biochemistry and Micellar drug carriers are commonly taken up by cells via endocytic path- Molecular Biophysics, University of Chicago, Chicago, IL, USA, ways. A particle that comes into contact with the cell surface becomes en- 5Department of Biochemistry and Molecular Biophysics, University of gulfed by the cell membrane, forming an invagination, which afterwards Chicago, Chicago, IL, USA, 6Department of Chemistry and James Franck separates from the membrane as a vesicle and is typically transported to ly- Institute, University of Chicago, Chicago, IL, USA. sosomes for degradation. This puts significant restrictions on the cargo

BPJ 8625_8627 Monday, February 19, 2018 279a delivered in this way, e.g., genetic material. Non-endocytic pathway typically wrapping, but as little as 5 or 6 closest receptor binding sites can do the requires specific functionalization, for instance with cell penetrating peptides. same, e.g., occupied by a single multivalent inhibitor. We also evaluated In this report, we present a polymeric drug delivery system based on amphi- possible disadvantage of multivalent inhibitors in aggregating and uptake of philic poly-N-vinyl-2-pyrrolidone containing a thiooctadecyl end-group more capsids together and proposed a solution to this phenomena by use of (PVP-OD). We showed that PVP-OD is capable of forming nanocarriers in limited valence or by ‘‘Janus’’ multivalent inhibitors, with part of the mole- aqueous media and can solubilize highly hydrophobic compounds such as cule being inert. To estimate the optimal valence, we derived an analytical the- curcumin, used as a model pharmaceutically active substance in this study. ory for estimation of the binding constant between two viruses/nanoparticles Two commonly used drug loading methods—emulsification and ultrasonic bridged by a multivalent inhibitor. Our findings could be useful in the design dispersion—were successfully applied and found to lead to different carrier of multivalent inhibitors preventing the uptake processes of viruses, bacteria, size distributions and, more importantly, a distinctively different biological or toxic nanoparticles. effect. Using the U87 glioblastoma and CRL 2429 fibroblast cells, we observed that larger drug carriers produced via the emulsion route penetrated 1406-Pos Board B315 cells by endocytic mechanisms. The uptaken drug was accumulated in endo- New, Non-axisymmetric Modes of Deformation in Endocytosis somes and did not penetrate into the nucleus. Furthermore, its uptake could be Yannick A.D. Omar1, Amaresh Sahu2, Roger A. Sauer1, blocked by Dynasore—a dynamin inhibitor. On the other hand, the smaller, Kranthi K. Mandadapu2. dispersion-produced drug carriers exhibited much faster kinetics and uni- 1AICES, RWTH Aachen University, Aachen, Germany, 2Department of formly penetrated throughout cellular compartments, including the nucleus. Chemical & Biomolecular Engineering, University of California at Berkeley, The uptake of dispersion-produced carriers could not be inhibited by Dyna- Berkeley, CA, USA. sore or sodium azide, indicating non-endocytotic penetration through the Endocytosis is a process in which cells form invaginations by internalizing cell membranes. Removal of the small-particle fraction from the parts of the plasma membrane for the purpose of bringing cargo into the cell. dispersion-produced nanocarriers also removed the ability of non-endocytic In particular, the cargo material can be various proteins but also pathogens, penetration. This is a fundamentally new result, potentially enabling direct thus making endocytosis an important mode of intercellular trafficking. Endo- delivery of pharmaceutically active substances into cell nuclei and establish- cytic events are mediated through a protein coat attaching to the lipid bilayer of ing a promising approach for gene therapy. the cell. The protein’s structure induces curvature into the lipid bilayer, thus forming an invagination and subsequently a bud which carries the material to 1404-Pos Board B313 be transported. When the bud is sufficiently constricted, scission occurs at A Holistic Approach to Study Interactions between Nanoparticles/Vesicles/ the bud’s neck and a vesicle is formed. Despite the fact that endocytosis has Viruses and Supported Lipid Bilayers using QCM-D, Dual-Wavelength been studied extensively, its mechanics are not yet fully understood. Lipid bi- SPR, and Neutron Reflectometry layers are usually modeled as liquid shells which do not have any shear resis- Antonius Armanious1, Yuri Gerelli2, Bjo¨rn Agnarsson1, Hudson Pace1, tance in their in-plane direction. There are few non-axisymmetric, continuum Samantha Micciulla2,3, Emanuel Schneck3, Fredrik Ho¨o¨k1. studies on lipid bilayers available. Recent findings for lipid bilayers based on 1Physics Department, Chalmers University, Gothenburg, Sweden, 2Institut a three-dimensional, C1-continuous Finite Element formulation showed that Laue-Langevin, Grenoble, France, 3Max Planck Institute of Colloids and non-axisymmetric solutions can be energetically preferable over axisymmetric Interfaces, Potsdam, Germany. solutions. In this work, we investigated under which conditions, non- Interactions of nanoparticles (NPs), lipid-based vesicles, and enveloped viruses axisymmetric solutions are favorable. Namely, we find that axisymmetric with cell membranes constitute the heart of countless biological processes. In shapes can branch out into non-axisymmetric shapes depending on factors most of these processes, the binding strength and the distance between the par- such as the surrounding tension, viscous dissipation and the shape of the protein ticle of interest and the cell membrane are two crucial parameters that deter- coat. We identify two different branches of solutions in which the mean curva- mine the fate of these particles. These two parameters are dynamic, i.e., they ture can take a value close to the prescribed spontaneous curvature. Firstly, this change with time according to the stage of the biological process being inves- condition is met by forming spherical shapes, i.e. shapes with two equal prin- tigated. Their dynamic nature and the small length scale on which they operate, cipal curvatures. Secondly, cylindrical shapes, i.e. shapes where one of the nano to a˚ngstrom scale, makes it extremely challenging to probe them. In this principal curvatures is close to zero, can satisfy the same condition. This result study, we adopted a holistic approach utilizing three different surface tech- and a set of factors determining the branching of solutions will be discussed in niques: quartz crystal microbalance with dissipation monitoring (QCM-D), detail. dual-wavelength surface plasmon resonance (SPR), and neutron reflectometry (NR), to study the kinetics and equilibrium state of interactions between silica 1407-Pos Board B316 NPs, silica NPs coated with supported lipid bilayer (nano-SLB) and lipid-based Low Affinity Receptors Can Enter Endocytic Pits by Binding to High Af- vesicles with flat supported lipid bilayers (flat-SLBs). The nano-SLBs and the finity Receptors vesicles were tethered to the flat-SLBs using two different DNAs: one which Chi Zhao1, Andre C.M. DeGroot1, Jeanne C. Stachowiak1,2. keeps them at a defined distance from the flat-SLB and the other which brings 1Biomedical Engineering, University of Texas at Austin, Austin, TX, USA, them in contact with the flat-SLBs. The QCM-D response allowed us to deduce 2Institute for Cellular and Molecular Biology, University of Texas at Austin, real-time qualitative information about the stiffness of the binding between the Austin, TX, USA. different particles and the flat-SLB. Using dual-wavelength SPR we could Clathrin-mediated endocytosis, essential to signal transduction from the quantitatively monitor both the adsorbed masses and thicknesses in real-time. cellular surface, gives cells precise temporal and spatial control over hun- NR offered the most detailed information about the z-profile of the adsorbed dreds of receptors on the plasma membrane surface. The biochemical motifs particles at equilibrium with a˚ngstrom accuracy. The combination of these responsible for recruiting individual receptors into clathrin-coated pits have three methods was crucial to obtain a detailed multifaceted picture of the inves- been thoroughly mapped. However to initiate a signaling cascade, membrane tigated systems and serves as a roadmap for conducting similar experiments receptors such as G-protein coupled receptors (GPRCRs) often assemble into on other biological systems and processes, such as virus-membrane heterogenous dimers and clusters. How does assembly of heterogeneous re- interactions. ceptor clusters impact the uptake of individual receptor species? To address this question, we created a simplified model system consisting of 2 chimeric 1405-Pos Board B314 transmembrane receptors. While both receptors are constitutively endocy- Multivalent Inhibitors Preventing Uptake of Virus-Like Particles tosed, one receptor contains a mutated AP2 binding motif, leading to severely Ivo Kabelka, Radim Brozek, Robert Vacha. reduced localization to clathrin-coated pits. Using quantitative imaging to CEITEC - Masaryk University, Brno, Czech Republic. precisely measure the distribution of receptors on the plasma membrane, Cellular entry of viruses can be inhibited by molecules adsorbed on the virus we demonstrate that when two receptors form a biochemical bond, the recep- capsid. However, apart from stronger binding little is known about the prop- tor with high affinity for endocytic structures can drive efficient internaliza- erties of such inhibitors that could increase their effectiveness. We investi- tion of the receptor of low affinity. Specifically, localization of low affinity gated the inhibition of capsid uptake during the process of spontaneous receptors to clathrin-coated pits increases as a function of high affinity recep- receptor-mediated endocytosis in the presence of monovalent and multivalent tor expression levels, suggesting that the binding interaction between the two inhibitors. Using computer simulations, we found that multivalent inhibitors receptors is the driving force behind internalization of the low affinity recep- are much more efficient in uptake inhibition not only due to the increased af- tor. In particular, our data demonstrates that the recruitment of low affinity finity but largely due to the spatially correlated (i.e., localized) inhibition. receptors can be further enhanced by increasing the overall binding affinity Roughly 1/3 of all the receptor binding sites on virus capsid have to be in- between the two receptors, achieved by incorporating serial repeats of the hibited by monovalent randomly distributed inhibitors to stop the membrane receptor-receptor interaction domain into our model receptors. Collectively,

BPJ 8625_8627 280a Monday, February 19, 2018 these results suggest that receptor uptake depends on both recruitment of in- the vATPase and blocks its activity. Such inhibition of vATPase by clathrin dividual receptors and recruitment of heterogeneous protein complexes. coat is likely fundamental for the proper timing of SV refilling that is driven Moving forward, this work suggests novel approaches for amplifying by the electrochemical gradient, as well as for the regulation of vATPase activ- signaling in therapeutically relevant pathways, such as GPCR and apoptotic ity during the endocytic process. signaling, by promoting interactions between target receptors and receptors undergoing active internalization. 1410-Pos Board B319 Role of Actin and Membrane Tension in Regulating Modes of Exocytosis 1408-Pos Board B317 Julian Hassinger1, David Drubin2, Padmini Rangamani3. Modeling the Flat to Curved Transition during Clathrin Mediated Endo- 1Biophysics, University of California, Berkeley, Berkeley, CA, USA, cytosis 2Molecular and Cell Biology, University of California, Berkeley, Berkeley, Felix Frey1, Delia Bucher2, Kem A. Sochacki3, Susann Kummer2, CA, USA, 3Mechanical and Aerospace Engineering, University of California, Hans-Georg Kr€ausslich2, Karl Rohr4, Justin W. Taraska3, Steeve Boulant2, San Diego, La Jolla, CA, USA. Ulrich S. Schwarz1. Exocytosis is a critical biological process in which secretory vesicles fuse 1Institute for Theoretical Physics, Heidelberg University, Heidelberg, with the plasma membrane, expelling their contents into the extracellular Germany, 2Department of Infectious Diseases, Virology, University Hospital space. Recent experimental evidence implicates a role for actin as a modu- Heidelberg, Heidelberg, Germany, 3National Heart Lung and Blood Institute, lator of local membrane tension and setting the balance between ‘‘kiss-and- National Institutes of Health, Bethesda, MD, USA, 4BioQuant-Center, run’’ vs. ‘‘full-fusion’’ modes of exocytosis. How exactly physical parame- Heidelberg, Germany. ters like vesicle size, spontaneous curvature, membrane tension, turgor pres- Clathrin-mediated endocytosis is essential for the cellular uptake of receptors sure and external forces come together to control the balance of these two and nutrients. Although under investigation since decades, the exact sequence exocytic modes is unclear. To address this question, we use a general of structural and molecular events remains elusive. There are two basic models continuum-mechanical model of lipid bilayers to simulate membrane shapes that have been suggested for the way it proceeds. In the constant curvature following the fusion of an exocytic vesicle with the plasma membrane. We model, it is assumed that clathrin-coated pits grow with constant curvature, find that a local increase in membrane tension will either cause the membrane determined by the geometry of clathrin triskelia. In the constant area model, to flatten or shrink the fusion pore as determined by a balance between vesicle it is assumed that clathrin triskelia first assemble into flat hexagonal arrays size, bending rigidity and magnitude of spontaneous curvature. Furthermore, that later invaginate at constant surface area. This second model implicitly as- an increase in turgor pressure can provide a complementary driving force for sumes that during bending, some hexagons are converted into pentagons. Here, flattening the vesicle membrane post-fusion which could compensate for a we integrate data sets from correlative electron and light microscopy and quan- lack of actin-induced membrane tension. We also show that dissipation of tify the sequence of ultrastructural rearrangements of the clathrin coat during spontaneous curvature as well as membrane tension contribute to shrinking endocytosis in mammalian cells with the help of some simple mathematical of an omega-shaped membrane profile even while the fusion pore is pinched growth laws. Our main assumption is that the clathrin domain can grow only by an external force. A localized pressure on an omega-profile is also suffi- over the boundary. In the case of flat arrays, this requires some balancing pro- cient to induce shrinking, indicating other hypotheses for the role of actin cess to prevent a run-away process, which we assume to grow in proportion to in compressing the exocytic vesicle to expel its contents are also plausible. the domain area. In the case of curved arrays, pit closure is sufficient to limit Importantly, the length scales over which these mechanisms are relevant pro- growth and thus an additional process is not required. Our analysis shows vides insight into why actin might differentially contribute to the mode of that clathrin-coated structures initially grow flat but start to acquire curvature exocytosis in different cell types. when 70% of the final clathrin content is reached. We find that this transition correlates with a change in the ratio of clathrin to adaptor protein AP2, and 1411-Pos Board B320 that membrane tension suppresses this transition. Hence, our analysis suggests Force Generation by Curvature-Generating Molecules in Cells with that elements of both suggested models are present and that mechanical and Turgor cellular factors will decide about the relative weights of growth versus curva- Jonah K. Scher-Zagier. ture formation. Washington University in St. Louis, University City, MO, USA. Curvature-generating molecules (CGMs) are central to a variety of biological 1409-Pos Board B318 processes. Proteins such as clathrin and BAR-domain proteins help provide Clathrin Coat Controls Vesicle Acidification by Blocking Vacuolar forces and moments to drive membrane bending, as in the case of endocy- ATPase Activity tosis. We develop a discrete equilibrium mechanical model of the shape of Zohreh Farsi1, Sindhuja Gowrisankaran2, Matija Krunic2, a small CGM-membrane complex that incorporates the effects of cell wall Burkhard Rammner3, Andrew Woehler1, Carsten Mim4, Reinhard Jahn5, elasticity and turgor pressure, as well as a simple continuous model that Ira Milosevic6. also incorporates the effects of the wall force. We calculate the dependence 1Light Microscopy Platform, Max-Delbruck€ Center for Molecular Medicine of the force generated by the CGPs on various parameters, including the in the Helmholtz Association, Berlin, Germany, 2European Neuroscience bending modulus, the patch size, and the turgor pressure, and find evidence Institute, Go¨ttingen, Germany, 3sciloop, Hamburg, Germany, 4School for of transitions as a function of external turgor pressure and intrinsic curvature Technology and Health, Huddinge, Sweden, 5Neurobiology, Max-Planck- that are discontinuous for the force and continuous for the displacement. We Institute for Biophysical Chemistry, Go¨ttingen, Germany, 6Synaptic Vesicle develop simple formulas for the maximum force generated by the CGM patch Dynamics, European Neuroscience Institute, Go¨ttingen, Germany. in terms of the patch size, the bending rigidity, and the preferred curvature. At neuronal synapses, exocytosed membrane and proteins are internalized pre- Comparison of the discrete model to previous continuous models of CGP dominantly by clathrin-mediated endocytosis. Newly formed synaptic vesicles forces reveals important corrections to the continuous models. We find that (SVs) are rapidly acidified by vacuolar ATPases, generating a proton electro- the spatial distribution of the forces depends on the strength of the turgor chemical gradient across the membrane that is subsequently used by vesicular pressure relative to bending energy, with forces being localized at the edges transporters to fill the vesicles with neurotransmitters. To date, it is unclear for high turgor pressure, and more widely distributed for low turgor pressure. when endocytosed vesicles acidify and refill at the synapse. While some In addition, the energy exhibits a minimum at small numbers of molecules. have suggested that endocytic coated vesicles do not have acidic internal pH, Further, for realistic values of the bending rigidity and curvature, CGPs alone other studies have shown that membrane-permeable pH-sensitive weak bases are insufficient to initiate endocytosis against turgor pressure, consistent with accumulate in the lumen of clathrin-coated vesicles (CCVs), indicating that previous findings. acidification occurs in the presence of the coat. To weigh in on this debate, and to inspect whether vATPase is active at the CCV, we isolated CCVs 1412-Pos Board B321 from the mouse brain and used our recently developed single-vesicle imaging Membrane Tension Dictates the Spatiotemporal Heterogeneity of Endo- assay (Farsi, et al. Science, 2016) to perform full characterization of the electro- cytic Clathrin Coat Dynamics in Cells chemical gradient in single CCVs. We have observed that the ATP-induced Nathan M. Willy1, Joshua Ferguson1, Scott Huber1, Spencer Heidotting1, acidification of CCVs was strikingly reduced in comparison to SVs. Remark- Esra Aygun2, Sarah Wurm1, Ezekiel Johnston-Halperin1, Michael Poirier1, ably, when the clathrin coat was removed from CCVs, uncoated vesicles re- Comert Kural1. gained ATP-dependent acidification, demonstrating unequivocally that CCVs 1Ohio State University, Columbus, OH, USA, 2Capital University, contain the functional vATPase, yet its function is inhibited by the clathrin Columbus, OH, USA. coat. Considering the known structures of the vATPase and clathrin coat, The dynamics of endocytic clathrin coat assembly can be widely divergent, not we propose a model in which the formation of the clathrin coat surrounds only among cells within the same culture but even within distinct regions of the

BPJ 8625_8627 Monday, February 19, 2018 281a same cell. While the full scope of causes for this diversity is unknown, tension new mechanistic insights into curvature stimulation of mechano-chemical ac- on the plasma membrane is a likely contributor, as each endocytosis event ne- tivity of Dyn1. cessitates induced membrane curvature. Our results demonstrate that increased membrane tension impedes clathrin-mediated endocytosis (CME) through inhi- 1415-Pos Board B324 bition of initiation, formation and dissolution of clathrin-coated structures, and The FERA Domain Is a Membrane-Binding Four-Helix Bundle Embedded endocytic dynamics rapidly and locally reflect changes in membrane tension within Ferlin Membrane Fusion Proteins due to pharmacological and mechanical stimuli. We found that spatial and tem- Faraz Harsini, Sukanya Chebrolu, Anne Rice, Roger Bryan Sutton. poral heterogeneities in clathrin coat dynamics mark the tension gradients Cell Physiology and Molecular Biophysics, Texas Tech University Health arising during cell spreading and migration. Spatiotemporal variations in Sciences Center, Lubbock, TX, USA. CME are also observed during morphological changes shaping development The ferlins are long, string-like proteins that bind to phospholipid membranes of multicellular organisms. Collectively, our findings suggest that tension gra- in the presence of Ca2þ, and have been implicated in membrane fusion. dients in tissues can lead to patterning and differentiation through mechano- Dysferlin, myoferlin, and otoferlin in particular have received considerable regulation of CME. attention due to their impact on human health. Dysferlin is involved in mem- brane repair, and lack of dysferlin function in skeletal muscle could lead to pro- 1413-Pos Board B322 gressive damage to muscle cells. Mutations in the dysferlin gene are linked to Energetics and Stability of Neck Formation in Yeast and Mammalian Limb-Girdle Muscular Dystrophy in humans. Myoferlin is involved in the Endocytosis initial formation of muscles by fusing myotubes into myocytes early in devel- Ritvik Vasan1, Julian Hassinger2, Haleh Alimohamadi1, David Drubin3, opment. Otoferlin is found exclusively in the ear, and may play a Padmini Rangamani1. synaptotagmin-like role in exocytosis. Mutations in otoferlin are linked to 1Mechanical and Aerospace Engineering, UC San Diego, San Diego, CA, non-syndromic deafness in humans. We have discovered that the FerA domain, USA, 2Biophysics Graduate Group, UC Berkeley, Berkeley, CA, USA, which is located at the center of each of the ferlin proteins, is a four-helix 3Molecular and Cell Biology, UC Berkeley, Berkeley, CA, USA. bundle that can bind to lipids in a Ca2þ-dependent manner. Although the The formation and constriction of membrane necks is a fundamental step that net charge of each FerA domain varies from highly negative to highly positive, immediately precedes fission in membrane remodeling processes like endo- each that we have studied shows a Ca2þ-dependent lipid association, suggest- cytosis, viral egress, and cytokinesis. Even in endocytosis, the formations ing a role in membrane fusion. Additionally, thermodynamic analysis of the of invaginations and buds are different in yeast vs mammalian cells. In dysferlin FerA domain and two clinically-identified mutations, L705M and mammalian cells, structural and biophysical studies have identified that P731R, shows that domain stability is compromised in these mutants. constriction of the neck by dynamin helices triggers membrane fission. How- Decreased stability and enhanced membrane binding of the FerA mutants sug- ever, yeast cells do not have dynamin. A detailed physical understanding of gest that an open, membrane bound form exists in addition to the closed, sol- the force and energetic requirements for neck constriction in yeast vs uble form. mammalian cells is difficult to ascertain using experimental techniques due to the small length scales nearing self contact. In this work, we apply a modi- 1416-Pos Board B325 fiedHelfrichtheoryoflipidmembranes to investigate membrane necking in MUNC18-1 Catalyzes Snare Assembly by Templating Snare Folding and yeast and mammalian endocytosis. Specifically, we seek to understand how Association applied forces and protein-induced spontaneous curvature contribute to Yongli Zhang. induce necking and energy distribution on the membrane. Application of a Cell Biology, Yale University, New Haven, CT, USA. radial constricting force, mimicking the action of dynamin, leads to a snap- Sec1/Munc18-family (SM) proteins are essential for SNARE-mediated mem- through instability in the shape of the membrane during budding. We observe brane fusion and associated with many human diseases. Yet, their conserved a large energy barrier that can be overcome by force to achieve a narrow neck. role in SNARE assembly is unknown. Using single-molecule force spectros- This instability is eliminated by applying a region of spontaneous curvature, copy, we found that Munc18-1 catalyzes the assembly of three synaptic contracting the radius or widening the force. Application of a pulling force, SNAREs into a four-helix bundle. Munc18-1 targets the N-terminal domain essential to yeast endocytosis, also leads to snapthrough instability for a rigid of syntaxin and binds to SNARE motifs of both syntaxin and VAMP2 to protein coat. Here, the instability is eliminated by ensuring uniform rigidity of nucleate SNARE assembly as a template. SNAP-25 rapidly binds to the tem- coat and membrane. We extend the analysis to images of clathrin coated buds plate complex to form a partially-zippered SNARE complex and then fully zip- from mammalian and yeast experiments and track the process of neck forma- pers to displace Munc18-1. We have quantified the stabilities, conformations, tion. To do this, we implement an image processing framework that was and kinetics of key intermediates and demonstrated their biological significance recently implemented by the authors. Our model sheds insight into the in membrane fusion and implications in diseases. Similar assembly pathway defining characteristics of membrane necking and related snapthrough has been observed in Vps33-vacuolar SNARE fusion machinery, suggesting instabilities. a conserved mechanism for all SM proteins.

1414-Pos Board B323 1417-Pos Board B326 Highly Charged Membrane Templates for Studying the Mechano- Conformational Changes of SNAP-25 due to Environmental Conditions Chemistry of Dynamin 1 Ani C. Nichol, Matt C. Pettit, Walker L. Johnson, Wade J. Whitt, Javier Vera Lillo1, Anna V Shnyrova1, Vadim A. Frolov1,2. Emily Campbell Whitt, Skyler F. Nichol, Robert E. Coffman, 1Biophysics Institute (CSIC, UPV/EHU) and Department of Biochemistry Dixon J. Woodbury. and Molecular Biology, University of the Basque Country, Leioa, Spain, Physiology and Developmental Biology, Brigham Young University, Provo, 2IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. UT, USA. Dynamin 1 (Dyn1) is a large GTPase mediating membrane fission during syn- The SNARE protein SNAP-25 plays a critical role in neuronal exocytosis by aptic vesicle recycling. Dyn1 converts the energy obtained from GTP- providing two of the four helical regions (SNARE domains) that form the hydrolysis into mechanical work required to constrict and cut the neck of en- coiled-coil complex required for neurotransmitter release. This complex is docytic vesicle. Mechano-chemical activity of Dyn1 is coupled to membrane continually formed and unwound as exocytotic vesicles fuse and recycle. Using curvature. Dyn1 preferentially binds to highly curved membrane surfaces, circular dichroism and fluorescent spectroscopy, we observe clear structural such as vesicle necks. The binding is followed by oligomerization of Dyn1 changes of SNAP-25 in response to pH, ionic strength, temperature, oxidation into helices augmenting membrane constriction. The helical arrangement state, and in response to small biological molecules. dramatically boosts the GTP hydrolysis that furthers constriction to the crit- Using circular dichroism to measure the secondary structure of SNAP-25B, we ical curvatures triggering fission. Molecular mechanisms behind this observe a decrease in alpha-helical structure when decreasing ionic strength or coupling, constituting the basis of Dyn1 function, remain a subject of debate. increasing temperature. (In vivo, helix formation is greatest during complex Membrane binding of Dyn1 is regulated by membrane charge. Here we pre- formation with syntaxin and VAMP.) SNAP-25B becomes more helical at sent highly charged membrane templates that allow uncoupling of membrane low temperatures (4C) and physiological ionic conditions (150 mM salt). binding and oligomerization of Dyn1 from its curvature and fission activities. Under these optimal conditions, addition of ATP or AMP decreases helicity. We reveal that even on flat templates Dyn1 self-assembles into clusters that Using fluorescently labeled ATP (TNP-ATP) we observe shifts in emission undergo GTP-dependent transformations. We further quantified the stoichi- wavelength and intensity consistent with its interactions with the protein. ometry and dynamics of such clusters using GFP-conjugated dynamin and These data suggest that SNAP-25 may be classified as an intrinsically disor- compared the effect of GTP on flat and curved templates. Our data provide dered protein that changes structure in response to environmental conditions.

BPJ 8625_8627 282a Monday, February 19, 2018

Observed conformational changes may alter SNAP-25’s ability to form Additionally they suggest the role of Synaptotagmin-1 is not confined to trig- a complex with syntaxin and VAMP or change the accessibility of the gering fusion pore opening; it also plays a post-fusion role in pore dilation in cysteines in SNAP-25B which may contribute to regulation of exocytosis the absence of additional factors. in vivo. 1420-Pos Board B329 1418-Pos Board B327 Structural Insight into the Interaction of Synaptotagmin-1 and Snare Single SNARE Complex Recycling By NSF Complex on Lipid Bilayer by Cryo-Electron Microscopy Ucheor Brandon Choi1, Minglei Zhao2, Kristopher I. White1, Kirill Grushin1, Jing Wang1, Jeff Coleman1, James E. Rothman1, Qiangjun Zhou1, Richard Pfuetzner1, Axel T. Brunger1. Charles V. Sindelar2, Shyam S. Krishnakumar1. 1Molecular and Cellular Physiology, Stanford University, Stanford, CA, 1Cell Biology, Yale University, New Haven, CT, USA, 2Molecular USA, 2Department of Biochemistry and Molecular Biology, University of Biophysics and Biochemistry, Yale University, New Haven, CT, USA. Chicago, Chicago, IL, USA. Synaptotagmin-1 (Syt1) is the primary Ca2þ sensor that triggers the release of NSF (N-ethylmaleimide sensitive factor), a member of AAAþ ATPase fam- neurotransmitters stored within synaptic vesicles (SV). It is well-established ily, together with aSNAP (alpha soluble NSF attachment protein) disas- that Syt1 acts on both SNARE proteins, which catalyze the SV fusion, and semble SNARE (soluble NSF attachment protein receptor) complexes for the lipid membrane itself to achieve rapid and synchronous release. However, recycling of the SNARE proteins for subsequent rounds of fusion. Neuronal the precise molecular mechanism underlying this process is still unknown. SNAREs (synaptobrevin, syntaxin, SNAP-25) are the minimal fusion ma- Recent X-ray studies have described the Syt1-SNAREs interfaces in great chinery, but require synaptotagmin, complexin, Munc13, and Munc18 for detail, but were carried out in the absence of lipid bilayers. Thus, the precise fast Ca2þ-triggered neurotransmitter release. The molecular mechanism of molecular architecture of these proteins at the site of docked vesicle and during NSF regulated SNARE assembly and disassembly is largely unknown. We the Ca2þ activation process is still unclear. Here, we used cryo-electron micro- developed a single molecule fluorescence approach of monitoring the sponta- scopy (Cryo-EM) to determine the relative positioning of Syt1, SNAREs and neous assembly and the NSF-mediated disassembly process of a single linked lipid membranes. We utilized a covalently linked Syt1 C2AB-SNARE com- SNARE complex (termed L-SNARE complex) for multiple rounds without plex, combined with lipid nanotubes made with physiologically-relevant lipid applying any external force. Similar to previous chimeric SNARE design composition to obtain the samples suitable for Cryo-EM imaging under Ca2þ- used in high resolution optical tweezer setup, we linked SNARE complex free conditions. The reconstruction of the helically organized Syt1-SNARE in the sequence of syntaxin-1A, SNAP-25A, and synaptobrevin-2 with spacer complex reveal that proteins partially preserve the previous reported ‘primary’ sequences, and an Avi-tag sequence at the C-terminal end of synaptobrevin-2. interface between Syt1 C2B domain and the SNARE complex with interaction We conducted single molecule FRET measurements with labeling sites on of C-terminal ends of SNARE-pins stabilizing its orientation on the membrane syntaxin-1A residue 249 and synaptobrevin-2 residue 82, determined by surface. However, in this organization, we find that the aliphatic loops flanking the known atomic structure to produce high FRET efficiency when the the Ca2þ-binding domain of both C2A and C2B domains are partially inserted SNARE complex was properly assembled. By observing proteins at work into the membrane. We are currently investigating if this represents a novel in- in single molecule level, we discovered co-factors (in particular, complexin) termediate state during the Ca2þ-activation process and its physiological that prime the SNARE complex to inhibit disassembly by NSF until fusion relevancy. has occurred. Additionally we report a new structure of NSF/aSNAP/L- SNARE (20S) supercomplex using single-particle cryo-EM, revealing that 1421-Pos Board B330 only two aSNAP molecules are bound. Interestingly the two aSNAP inter- The Synaptotagmin Calcium-Binding Loops Modulate the Rate of Fusion face overlapped with the binding site of complexin on the SNARE complex, Pore Expansion supporting the new role of complexin shielding the trans SNARE complex Mounir Bendahmane1, Kevin P. Bohannon1, Tejeshwar C. Rao1, from NSF mediated disassembly until fusion has occurred. Furthermore Schmidtke W. Michael1, Prabhodh Abbineni1, Alexandra H. Ranski1, NSF was able to disassemble improperly assembled or misfolded SNARE Mazdak M. Bradberry2, Sherleen Tran3, Nara L. Chon3, Jefferson D. Knight3, complexes, thus acting as a protein quality control mechanism for efficient Hai Lin3, Edwin R. Chapman2, Arun Anantharam1. membrane fusion. 1Pharmacology, The University of Michigan, Ann Arbor, MI, USA, 2Neuroscience, Howard Hughes Medical Institute, University of Wisconsin, 1419-Pos Board B328 Madison, WI, USA, 3Chemistry, University of Colorado, Denver, CO, USA. Dilation of Fusion Pores by Synaptotagmin-1 C2AB Domains In adrenomedullary chromaffin cells, the kinetics of fusion pore expansion can Zhenyong Wu1,LuMa2, Yongli Zhang2, Erdem Karatekin1. vary depending on which synaptotagmin isoform (Syt-1 or Syt-7) drives 1Cellular and Molecular Physiology, Yale University, New Haven, CT, USA, release. Our recent studies have shown that fusion pores of granules harboring 2Cell Biology, Yale University, New Haven, CT, USA. Syt-1 expand more rapidly than those of granules harboring Syt-7. In this study, Synaptotagmin-1 is the calcium sensor for rapid, synchronous neurotrans- our goal was to define the structural specificity of synaptotagmin isoform action þ mitter release, but how calcium binding leads to fusion is not understood. at the fusion pore by manipulating properties of the Ca2 -binding C2B module. We tested the effects of Synaptotagmin’s cytoplasmic C2 domains (C2AB) We generated a chimeric Syt-1 protein in which one, two, or all three of its C2B þ on fusion pore opening and dilation using a recently established single- Ca2 -binding loops had been exchanged for those of Syt-7. The chimeric Syt-1 pore conductance assay which probes fusion between nanodiscs reconstituted proteins were expressed in chromaffin cells and were sorted to dense core gran- with neuronal v-SNAREs and engineered cells that express the complemen- ules. Fusion pores of granules harboring a Syt-1 C2B chimera with all three þ tary t-SNAREs in a flipped configuration on the cell surface. Fusion results in Ca2 -binding loops of Syt-7 (Syt-1:7 C2B123) exhibited significantly slower a nm-sized fusion pore that can be detected in cell-attached, voltage-clamped rates of fusion pore expansion and neuropeptide cargo release relative to WT recordings. Compared to fusion induced by SNAREs alone, in the presence of Syt-1. After fusion, this chimera also dispersed more slowly from fusion sites C2AB the fusion rate, the mean single-pore conductance, and the pore life- than WT protein. Based on prior work, we speculated that the Syt-1:7 C2B123 time increased, but only in the presence of calcium and PI(4,5)P2. The assay and WT Syt-1 are likely to differ in their interaction with Ca2þ and mem- recapitulated effects of previously characterized mutations on Synaptotag- branes. Stopped-flow fluorescence spectroscopy revealed the dissociation ki- min. In particular, abolishing binding of Synaptotagmin to the SNARE com- netics of the three loop C2B chimera from anionic phospholipids to be at plex or calcium via mutagenesis eliminated Synaptotagmin’ss effects on pore least two-fold slower than WT Syt-1. Similarly, molecular dynamics simula- dilation. tions suggested an overall higher affinity of the chimera for negative charges Binding of calcium to Synaptotagmin does not alter its interactions with the than WT Syt-1. We conclude that the affinity of synaptotagmin for the plasma þ SNARE complex, suggesting rapid calcium-dependent Synaptotagmin-bilayer membrane in the presence of Ca2 and, by extension, the rate at which the interactions may be key to triggering fusion. We probed interactions between plasma membrane is ‘‘released,’’ are important predictors of the rate of fusion single C2AB domains and lipid bilayers using high-resolution optical tweezers. pore expansion. It was recently shown that in the presence of calcium, Synaptotagmin-1 C2AB domains bound and unbound the membrane rapidly, releasing 8-13 kT per 1422-Pos Board B331 C2AB molecule, depending on the lipid composition. Here we show that dis- Structure-Based Enhancement of Gossypium Synaptotagmin to Modify rupting binding of C2AB to PI(4,5)P2 through mutagenesis reduced the binding Tolerance to Environmental Stress energy, while disrupting binding to calcium abolished C2AB-membrane Anthony A. Bui, Faraz M. Harsini, Roger Bryan Sutton. interactions. Texas Tech University Health Sciences Center, Lubbock, TX, USA. Together, our results indicate that Synaptotagmin-1’s effects on fusion pore Cotton is primarily a tropical plant; consequently, it is not well adapted to opening and dilation require it to interact both with membranes and SNAREs. freeze-thaw damage, nor is it adapted to thrive in soil with high salinity. It

BPJ 8625_8627 Monday, February 19, 2018 283a has been shown that the phospholipid binding activity of the Arabidopsis thali- ing to the fusion of 370-3800 vesicles (n=38). Presynaptic potentiation was also ana synaptotagmin protein was critical to repair cell membranes that have been seen at this synapse after 4 minutes of treatment of 1 mM cAMP (n=22). damaged either by exposure to freezing conditions or high soil salinity. Following a 50 ms depolarizing step, we saw a gradual decay in Cm, indicative Mammalian syt is a Caþ2-dependent/phospholipid binding protein that has of vesicular membrane retrieval (endocytosis). Tau ranged from 3.90 to 7.11 s been identified as the Caþ2 sensor for neuronal exocytosis. The domain struc- (n=10), suggesting that AIIs are suited for fast membrane recovery. We propose ture of all syt proteins shows similar overall organization containing C2A and that the variability in membrane dynamics of these interneurons is due to their C2B. Phospholipids interact with two hydrophobic residues and mutation of diverse cell-to-cell lobular morphologies. In order to test this hypothesis, we both residues to Trp in mammalian syt increases the rate of vesicle fusion in performed Cm recordings in conjunction with wide-field optical sectioning of mammalian neurons by changing how the protein interacts with phospholipid. dye filled AIIs. We were able to compare Cm jumps to 3D reconstructions of Our hypothesis is that by modifying the overall avidity for phospholipids in the lobular varicosities from individual AIIs. Our results show that the magni- Gossypium syt, we can alter how the cells respond to environmental stresses. tude of glycine release positively correlates to the number and overall size of Pairwise comparison of mammalian and cotton syt proteins shows that the these lobules (n=10). These results suggest that AIIs with larger lobules have C2A domain is similar to the mammalian syt C2A domain. We have success- more active zones and make more synaptic connections with OFF BCs and/ fully expressed and purified both cotton synaptotagmin C2A and C2B. In this or ganglion cells. study, we present new structural information about cotton synaptotagmin and test a series of mutations in Gossypium syt C2A and C2B with progressively 1425-Pos Board B334 larger hydrophobic amino acid surface areas. Next, we measure how the Single Molecule Fluorescence Studies Reveal Otoferlin as a Multivalent various mutations interact with artificial bilayers at different temperatures Scaffold Linking SNAREs and Calcium Channels for Exocytosis at Ribbon and salt concentrations. By enhancing the cotton’s natural membrane repair Synapses mechanisms, we hope to use this information to develop strains of cotton Colin P. Johnson1, Nicole Hams1, Weihong Qiu2. that have wider growth ranges, as well as reducing plant damage caused by 1Biophysics, Oregon State University, Corvallis, OR, USA, 2Physics, Oregon exposure to freezing temperatures. Further, more arid, higher salt soils could State University, Corvallis, OR, USA. be utilized for planting new cotton crops. Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and en- coding of sound preferentially over the neuronal calcium sensor synaptotagmin. 1423-Pos Board B332 Although it is established that synaptotagmin cannot rescue the otoferlin KO Optical Monitoring of Individual Release Sites Tests a New Mechanism for phenotype, the large size and low solubility of otoferlin have prohibited direct Synaptic Depression biochemical comparisons that could establish functional differences between Hua Wen, Paul Brehm. these two proteins. To address this challenge, we have developed a singlemo- Vollum Institute, Oregon Health and Sciences University, Portland, OR, lecule colocalization binding titration assay (smCoBRA) that can quantitatively USA. characterize full-length otoferlin from mammalian cell lysate. Using smCo- We recently used paired motoneuron muscle recordings in zebrafish to BRA, we found that, although both otoferlin and synaptotagmin bind mem- explore frequency-dependent synaptic depression. Most models for synaptic brane fusion SNARE proteins, only otoferlin interacts with the L-type depression focus on vesicle heterogeneity. However, our variance analysis of calcium channel Cav1.3, showing a significant difference between the synaptic steady state endplate current (EPC) amplitudes has turned up a new mecha- proteins. Furthermore, otoferlin was found capable of interacting with multiple nism that assigns depression to the dropout of a release site class with SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer com- ultra-slow vesicle reloading rates. This assignment was made on the basis plex. We also found that a deafness-causing missense mutation in otoferlin at- of 1) high release probability of all release sites at low frequency; 2) low vari- tenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in ance of EPC amplitude at steady state transmission, suggesting that only a this interaction may form the basis for otoferlin-related hearing loss. Based on subset of release sites can sustain fast release at high frequencies; 3) greater our results, we propose a model in which otoferlin acts as a calcium-sensitive than 60 fold difference in vesicle reloading rate of for the two classes of scaffolding protein, localizing SNARE proteins proximal to the calcium chan- release sites. As a direct test of these processes, we generated a fish line where nel so as to synchronize calcium influx with membrane fusion. Our findings the calcium indicator GCaMP6f was fused to postsynaptic rapsyn to optically also provide a molecular-level explanation for the observation that synaptotag- monitor the activity of postsynaptic ACh receptors. Both the density and cal- min and otoferlin are not functionally redundant. This study also validates a cium permeability of the receptors provide detection of single release events. generally applicable methodology for quantitatively characterizing large, Because the 15 synaptic boutons are physically separated along the length multivalent membrane proteins. of the muscle, each can be individually monitored for optical response to a motoneuron action potential. Data thus far indicates that stimulus frequencies 1426-Pos Board B335 that produce no depression (e.g., 0.2 Hz) show faithful GCaMP6f fluores- Comparison of Deterministic and Stochastic Approaches for Calcium cence responses to each stimulus. Stimulation at frequencies associated Dependent Exocytosis with synaptic depression has been more difficult to analyze due to the slow Victor Matveev. kinetics of GCaMP6f. However, preliminary recordings obtained at 10 Hz re- Mathematical Sciences, New Jersey Institute of Technology, Newark, NJ, vealed all synapses responding to the first stimulation and with select synap- USA. ses consistently dropping out during the stimulus train. These results are A distinguishing feature of Ca2þ-triggered synaptic and endocrine vesicle consistent with two classes of active sites bearing distinct release kinetics un- exocytosis is its high degree of variability. Stochastic Ca2þ channel gating derlying frequency-dependent depression. Firm conclusions require further is the primary source of this stochasticity. Another reason for high variability testing at high frequencies. is that only a small number of Ca2þ ions enter the cell through a single chan- nel during an action potential, as well as the stochasticity in the Ca2þ binding 1424-Pos Board B333 to Ca2þ buffers and sensors. This leads to a widely-held preference for sto- Exo- and Endocytosis at a Retinal Inhibitory Synapse during Crossover chastic Monte-Carlo approaches for accurate modeling of Ca2þ dependent Inhibition cell processes. However, several recent comparative studies show a surpris- Marc A. Meadows1, Margaret Veruki2, Espen Hartveit2, ing close agreement between deterministic and trial-averaged stochastic sim- þ þ Henrique von Gersdorff1. ulations of Ca2 diffusion, buffering and binding, as long as Ca2 channel 1Vollum Institute, OHSU, Portland, OR, USA, 2University of Bergen, gating is not strongly Ca2þ dependent. We extend these prior studies, Bergen, Norway. focusing on Ca2þ dynamics downstream of Ca2þ channel gating. We show The AII amacrine cell (AII) receives chemical and electrical synapses from rod that under physiological constraints on the rate of Ca2þ binding, buffering bipolar cells (BC) and ON cone BCs. AIIs form glycinergic synapses at lobular and diffusion, discrepancy between deterministic and stochastic approaches varicosities near the soma, providing inhibitory output to the OFF pathway; this can be surprisingly small even when only 30 ions enter per single channel- is called crossover inhibition. We performed whole-cell voltage clamp record- vesicle complex. One reason for this is that it is correlations rather than vari- ings and used the ‘‘sine þ DC’’ method to make measurements of membrane ance in reactant molecule numbers that reduce the accuracy of deterministic 2þ capacitance (Cm) with high temporal resolution. With this approach we were description in the presence of bimolecular Ca -sensor interactions. Further, able to investigate the cell membrane dynamics of this inhibitory synapse. contrary to naı¨ve intuition, reaction-diffusion description provides informa- AIIs were held at 80 mV and L-type Ca2þ currents were evoked with a tion about the full Ca2þ binding latency probability distribution, since the 100 ms, 70 mV depolarizing step, triggering Ca2þ dependent vesicle fusion standard deterministic chemical reaction system can be interpreted as a sys- (exocytosis). Our observed Cm jumps ranged from 14.81-152.3 fF, correspond- tem of master equations for the underlying stochastic process under an

BPJ 8625_8627 284a Monday, February 19, 2018 assumption of negligible correlations. This explains the close match between stimulation sheds new light on the nature of the morphological changes dur- stochastic and reaction-diffusion simulations of the Ca2þ-sensor biding la- ing WPB exocytosis. tency time distribution, despite the high variability of the underlying processes. The reason for the small magnitude of molecule number correla- 1429-Pos Board B338 tions will be also discussed. Supported by NSF DMS-1517085. Dynamin-1 Restrains Vesicular Catecholamine Release to a Sub-quantal Mode in Mammalian Adrenal Chromaffin Cells 1427-Pos Board B336 Qihui Wu, Quanfeng Zhang, Yinglin Li, Bin Liu, Xi Wu, Changhe Wang, The Interplay of Dopamine Receptors in the Pancreatic Islet Regulates Feipeng Zhu, Zhuan Zhou. Hormone Secretion Institue of Molecular Medicine, Peking University, Beijing, China. Alessandro Ustione, David W. Piston. Dynamin 1 (dyn1) is required for clathrin-mediated endocytosis in most secre- Cell Biology and Physiology, Washington University in St. Louis, Saint tory cells, including neurons and neuroendocrine cells. In 1996, we first re- Louis, MO, USA. ported two modes of Ca2þ-dependent catecholamine (CA) release from The regulated release of hormones from the islets of Langerhans is funda- single large dense-core vesicles, quantal by ‘‘full-fusion-like’’ (FFL) and mental to achieve glucose homeostasis. The balance between insulin and sub-quantal by ‘‘kiss-and-run’’ (KAR) in adrenal chromaffin cells (ACCs), glucagon output signals to multiple tissues in the body to uptake or to release which have been confirmed by others. Quantal size (QS, CA release per exocy- glucose. Any acute impairment of this function has dangerous and potentially totic event) is increasedby acute pharmacological manipulation of dyn1, lethal consequences, such as hyperglycemia, which can lead to diabetic ke- implying that some of the previously-assumed full-quanta are probably sub- toacidosis and coma, or severe hypoglycemia that often leads to seizures, quanta. Using genetic dyn1-knockout (KO) mouse ACCs, here we showed loss of consciousness or death. A chronic imbalance in the insulin/glucagon that 1) KO increased QS (both average and largest QS) by R 200% without output is the defining trait of diabetes. This medical condition is characterized increasing the vesicle size; 2) the KO-induced increasement in QS was rescued by chronic hyperglycemia which in turn leads to damage to the microvascu- by overexpressing WT-dyn1 (but not its deficient mutant); 3) the ratio of FFL lature in the body. Given the high prevalence of diabetes, the cost of care for versus KAR events was substantially increased by dyn1-KO; and 4) the fusion diabetic patients is skyrocketing around the globe, and better treatments are pore size dp was increased from % 9nmtoR 9 nm by dyn1-KO. Thus, most (if constantly needed.One potential treatment target is the neurotransmitter not all) Ca2þ-induced exocytotic events occur in the sub-quantal/KAR release dopamine, which has been shown to exhibit autocrine and paracrine signal mode in native ACCs, implying that the dyn1-dependent sub-quantal mode between islet cells that can fine tune hormone output via a tonic inhibition plays an essential role in neurotransmission and hormone signaling (inset on the secretory activity. The dopamine receptors D3 and D2 are present graph). and active in islet cells In particular, and we are characterizing the complex interplay between the dopamine receptors that are active in the various islet 1430-Pos Board B339 cell types. We hypothesize that their signaling cascades are acting on distinct Secretory Granule Lumenal Proteins Have Highly Limited Mobility 1 1 1 pathways to differentially regulate insulin and glucagon. To test our hypoth- Prabhodh S. Abbineni , Kevin P. Bohannon , Mary A. Bittner , 2 1 esis, we use in situ hybridization to test cell specific receptor expression in the Daniel Axelrod , Ronald W. Holz . 1Department of Pharmacology, University of Michigan, Ann Arbor, MI, islets. We use adeno viruses to deliver genetically encoded fluorescent re- 2 porters to mouse and human islets, and confocal microscopy to measure USA, Departments of Pharmacology, Physics and LSA Biophysics, the different responses triggered by selective activation of D2 or D3 recep- University of Michigan, Ann Arbor, MI, USA. tors. These experiments allow us to define a model for dopaminergic regula- The lumen of secretory granules is a poorly understood, highly unusual envi- tion of hormone secretion. ronment. Chromaffin granules store 500 mM catecholamines, 125 mM ATP, and 250 mg/ml of proteins, likely leading to a high degree of molecular 1428-Pos Board B337 crowding. Although many of the proteins of secretory granules have been iden- Ultrastructural and Functional Analysis of Weibel-Palade Bodies tified, little is known about their organization and macromolecular structure. James Streetley1, Ana-Violeta Fonseca2, Jack Turner3, Nikolai I. Kiskin3, Granules’ contents are discharged following fusion at highly variable rates Tom Carter4, Peter Rosenthal3. (milliseconds to seconds); the mobility of soluble granule proteins, in part, 1MRC Center for Virus research, Glasgow University, Glasgow, United could determine the variable discharge rate. To address these issues, we are Kingdom, 2MRC NIMR, London, United Kingdom, 3The Francis Crick measuring the mobility of specific (fluorophore-tagged) soluble proteins. We Institute, London, United Kingdom, 4Molecular & Clinical Sciences are using a method that is not limited by standard optical resolution. A bright Research Institute, St George’s University London, London, United flash of strongly decaying evanescent field (100 nm exponential decay con- Kingdom. stant) produced by total internal reflection (TIR) selectively bleaches mCherry The adhesive glycoprotein von Willebrand factor (VWF) is stored for secre- or GFP-labeled protein proximal to the glass coverslip within individual gran- tion in specialized endothelial cell secretory organelles called Weibel-Palade ules, imprinting a gradient of fluorescence in the granule. Fluorescence recov- bodies (WPBs). VWF plays a crucial role in hemostasis and defects in the ery occurs as unbleached protein from distal regions within the 300 nm granule protein result in the bleeding disorder Von Willebrands disease. Previously diffuses into the bleached proximal regions. We consider the influence of noise, we used Fourier analysis of cryo-EM images and tomographic reconstruc- light scattering, reversible bleaching, and the fluorophore on the measurements. tions of vitrified human umbilical vein endothelial cells (HUVEC) to show The studies reveal, with 100 ms resolution, highly limited mobility of the that VWF is stored as a series of helical tubules that tight pack to a paracrys- lumenal proteins neuropeptide Y (NPY), tissue plasminogen activator (tPA), talline matrix in the WPB lumen (Berriman et al 2009;106:17407-17412). brain derived neurotrophic factor (BDNF), and chromogranin A (CgA). WPB fusion with the plasma membrane is associated with disassembly of Furthermore, we targeted mCherry alone to the granule lumen using the the paracrystalline array of tubules and appearance of a membranous exten- NPY signal sequence, and found similar limited mobility. These results suggest sion of the WPB that we speculated might represent a connection to the that the environment in the granule lumen, not specific protein chemistry, plasma membrane. Here we extend our ultrastructural studies of WPBs in greatly restricts protein mobility. Furthermore, the very rapid post-fusion situ in vitrified HUVEC to reveal a novel structural feature of the organelle. discharge of NPY (often within a second) and the very slow discharge of Using light microscopy of live and fixed cells we identify a marker of this tPA (over tens of seconds) is not explained by the mobility of the protein in novel structural feature. Direct visualization of these features during cell the granule.

BPJ 8625_8627 Monday, February 19, 2018 285a

Posters: Calcium Signaling I store depletion is driven by direct interactions between the Orai1 subunits with the endoplasmic reticulum Ca2þ sensor, STIM1, but how STIM1 binding transduces 1431-Pos Board B340 into opening of the Orai1 channel pore remains unclear. At the structural level, Store-Operated Calcium Entry in Adult Wild Type Ventricle Cardio- each Orai1 channel consists of six subunits with the transmembrane helices myocytes (TMs 1-4) of each subunit arranged in three concentric rings around a central Dmitry Grekhnyov, Konstantin Gusev, Vladimir Vigont, aqueous pore formed by TM1. A prevailing model of channel gating postulates Elena Kaznacheyeva. that STIM1 binding to the peripherally located Orai1 C-terminus is followed by Institute of Cytology RAS, Saint-Petersburg, Russian Federation. a second, weaker STIM1 binding step to the centrally located N-terminus to drive Calcium is a one of the most important second messenger in cell, controlling a pore opening. However, recent reports have cast doubt on the existence of a N-ter- wide range of intracellular processes such as activation of transcriptional fac- minal STIM1 binding site, raising questions about how the gating signal is trans- tors, metabolism, muscle contraction, apoptosis, proliferation etc. It has been mitted to the central pore. Here, we investigated the role of the transmembrane reported that alterations in calcium homeostasis are associated with number domains in this process and identified sixteen mutations in the non-pore lining of pathologies including neurodegenerative disorders, cardiac pathologies, TMs 2-4 that activate Orai1 channels in the absence of STIM1. Cysteine accessi- muscular dystrophies and allergies. Store-operated calcium entry (SOCE) has bility analysis and MD simulations indicate that the most robust of these activating been established as one of the most ubiquitous pathway of calcium entry in mutations, H134S, induces rotation of the pore helix to move the side-chains of the cells. The presence of SOCE has been shown both in excitable and non- gate formed by F99 aside, analogous to gating by STIM1. Further, atomic packing excitable cells. It was discussed that SOCE may play a significant role in early and mutational analysis revealed critical contact sites betweenTM1 and the TM2/3 development of postnatal cardiomyocytes. However, existence of functional helices, whose disruption by mutagenesis abrogated channel activation by STIM1 SOCE in adult wild type cardiomyocytes remains controversial. Using a binding. We conclude that specific interactions between the transmembrane do- patch-clamp technique in whole-cell mode we recorded caffeine- and mains of Orai1 are critical for relaying the STIM1 activating signal from the C-ter- thapsigargin-induced calcium currents in primary culture of wild type ventricle minus to the pore. cardiomyocytes isolated from adult mice. These currents could be partially 1434-Pos Board B343 blocked by classical SOCE inhibitor 2-APB at concentration of 50 mkM. STIM Proteins Cluster Orai1 Channels and Modulate Receptor-Mediated Current-voltage relationships of 2-APB-sensitive fraction of the currents had Calcium Signals þ þ a strong inward rectification that is typical for Ca2 release-activated Ca2 Robert M. Nwokonko, Yandong Zhou, Xiangyu Cai, Natalia Loktionova, (CRAC) channels. The electrophysiological results were confirmed by fluores- Mohamed Trebak, Donald L. Gill. cent measurements with calcium dye Fura-2AM. Thus we for the first time ob- Cellular and Molecular Physiology, Penn State University, Hershey, PA, tained electrophysiological recordings of 2-APB-sensitive store-operated USA. currents in adult wild type cardiomyocytes. These results can be used in the Ca2þ entry through Ca2þ release-activated Ca2þ (CRAC) channels plays an future studies for comparison of normal SOCE and SOCE in pathologies to indispensable role in receptor-evoked Ca2þ signaling. The two main CRAC establish SOCE as a possible novel target for medical treatment. The study channel facilitators are the PM Orai channels, and the ER Ca2þ sensors, was supported by RSF grant No 14-04-00720-P. STIM1 and STIM2. The molecular mechanism of STIM1/Orai1 coupling re- mains unsolved. Our recent work provided insight into the coupling mechanism 1432-Pos Board B341 2D between the Orai1 activating domain of STIM1 (SOAR) and the Orai1 C-termi- STIM1 Function Is Controlled by Multiple Ca Binding Sites in its nus. Using rationally-designed mutational screens of the purported Orai1 interac- Luminal Domain tion site within SOAR, we determined that mutating residue F394 to histidine 1 1,2 1 Aparna Gudlur , Ana Eliza Zeraik , Nupura Hirve , completely prevents binding of full-length STIM1. We developed concatenated 1 3 4 5 Rajanikanth Vangipurapu , Andrey Bobkov , Guolin Ma , Sisi Zheng , SOAR dimers containing one or two F394H mutations, and demonstrated that Youjun Wang5, Yubin Zhou4, Elizabeth Komives6, Patrick G. Hogan1. 1 2 only a single functional subunit within a SOAR dimer is necessary for Orai1 La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA, Instituto binding. Current studies reveal that concatenated SOAR heterodimers (S -S) de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, SP, Brazil, H 3 are unable to form Orai1 clusters, whereas cells expressing wild-type dimers Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA, (S-S) retain this ability. The recent discovery of the splice variant of STIM2 4Center for Translational Cancer Research, Texas A & M University, 5 (STIM2.1), which has an abrogated binding capacity similar to STIM1-F394H, Houston, TX, USA, College of Life Sciences, Beijing Key Laboratory of provided us with another tool to investigate STIM/Orai1 coupling and its phys- Gene Resource and Molecular Development, Beijing Normal University, iological role in receptor-evoked Ca2þ signals. Using concatenated heterodimers Beijing, China, 6Department of Chemistry and Biochemistry, University of (SH-S or S2.1-S), we observed profound effects on dimer mobility and generation California-San Diego, La Jolla, CA, USA. of I as compared to wild-type SOAR1 homodimers (S-S), in cells stably ex- STIM1 is a Ca2þ-sensing ER protein that undergoes a resting-to-active state CRAC 2þ pressing Orai1. The physiological role of clustering Orai1 channels is suggested transition following depletion of ER Ca stores. Activated STIM1 gates the from co-expressing full length STIM1 and STIM2.1. We observe profoundly plasma membrane Ca2þ channel, ORAI1. The Ca2þ sensing EFSAM domain 2þ altered oscillatory responses in HEK cells induced by physiological levels of of STIM1, studied as an isolated fragment, shows one Ca ion bound to the CCh. Our studies suggest that clustering may affect the ability for Orai1 channels EF-hand motif. Since the native EFSAM domain is linked via a transmembrane 2þ 2þ to generate fully functional Ca entry microdomains within ER-PM junctions. helix to the dimeric cytoplasmic domain of STIM1, we studied Ca binding This provides strong evidence supporting the ‘‘unimolecular model’’ of and associated functional responses of EFSAM domain linked to a similar sol- 2þ STIM1-Orai1 coupling, which appears to be critical for channel clustering, ICRAC uble dimer tag. This form of EFSAM is a soluble dimer in both Ca -bound and generation, and maintenance of Ca2þ oscillations. Ca2þ-free states, and responds to Ca2þ concentration changes with a conforma- tional switch that is comparable to the response of full-length STIM1 following 1435-Pos Board B344 store depletion. Transition to the active state involves a limited rearrangement EVP4593 Compound Decreases Abnormal Store-Operated Calcium Entry of the EFSAM structural elements, indicating that both resting and active states In Ipscs-Based Model of Huntington’s Disease are well-folded. Several lines of evidence show that resting EFSAM can bind Vladimir Vigont, Konstantin Gusev, Elena Kaznacheyeva. multiple Ca2þ ions at sites other than the EF-hand. We demonstrate that the Institute of Cytology RAS, Saint-Petersburg, Russian Federation. additional sites are functional in the physiologically relevant range and essen- Compound EVP4593 was initially described by Tobe and colleagues in 2003 as an tial for a normal STIM1 response to store depletion. inhibitor of activation of NF-kB signaling pathway. However there were no reports about direct interactions of EVP4593 with key proteins of this pathway. At the 1433-Pos Board B342 same time it is well known that for activation of NFAT and NF-kB signaling path- Helix-Helix Contacts between the ORAI1 Pore Segment and the TM2/3 ways store-operated calcium entry (SOCE) is required. SOCE is an important and Ring Regulates STIM1-Mediated CRAC Channel Activation ubiquitous part of the calcium signaling controlling a number of intracellular pro- Priscilla S.-W. Yeung1, Megumi Yamashita1, Christopher E. Ing2, cesses. Alterations in SOCE were observed in different pathologies including Regis Pome`s2, Douglas M. Freymann3, Murali Prakriya1. neurodegenerative disorders such as Huntington’s disease (HD), Alzheimer’s 1Department of Pharmacology, Northwestern University, Chicago, IL, USA, disease etc. We supposed that EVP4593 could act upstream preventing initial steps 2Molecular Structure and Function, Hospital for Sick Children, University of of NF-kB activation by blocking calcium entry through store-operated channels. Toronto, Toronto, ON, Canada, 3Department of Biochemistry and Molecular Our previous studies indicated that SOCE is abnormally increased in different Genetics, Northwestern University, Chicago, IL, USA. cellular models of HD and may underlie the HD pathogenesis. EVP4593 was Store-operated Orai1 channels mediate many critical cellular functions in animal able to decrease SOCE and also had a neuroprotective effect in fly and mice HD cells ranging from gene expression to exocytosis. Activation of Orai1 channels by models. Here we investigated new HD model based on genetic reprogramming

BPJ 8628_8631 286a Monday, February 19, 2018 of patient-specific fibroblasts into induced pluripotent stem cells (iPSCs) and direct drug resistance, the search for a viable vaccine is dependent on a better understand- differentiation of iPSCs into striatal neurons. We confirmed a pathological SOCE ing of the molecular and physiological processes through which the parasite prolif- increasing in HD neurons compared to wild type neurons. Then we showed that erates inside the blood stream. One of the most crucial, yet least understood, stages in HD neurons are more vulnerable to treatment by proteasome inhibitor MG-132. the erythrocytic reproduction cycle is the brief invasion period, during which para- EVP4593 not only efficiently decreased SOCE in HD neurons at high nanomolar sites are exposed to the adaptive immune system and represent a potential drug concentration but also protected HD neurons from MG-132-induced death. Thus target. Parasites egress from a mature infected cell and, after contacting a nearby we can conclude that EVP4593 compound represent a promising medical sub- red cell, form a tight junction that leads to parasite penetration. stance for anti-HD drug design. The study was supported by RSF grant No 17- Red blood cell deformations associated with this process were suggested to be 74-20068 and the fellowship of the President of RF. active plasma membrane responses mediated by transients of elevated intracel- lular calcium. Few studies though have imaged these events at single cell res- 1436-Pos Board B345 2D D olution, and a number of biophysical questions remain unanswered. Interplay of CRAC Channels with Ca Activated K Channels In our work, the fluorescent calcium indicator Fluo-4 was employed to inves- Adela Krizova, Romana Schober, Sonja Lindinger, Carmen Butorac, tigate the distribution of the dye in red blood cells used as parasite targets in Christoph Romanin, Isabella Derler. egress-invasion assays. The invasion dynamics was observed under bright- JKU, Linz, Austria. field and fluorescence microscopy simultaneously. Calcium signals were found Cell survival and normal cell function requires tight control of basal cytosolic Ca2þ 2þ absent throughout this interval in over half the records, and totally absent dur- concentration. Fine-tuned regulation of cytosolic Ca concentrations is essential ing the pre-invasion period regardless of deformation strength. These results for various signaling pathways, controlling among others gene transcription, pro- argue against a role of elevated intracellular calcium during the invasion stages. liferation and cell migration. My studies focus especially on the understanding of We suggest an alternative mechanism of parasite-induced deformations based the structural requirements triggering the co-regulation of the molecular key 2þ on passive red cell responses to transient agonist-receptor interactions associ- players of the CRAC channel STIM1 and Orai1 with small conductance Ca acti- ated with the formation of adhesive coat filaments. vated Kþ channels (SK channels). Several publications have already provided ev- idence, that the interplay of Orai1 with SK3 is essential in the control of cancer cell 1439-Pos Board B348 growth, as their downregulation reduces cancer cell proliferation and migration. Expression Level of STIM Proteins Alter Electrophysiological Properties SK3 channels are activated by intracellular Ca2þ in the range of 0.1-0.25 mM. I of Endogenous Calcium Channels discovered in electrophysiological studies in the absence of intracellular Ca2þ Alexey V. Shalygin, Dmitrii Kolesnikov, Anton Skopin, that a co-expression of Orai1 and SK3 in HEK cells initiates the activation of Anastasia Perevoznikova, Lyubov Glushankova, Elena V. Kaznacheyeva. SK3-like currents, in contrast to SK3 alone which remains inactive under these Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, conditions. A combined approach of Ca2þ imaging, electrophysiological and fluo- Russian Federation. rescence microscopy will uncover the so far unresolved molecular key determi- STIM proteins are the main regulators of store-operated channels. Enhance- nants regulating the interplay and co-localization of the channels, potentially ment of calcium selectivity is observed in overexpressed ORAI1 channels providing more specific target sites for cancer treatment. (Supported by Austrian when relative expression of STIM1 is increased (McNally et al, 2012). Here Science Fund FWF P25210 and P27641 to I.D. and P27263 to C.R.). we describe how STIM proteins change properties of endogenous channels. Imin channels have single-channel conductance of 1.2 pS and high selectivity 1437-Pos Board B346 to divalent cations (P Ba/K =20). They are activated upon calcium store deple- Characterization of the Calcium Release-Activated Calcium (CRAC) tion with Tg, BAPTA-AM, TPEN and inhibited by blocker SKF95365 or low Channel From the Human Pathogen Schistosoma mansoni concentrations of Gd 3þ in A431 cells. In our experiments in HEK293 cells Ana Eliza Zeraik1,2, Aparna Gudlur1, Ricardo DeMarco2, Imin channels were induced by selective STIM2 activation with 10 nM Tg, Ana Paula U. Araujo2, Patrick Hogan1. but not 1 mM Tg. In HEK S4 cells with stable knockdown of STIM1 Imin chan- 1La Jolla Institute for Allergy and Immunology, San Diego, CA, USA, nels were activated by 1 mM Tg. In A431 cell line Imin channels were activated 2Instituto de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, SP, by either 10 nM Tg or 1 mM Tg. Thus, different expression levels of STIM1 Brazil. protein in HEK 293, HEK S4 and A431 cells underline the changes in channel Schistosoma mansoni is the main etiologic agent of schistosomiasis in America, a properties. Another endogenous store-operated channels are Imax TRPC1- disease that is widespread in Africa, Asia and south America, affecting over 240 composed channels with moderate selectivity and single channel conductance million people. The success in the eradication of this parasite is limited. To date, 17 pS. In HEK293 STIM1 and STIM2 proteins did not change selectivity of the there is only one commercially available drug (praziquantel), and thus the search endogenous TRPC1 channels as opposed to ORAI channels. In our whole-cell for new targets is desirable. Calcium channel proteins are potential targets to new experiments with HEK 293 cells we observed that STIM2 knockdown changed interventions, therefore this work is focused on S. mansoni STIM (SmSTIM) and current-voltage relationship shape resulting in CRAC-like shape . We suggest Orai (SmOrai) proteins, which form the calcium release-activated calcium chan- that different STIM1 and STIM2 expression in various cell lines and tissues re- nel. SmSTIM and SmOrai were cloned into mammalian vectors and expressed in sults in diversity of cell calcium responses to a physiological stimulus. This HeLa and HEK293 cell lines, in order to assess their functionality as a calcium work was supported by RSF 14-14-00720 (to D.K., E.K.); RFBR 16-04- channel. Single-cell imaging using the calcium indicator Fura2 demonstrates 01792 (to L.G., A.Sk., A.P.); Fellowship of the President of Russia (A.Sh.) that SmOrai can conduct calcium, and requires STIM for activation. The schis- tosome and human channels appear to employ broadly similar mechanisms. 1440-Pos Board B349 Thus, human STIM1 can gate SmOrai, and a chimera of SmSTIM ER-luminal Tuning the Lateral Range of L-Type Calcium Channel-Dependent Cal- domain and human STIM1 cytoplasmic domain triggers store-dependent calcium cium Signals in Dendrites of Hippocampal Neurons influx through the human ORAI channel. However, there are also some promi- William A. Sather, Mark L. Dell’Acqua, Philip J. Dittmer. nent differences. SmSTIM localizes both throughout the ER and near the plasma Pharmacology, University of Colorado School of Medicine, Aurora, CO, USA. membrane in resting cells, and constitutively activates SmOrai independent of 2þ calcium store depletion, suggesting a distinct regulatory mechanism between In cultured hippocampal neurons, voltage-gated L-type Ca channel (LTCC) activity is subject to potentiation by protein kinase A (PKA) and to feedback mammalian and schistosome cells. Further, SmSTIM is unable to gate human 2þ ORAI, even after store depletion. The differences observed so far encourage inhibition by STIM1, a sensor of endoplasmic reticulum (ER) [Ca ]. Feedback by STIM1 is engaged following NMDA receptor-dependent LTCC activation: further studies with these proteins aiming to understand the mechanistic differ- 2þ 2þ ences between human and schistosome CRAC channels as well as the feasibility LTCCs allow influx of Ca that induces Ca release (CICR) from ER stores, of using these proteins as potential drug targets. and CICR-driven stores depletion activates STIM1, which is able to span the gap between cortical ER and plasma membrane to contact and inhibit LTCCs. 1438-Pos Board B347 PKA potentiation of LTCC activity relies upon anchoring of the kinase to No Role for Elevated Intracellular Calcium during Malaria Invasion LTCCs by the scaffolding protein AKAP150; AKAP150 co-anchoring of the Viola Introini, Alex J. Crick, Teresa Tiffert, Jurij Kotar, Yen-Chun Lin, phosphatase calcineurin (CaN) provides a counterbalance on LTCC activity. Pietro Cicuta, Virgilio L. Lew. Uncaging of MNI-glutamate adjacent to a dendritic spine triggers a rise in 2þ 2þ Physics, University of Cambridge, Cambridge, United Kingdom. [Ca ]cytosol and a drop in [Ca ]ER. A substantial component of each of these With one-third of the world’s population at risk of infection and half a million changes in [Ca2þ] is dependent upon LTCC activity, as measured by sensitivity deaths every year, malaria is a devastating health problem and socio- to the LTCC antagonist nimodipine. For a stimulated spine, the nimodipine- 2þ 2þ economical burden in many countries. sensitive component of both changes in [Ca ]cytosol and [Ca ]ER extends in Severe malaria is primarily caused by Plasmodium falciparum parasites during their exponential fashion along the adjoining dendrite, with length constants of asexual reproduction cycle within red blood cells. With continual rise of parasite 5 mm. AKAP150 knockdown and replacement with AKAP150 lacking its

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PKA binding domain reduces the spread of both the cytosolic and ER Ca2þ is not understood but it is accepted that it is not of thermal origin. Different bio- changes, whereas knockdown and replacement with an AKAP150 mutant lack- logical effects have been observed in vivo that could explain pain relief, however, ing the CaN site increases the lateral range of Ca2þ signaling. The effects on the biophysical mechanisms by which PRF causes those effects are unknown. 2þ 2þ [Ca ]cytosol and [Ca ]ER of delocalizing PKA or CaN are attributable, respec- Interestingly, most of those biological effects correspond to effects that could tively, to depotentiation of LTCC Ca2þ influx and hence decreased CICR or to be triggered by calcium influx into the neurons. We hypothesize that PRF causes unopposed potentiation and increased CICR. These results suggest that the mild electroporation of the cell membrane leading to extracellular calcium up- spatial extent of LTCC-dependent Ca2þ signals in dendrites may be tuned in take. In the present study, two cell lines (HEK-293 and SH-SY5Y) were exposed accord with the local balance of PKA and CaN activity. to fields with the same time patterns as in clinical PRF treatments and free cyto- solic calcium concentration was monitored by fluorescence microscopy. An in- 1441-Pos Board B350 crease in free cytosolic calcium was observed when cells were exposed to Store-Operated Ion Channels are Activated after Chronic ER Stress in electric fields with amplitudes of 20 kV/m and above. After adding Gadolinium Beta Cells (a non-specific calcium channel blocker) to the buffer, the same results were ob- Benjamin M. Thompson, Suryakiran Vadrevu, Leslie Satin. tained. In the absence of extracellular calcium, the calcium concentration in- Pharmacology, University of Michigan, Ann Arbor, MI, USA. 2þ crease after the treatment was reduced by an 80% in HEK cells and by a 70% ER stress has been linked to the depletion of ER Ca , which in turn induces a in SH cells. The results show that PRF treatments cause an influx of calcium number of compensatory responses; unmitigated ER stress leads to beta cell 2þ into the cells. This influx is independent of the membrane calcium channels death. To test the hypothesis that increased Ca influx into the cell through Store and it is presumably related to the electroporation phenomenon. A numerical Operated Channels (SOC) following ER stress, isolated mouse islets were treated study indicates that the transmembrane voltages reached values above 100 mV with tunicamycin (TM) for 16-18 hours to induce stress through inhibition of pro- 2þ 2þ during PRF delivery. Our results support the hypothesis that the mechanism of tein glycosylation. Cytosolic Ca was measured using fura-2 and ER Ca was action of PRF is related to a calcium signaling process after extracellular calcium > measured using the FRET probe D4ER. In control islets, 7 mM glucose induces influx caused by electroporation. robust Ca2þ oscillations having periods ranging from 3-5 minutes. TM-treated is- 2þ 2þ 2þ lets had elevated baseline cytosolic Ca , decreased ER Ca and Ca oscilla- 1444-Pos Board B353 tions that were active even in 5 mM glucose, a concentration that is usually GCAMP Calcium Imaging Reveals Kinetics and Location of MET Chan- subthreshold for islet electrical activity. In response to cyclopiazonic acid nels in Mammalian Semicircular Canal Hair Cells (CPA), a reversible SERCA inhibitor, control and TM-treated islets had acutely 2þ 2þ Holly A. Holman, Micah D. Frerck, Richard D. Rabbitt. elevated cytosolic Ca and a parallel drop in ER Ca . However, the cytosolic Bioengineering, University of Utah, Salt Lake City, UT, USA. 2þ < Ca oscillations were larger and more frequent in TM-treated islets (p 0.05). Sensation of angular head movements by the semicircular canals relies upon me- To test whether SOC were activated by ER Ca2þ depletion, we tested the selec- 2þ chanical gating of cation channels located in specialized microvilli at the apical tive SOC inhibitor YM58483 (10 uM). YM58483 blocked the Ca oscillations end of sensory hair cells. We examined the location, kinetics, and sequential and prevented ER refilling in TM-treated islets but had no effect on controls. We recruitment of mechanoelectrical transducer (MET) channels in stereocilia of propose that the potentiation of cytosolic Ca2þ oscillations we observed 2þ the crista ampullaris using a transgenic mouse expressing the calcium indicator following ER stress results from the activation of SOC secondary to ER Ca GCaMP5G. Expression of GCaMP was achieved through a Cre driver line target- depletion. If a similar mechanism is activated by a high fat diet or exogenous fatty ing GABAergic cells, which revealed a preferential expression in the cell body acids it may provide a novel pathway for pharmacologic intervention. and stereocilia of a subpopulation of hair cells located in peripheral zones of 1442-Pos Board B351 the crista. We examined calcium modulation in stereocilia by imaging changes New Fluorescent Tools to Identify Stressed Cells and Interrogate Second in GCaMP fluorescence in response to opening MET channels with controlled Messenger Signaling in Neurodegeneration mechanical deflection of the stereocilia bundle. Stereociliary bundles in the semi- Thomas Hughes, Kevin Harlen. circular canal are considerably longer relative to those in the auditory system and m Montana Molecular, Bozeman, MT, USA. canextend40to60 m in length. Swept field confocal microscopy was used to Neurodegenerative diseases are slow processes that only involve a few cells at a image calcium modulation in stereocilia using a semi-intact excised preparation. time. Imagine being able to find these cells, while they are still alive and under- GCaMP fluorescent puncta near the tips of the bundle increased intensity during going stress. One could ask: are the cells still responsive to neurotransmitters and excitatory bundle deflection when MET channels opened and decreased intensity drugs? Are their second messenger signaling systems still functional? There are when MET channels closed, reflecting modulation of calcium in the stereocilia. good reasons to ask these questions because abnormal Ca2þ signaling and cAMP Additional GCaMP puncta were recruited lower in the bundle as the stimulus levels appear to be grossly affected in Alzheimer’s, Parkinson’s, and Hunting- strength was increased. Adaptation or desensitization was not observed over ton’s disease. To address these questions we developed a two-color, multiplex the time course of bundle deflections studied. GCaMP modulation was restricted sensor system that produces one color in only the stressed cells while a different to a small domain in the vicinity of each channel, demonstrating that free calcium colored sensor detects cell signaling in both stressed and healthy cells. is rapidly buffered and transported out of each stereocilium. Results demonstrate To detect cellular stress brought on by endoplasmic reticulum stress we de- specializations in GABAergic hair cells in crista relative to other hair cell types, signed synthetic transcripts based on the non-canonical splicing of the XBP1 putatively driven by the need to sense tonic or slowly modulating mechanical sig- intron. Stressed cells splice the XBP1 intron, introduce a frameshift in the cod- nals over a very broad dynamic range. ing region, and produce a fluorescent protein. These constructs generate strong fluorescence in living HEK 293 cells when they are stressed with tunicamycin 1445-Pos Board B354 and thapsigargin. These sensors are also sensitive enough to detect the differ- Fast Decay Variants of Red Fluorescent Genetically-Encoded Calcium ence between overexpression of normal rhodopsin or the P23H mutant that Indicators causes retinitis pigmentosa. We then multiplexed this stress sensor with addi- Silke Kerruth, Catherine Coates, Katalin To¨ro¨k. tional, differently colored, sensor for Ca2þ or cAMP. In this case the expres- Molecular and Clinical Sciences Research Institute, St. George’s University þ of London, London, United Kingdom. sion of the P23H mutant rhodopsin raised the resting level of cytosolic Ca2 in 2þ the stressed cells, and blunted the amplitude of Ca2þ release from their intra- The time-course of intracellular Ca transients is hard to assess due to buff- cellular stores in response to activation of a Gq pathway. In the case of cAMP, ering and signal integrating interactions. Genetically-encoded calcium indica- tors (GECI) have proven useful for monitoring Ca2þ transients in living cells the resting levels of cAMP in the stressed cells was reduced, though the abso- 2þ 2þ lute amplitude of the response to Gs stimulation (DF/F) was not altered. and organisms. However, Ca indicators with high Ca affinity and slow decay kinetics themselves integrate Ca2þ signals, and furthermore may become þ 1443-Pos Board B352 saturated before peak [Ca2 ] is reached. Thus, for more faithful tracking of þ Pulsed Radiofrequency for Chronic Pain: An Electroporation Mediated rapid Ca2 dynamics, probes with faster off-kinetics are required1,2. Red- Calcium Signaling Process? fluorescent GECI have been developed with the view of multicolour imaging Borja Mercadal1, Ruben Vicente2, Antoni Ivorra1. and optogenetic applications3. Here we report novel fast-decay variants of þ 1Department of Information and Communication Technologies, Universitat red-fluorescent genetically-encoded Ca2 indicators jRGECO1a and 2 3 Pompeu Fabra, Barcelona, Spain, Laboratory of Molecular Physiology, jRCaMP1a with up to 8-fold (t1/2 of 6.4 ms) and 13-fold (t1/2 of 33 ms) faster Faculty of Health and Life Sciences, Universitat Pompeu Fabra, Barcelona, in vitro decay kinetics (37 C), respectively. Fast-decay jRGECO1a and Spain. jRCaMP1a variants retain comparable fluorescence brightness and dynamic Pulsed radiofrequency (PRF) treatment consists in the delivery of a series of range values to their parent proteins. The fluorescence dynamic range of the bursts (5 to 20 ms) of sinusoidal current (500 kHz) to the targeted nerve. This brighter mApple-based jRGECO1a variants is stable between pH 6.5 and þ clinical treatment has proved to effectively mitigate pain. Its mechanism of action 7.5, but declines above pH 7.5 to a Ca2 -independent fluorescent state. In

BPJ 8628_8631 288a Monday, February 19, 2018 contrast, the less bright jRCaMP1a variants, based on mRuby, are stable over We electrically paced hiPSC-CM and found that our maturation method the pH range of 6.5 to 10. Red-fluorescent GECI, like their green-fluorescent significantly improved the uniformity of calcium release, indicating that counterparts, are characterised by high cooperativity to Ca2þ, and complex ki- the t-tubules functionally connect the L-type calcium channel with RyR2 cal- netic patterns of Ca2þ-dependent fluorescence response with a limiting on-rate. cium release throughout the cell. These results were also verified in a second However, the fast-decay variants of jRGECO1a and jRCaMP1a reveal 8-fold hiPSC line, suggesting that we can mature hiPSC-CM to improve calcium faster ATP-evoked Ca2þ transients compared to their parent proteins in handling and calcium release properties. Our maturation method provides a HEK293T cells, showing the benefits of fast-decay red-fluorescent GECI indi- good model for examining cardiac arrhythmias and calcium release in human cators for monitoring Ca2þ dynamics in living cells. cardiomyocytes. References: This work is funded by BBSRC grant BB/M02556X/1 to K.T. 1448-Pos Board B357 [1] Helassa et al., 2015 Scientific Reports, 5:15978. TRPV4 Increases Cardiomyocyte Calcium Transients and Contributes to [2] Helassa et al., 2016 Scientific Reports, 6:38276. Cardiac Damage Following Ischemia-Reperfusion in Hearts of Aged Mice [3] Dana et al., 2016 eLife, 5:e12727. Deborah Peana, John L. Jones, Adam B. Veteto, Michelle D. Lambert, Timothy L. Domeier. 1446-Pos Board B355 University of Missouri, Columbia, MO, USA. The Bright Calbryte 520 Enables the Probenecid-Free Intracellular Cal- Transient Receptor Potential Vanilloid 4 (TRPV4) is an osmotically-activated cium Assays cation channel, and expression is increased in cardiomyocytes of Aged mice. Qin Zhao, Muhua Yang, Haitao Guo, Ruogu Peng, Jinfang Liao, The goal of this investigation was to determine the role of TRPV4 in calcium Zhenjun Diwu. handling following hypoosmotic stress and in cardiac function following AAT Bioquest Inc., Sunnyvale, CA, USA. ischemia-reperfusion (I-R). Hypoosmotic stress (250 mOsm pretreatment) The intracellular calcium flux assay is a widely used method in monitoring induced an increase in action potential-induced calcium transient amplitude signal transduction pathways and high throughput screening of G protein- (fluo-4) in cardiomyocytes isolated from Aged (24-26 month) mice. This effect coupled receptors (GPCRs) and calcium channel targets. Followed by was prevented by TRPV4 inhibition with HC067047 (1mM), and was absent in Fluo-3, Fluo-4 was developed with improved signal/background ratio, and 2þ cardiomyocytes from Young (3-6 month) mice. The elevation in calcium tran- became one of the most widely used Ca indicators. However, there are sients in Aged associated with an increase in ryanodine receptor-mediated cal- still a few severe problems with Fluo-4. For example, the use of probenecid cium sparks (3.450.7 sparks/s/100mm hypoosmotic versus 1.150.3 control, with Fluo-4-based calcium assays compromise the assay results since pro- P<0.05). Cardiac contractile function was examined in Langendorff-perfused benecid is well-documented to have a variety of complicated cellular effects. hearts prior to and following global I-R (45 minutes ischemia, two hours reper- CalBryte 520, which has the identical excitation and emission wavelength as fusion), and hearts of Aged responded to I-R with an initial increase in contrac- Fluo-4, overcomes this problem. Like other AM cell loading principles, Cal- tile function during reperfusion (DdP/dtMax: gain of 6745226 mmHg/s from Bryte 520 AM ester is non-fluorescent and once gets inside the cell, it is hy- baseline). The enhanced contractility during reperfusion of Aged was prevented drolyzed by intracellular esterase and activated to a polar molecule that is by TRPV4 inhibition with HC067047 (DdP/dtMax: loss of 2905102 mmHg/s well trapped inside the cell. Upon binding Ca2þ ions, CalBryte 520 produces from baseline) and was absent in hearts of Young (DdP/dtMax: loss of bright signal, extremely high signal to noise ratio, and also demonstartes 6215400 mmHg/s from baseline). Following two hours of reperfusion, meta- greatly improved intracellular retention. In this study, the performance of bolically active tissue was assessed using triphenyltetrazolium chloride. In CalBryte 520 AM was evaluated with different receptor signaling pathways hearts of Aged, TRPV4 inhibition with HC067047 decreased the percentage using several cell lines including HEK-293, CHO-M1 and CHO-K1 cells in of damaged tissue compared to untreated conditions (753% HC067047 versus microscope imaging and microplate assays. The microscope imaging data 2155% untreated, P<0.05), revealing a potential pathological role of TRPV4 showed that brightness of CalBryte 520 is 5 to 10 times brighter than that 2þ in cardiomyocyte viability. Taken together, our data suggest that while TRPV4 of Fluo-4 AM upon binding Ca , and requires and no organic-anion trans- may increase calcium transients and contractile function, it also contributes to porter inhibitors (such as probenecid) present in the assay system and has cardiomyocyte damage following I-R. around 1000 % of response over background signal in microplate assays. In conclusion, CalBryte 520 AM is a new generation of fluorescent indicator 1449-Pos Board B358 for the measurement of intracellular calcium with high signal-to-noise ratio Early Diastolic Ca2D Sparks Alter Repolarization Rate of Rabbit Cardio- and intracellular retention properties, is a superior indicator for evaluating myocytes GPCR and calcium channel targets as well as for screening their agonists Priyanka Saxena, Godfrey Smith, Niall Macquaide. and antagonists. Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom. Posters: Intracellular Calcium Channels and Cardiac contraction is controlled by the release of Ca2þ from the sarcoplasmic Calcium Sparks and Waves II reticulum (SR) during systole. During the release process, a host of membrane currents are both activated and inactivated by the elevated Ca2þ levels in sub- 1447-Pos Board B356 cellular microdomain between SR and sarcolemma called the dyad. This dy- Improved Calcium Handling in Human Induced Pluripotent Stem Cell namic process may have ramifications for determining the rate of Cardiomyocytes repolarisation of the cardiac action potential (AP). The current study aims to Daniel Blackwell, Shan S. Parikh, Nieves Gomez-hurtado, understanding how these currents are affected by under conditions of altered þ Bjorn C. Knollmann. RyR sensitivity and effects on AP morphology. Intracellular Ca2 measure- þ Vanderbilt University, Nashville, TN, USA. ments using 2D confocal microscopy allowed detection of Ca2 sparks, which Human induced pluripotent stem cell cardiomyocytes (hiPSC-CM) are being were detected and quantified every 100ms after subtraction of the moving dia- increasingly used to model cardiac disease. However, investigation of cal- stolic signal. After the initial 50ms of systolic release, spark frequency re- cium handling and arrhythmias has been hampered by lack of t-tubule devel- mained constant over the diastolic period. In the steady state, tetracaine did opment and poor calcium-induced calcium release (CICR). We recently not affect this relationship. However, caffeine (500mM) caused spark frequency developed a hormone maturation protocol (addition of T3, Dexamethasone, to increase by 133560% over the initial 100ms, but reduced by 49.8512.1% and Matrigel substrate) that promoted t-tubule genesis in hiPSC-CM. over the remainder of diastole. In current clamped patched myocytes, AP þ þ To investigate whether calcium handling was improved in these cells, we morphology and Ca2 was measured with Fura-2. No alteration in Ca2 tran- utilized a saponin-permeabilized cell spark assay to measure sarcoplasmic re- sient amplitude or rate of decline was detected on addition of tetracaine ticulum (SR) calcium release via the ryanodine receptor (RyR2). Hormone- (N=12) or caffeine (N=11) in the steady state. AP duration at 30- 90% repolar- treated cells showed a significant improvement in the frequency of calcium isation levels(APD30-90) were reduced by >25% (N=11). Addition of a spe- 2þ release (8.31 vs 4.02 sparks/100mm/s), time to peak (21.69 vs 16.24 ms), cific Ca -activated-chloride current (ICa-A-Cl) blocker (9AC-0.5mM) and the amount of calcium released as assessed by spark mass (33.36 vs reversed this shortening. Measuring of tail current of repolarisation at 100 6.62 DF/F0*mm^3), without altering total SR calcium content. Notably, these ms compared to 600ms, no difference in amplitude was observed indicating values were similar to those observed in isolated mouse ventricular cardio- similar levels of NCX activation at early and end diastolic periods. Sensitisa- myocytes, indicating mature RyR2 clustering and localization. To examine tion of RyR leads to more early-diastolic sparks and fewer over the remaining this, we performed immunostaining of RyR2 and found that our hormone- diastolic period. This shift in spark kinetics shortens the AP. Blockade of treated hiPSC-CM had a striated pattern consistent with dyadic localization, ICa-A-Cl reverses this, indicating a major role of ICa-A-Cl activation upon dia- þ in contrast to diffuse perinuclear staining from vehicle-treated hiPSC-CM. stolic Ca2 -dependant AP.

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1450-Pos Board B359 transfected with fluorescently labeled cardiac SERCA2a revealed that SERCA D D Re-triggerability of Ca2 Sparks Following Evoked Ca2 Release forms homodimers in a dose-dependent manner. In parallel experiments, proper- Ewan D. Fowler, Cherrie H.T. Kong, Jules C. Hancox, Mark B. Cannell. ties of endoplasmic reticulum (ER) Ca uptake were studied at different levels of School of Physiology, Pharmacology & Neuroscience, University of Bristol, SERCA2a expression. Intra-ER [Ca] ([Ca]ER) was measured in HEK293 cells Bristol, United Kingdom. transfected with SERCA2a (tagged with cerulean), cardiac ryanodine receptor þ The systolic Ca2 transient in cardiac myocytes arises from the near- (RyR2 tagged with GFP) and the ER-targeted Ca sensor Cepia-1er. The plasma þ synchronous activation of myriad Ca2 sparks in response to an action poten- membrane was permeabilized with saponin to control cytosolic milieu. Activa- 2þ tial. The consequent depletion of sarcoplasmic reticulum (SR) Ca is believed tion of RyR2 with caffeine was used to completely deplete [Ca]ER. Once caffeine 2þ 2þ to be important in terminating further Ca -induced Ca release. It was re- was removed, RyR2 inhibitors were applied to measure the rate of [Ca]ER recov- þ ported that in rat cardiac myocytes following spontaneous diastolic Ca2 ery. At the end of each experiment, RyR2-independent Ca leak was measured sparks, ryanodine receptor (RyR) release sites enter a refractory period which during SERCA inhibition with thapsigargin. For each individual cell, ER Ca up- 2þ is likely due to depletion of junctional SR Ca . It is not known whether the take and leak as a function of [Ca]ER were analyzed to determine maximum ER þ same refractory mechanism occurs following normal evoked Ca2 release. Ca uptake rate and maximum ER Ca load. SERCA2a expression level was esti- Rabbit ventricular myocytes were isolated using standard enzymatic dissocia- mated from the cerulean fluorescence. This analysis revealed that ER Ca uptake tion methods. Cells loaded with Fluo-4-AM were placed in a recording cham- increased as a function of [SERCA], with a particularly steep increase at high þ ber on the stage of a confocal microscope. Ca2 sparks and transients were SERCA expression level. We also found that maximum ER Ca load (or SERCA recorded in line scan mode in modified Tyrode’s solution containing 1.8 mM thermodynamic limit) increased with an increase of SERCA expression. Partial þ þ Ca2 .Ca2 transients were elicited at 0.5 Hz by field stimulation electrodes. inhibition of SERCA with thapsigargin reduced not only ER Ca uptake rate The interval between spontaneous and evoked release was analysed to deter- but also maximum ER Ca load. These results suggest that SERCA dimerization þ mine the effective refractory period and amplitude restitution of Ca2 sparks can facilitate ER Ca uptake and improve the efficacy of coupling between ATP þ following evoked release. The probability density function revealed Ca2 hydrolysis and Ca transport. Malfunction of this mechanism (due to SERCA release in quick succession occurred infrequently. In such cases, the relative downregulation) can contribute to reduced SR Ca uptake in the failing heart. amplitude of the second Ca2þ release was depressed. The time-course of recov- þ ery of Ca2 spark amplitude was comparable to that reported in rat myocytes 1453-Pos Board B362 with chemically modified RyR and similar to that expected for SR refilling. Sarcoplasmic Reticulum Calcium Leak in Cardiomyocytes: A Contribu- Our data shows a refractory period due to SR depletion reduces repeated acti- tion of TRPC1 Channels þ vation of RyR clusters by the increased cytosolic [Ca2 ] present during the Azmi A. Ahmad, Chris Hunter, Frank B. Sachse. þ þ normal cardiac Ca2 transient, although some late Ca2 release may still occur. Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT, USA. 1451-Pos Board B360 The sarcoplasmic reticulum (SR) plays an important role as calcium store in Subcellular Calcium Events and Calcium Waves in Leg Skeletal Muscle cardiac myocytes. The SR calcium concentration is a major determinant of Fibers Isolated From the Honey Bee APIS Mellifera the calcium transient. Leak of calcium from the SR is thought to occur through Claude Collet1, Cecilia Simut2, Marianna Takacs3, Laszlo Szabo4, ryanodine receptors and to be increased in some cardiac diseases including Peter Szentesi5, La´szlo´ Csernoch5. heart failure. Here, we test the hypothesis that the transient receptor potential 1Research Unit INRA UR406 Bees and Environment, Avignon, France, cation (TRPC) 1 channels contribute to SR calcium leak and modulate the cal- 2University of Oradea, Oradea, Romania, 3University of Debrecen, Debrecen, cium transient in cardiomyocytes. Hungary, 4Department of Electrical Engineering, Sapientia Hungarian We studied isolated neonatal rat ventricular myocytes infected with adenoviral University of Transylvania, Targu Mures, Romania, 5Department of eGFP (control), TRPC1-eGFP and shRNA-TRPC1-eGFP constructs. Cultured Physiology, University of Debrecen, Debrecen, Hungary. cells were loaded with the calcium sensitive dye Rhod-3 at 4-5 days after infec- The domestic honey bee participates in vegetal biodiversity maintenance and tion. Cytosolic calcium concentrations and eGFP signals were measured using food production, since it is one of the most efficient pollinators. These late rapid scanning confocal microscopy (Leica TCS SP8 equipped with GaAsP- years, beekeepers happen to face an abnormal level of colony mortality. Neuro- HyD detectors). The eGFP signals served as marker of successful infection muscular studies are needed to better characterize the impact of insecticides on and protein location. Our experimental protocol involved pacing of cells super- honey bees, since most of active substances on the market are targeting ion fused with modified Tyrode solution followed by superfusion with 0 sodium/ channels from insect nerves and muscles, including those acting on the Ryano- 0 calcium solution for 2 min. We then applied caffeine (20 mM) to trigger dine receptor. Electron microscopy and confocal imaging has shown that ultra- SR calcium release. Calcium signals were averaged and self-ratioed. structural characteristics of bee skeletal muscle cells resemble those of We found a decreased SR calcium content in cells infected with TRPC1-eGFP mammals. Here, excitation-contraction coupling and intracellular calcium (1.8650.36) versus control (3.0650.34). SR calcium content was increased in signaling was further characterized at the subcellular level in intact skeletal cells infected with shRNA-TRPC1-eGFP constructs (6.1951.1) versus control. muscle fibers enzymatically isolated from legs of bees aged 1-3 days. Confocal Analysis of eGFP signals in TRPC1-eGFP infected cells revealed that the x-y and line-scan observations were taken in a physiological Tyrode’s solution construct is primarily localized in the cell interior and not in the sarcolemma. containing calcium, after loading with the calcium indicator fluo-8 AM. Spon- The studies support our hypothesis that the TRPC1 channels act as SR calcium taneous localized calcium release events (CRE) were frequently detected. Oc- leak channels and modulate calcium transients in cardiac myocytes. Further- casionally, propagating calcium waves were observed. In fibers showing more, our studies indicate that TRPC1 channels are not involved in sarco- spontaneous activity, event frequency was calculated from x-y sequences taken lemmal electrophysiology of rat ventricular myocytes. Our findings suggest at 10 Hz and was 22005470 Hz/mm2 (n=15 cells). Images were automatically that TRPC1 channels are physiological modulators of intracellular calcium processed and gave a preliminary characterization of CRE. They looked ‘wide’ and contractility in cardiac myocytes. as spatial spread at half maximum was 3.7150.02 and 3.2850.02 mm (n=5174 events) parallel with and perpendicular to the fiber axis, respectively. The mean 1454-Pos Board B363 amplitude of the events was 0.22050.001. Frequency and spatial spread were Downregulation of NCX and RYR Drives Changes in Complementary thus different from events (sparks, embers) detected previously in cardiac my- Channels to Regulate Calcium Transients in Cultured Neonatal Ventricu- ocytes, batrachian and mammalian skeletal muscle fibers. For the first time, lar Myocytes subcellular calcium events were monitored in isolated skeletal muscle cells Esteban Vazquez-Hidalgo, Paul Paolini, Parag Katira. from an arthropod and this new approach may help in understanding their San Diego State University, San Diego, CA, USA. role and regulation in muscles and the myotoxicity of insecticides. Cytosolic [Ca2þ] is regulated by 3 key proteins: RyR, NCX, and SERCA. Expression levels of these proteins are a function of development. As such, 1452-Pos Board B361 neonatal rat ventricular myocytes are in a transitional stage with protein expres- The Functional Significance of Cardiac SERCA Dimerization sion shifting toward the adult phenotype. Additionally, other factors such as Elisa Bovo, Siddharth Bhayani, Roman Nikolaienko, Daniel Kahn, culturing, affects morphology, protein expression, and subsequently, cytosolic Seth Robia, Aleksey Zima. [Ca2þ]. With cultured neonatal ventricular myocytes being widely used to Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, study different aspects of cardiovascular health, it remains important to under- USA. stand how the relationship between the expression levels of RyR, NCX, and It has been shown that the sarcoplasmic/endoplasmic reticulum Ca-ATPase SERCA affect the Ca2þ transients in the immature cell. We have investigated (SERCA) can form homodimers. However, the functional significance of how independent downregulation of RyR and NCX affects the expression SERCA dimerization remains unclear. FRET measurements in HEK293 cells levels of the complimentary proteins and their respective Ca2þ transients.

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We have also incorporated the protein expression measurements into a mathe- #P<0.05 human vs rabbit control frequency). Average ensemble of the LCR matical model to predict the behavior of the [Ca2þ] transient. Our experimental Ca2þ signal (integral LCR Ca2þ signals that activate NCX during diastole, results show that downregulating RyR decreased SERCA and increased NCX log(mmxmsxDF/F0)) in human, rabbit and mouse SANC, respectively was protein levels. The associated [Ca2þ] transient is altered: decreased amplitude, 3.350.12*#, 3.850.07* and 4.850.08. We conclude that species differences increased time-to-peak, 50%, and 90% Ca2þ removal. NCX downregulation in- in Ca2þ clock Ca2þ signals are proportional to the species differences in HR. creases SERCA production without significantly changing the expression Thus, Ca2þ clock signaling is a general control mechanism of normal automa- levels of RyR. The corresponding [Ca2þ] transient is altered: increased ampli- ticity in SANC of all species studied to date. tude, no change in time-to-peak and 50% Ca2þ removal, and increased 90% Ca2þ removal. Computational simulations incorporating the appropriate 1457-Pos Board B366 changes in the parameters associated with RyR, NCX, and SERCA activity Action Potential Shortening Prevents Atrial Calcium Alternans show that these three key proteins are sufficient to predict alterations in the Giedrius Kanaporis1, Jaime DeSantiago1, Zane M. Kalik1, Kathrin Banach2, [Ca2þ] transient of the respective knockdown experiments. Lothar A. Blatter1. 1Physiology & Biophysics, Rush University Medical Center, Chicago, IL, 1455-Pos Board B364 USA, 2Medicine/Cardiology, Rush University Medical Center, Chicago, IL, Protein Phosphatase-1 Modulates Basal Spontaneous Beating Rate of USA. Sinoatrial Node Cells (SANC) Alternans is a risk factor for cardiac arrhythmia, including atrial fibrillation. At Tatiana M. Vinogradova, Ihor Zahanich, Yevgeniya O. Lukyanenko, the cellular level alternans manifests as beat-to-beat alternations in contraction Syevda Sirenko, Daniel R. Riordon, Kirill V. Tarasov, Yue Li, strength, action potential (AP) duration and magnitude of the Ca transient (CaT). Alexey E. Lyashkov, Dongmei Yang, Edward G. Lakatta. Pacing-induced AP and CaT alternans were studied in rabbit atrial myocytes using Lab Cardiovascular Sciences, NIA, NIH, Baltimore, MD, USA. combined Ca imaging and electrophysiological measurements. Development of Basal PKA- and CaMKII-dependent phosphorylation in SANC exceeds that in CaT alternans was strongly affected by AP morphology. APs of longer duration ventricular myocytes and is crucial for ryanodine receptor (RyR)-generated and beat-to-beat alternations in AP morphology lowered the pacing frequency local subsarcolemmal Ca2þ releases (LCRs), which regulate spontaneous firing threshold and increased the degree of CaT alternans. AP morphology contributed of SANC. Since protein phosphorylation is determined, in part, by the balance to the development of CaT alternans by affecting diastolic sarcoplasmic reticulum between activity of protein kinases and protein phosphatases (PP), which de- (SR) Ca levels and the efficiency of L-type Ca currents to trigger SR Ca release. phosphorylate proteins, we hypothesized that basal PP activity in SANC plays Pharmacological modulation of AP morphology determined the severity of Ca al- a key role in the regulation of normal SANC automaticity. Rabbit SANC ex- ternans. Inhibition of Ca-activated Cl channels reduced beat-to-beat AP alterna- pressed (RT-qPCR, western blot) both PP1 and PP2A. Dual inhibition of tions and prolonged AP duration, but failed to suppress Ca alternans. In PP1 and PP2A by calyculin A (CyA, 100-500 nmol/L) markedly increased contrast, AP shortening induced by K channel agonists ML277 and NS1643, acti- phospholamban phosphorylation by2-fold and 3-fold at the PKA- vating Kv7.1 and Kv11.1 channels respectively, significantly reduced the degree dependent Ser16 and CaMKII-dependent Thr17 sites, respectively, indicating of Ca alternans in field-stimulated and current-clamped atrial myocytes. K channel basal PP activity in SANC. In freshly isolated single rabbit SANC, CyA (100 activators had no effect on the degree of Ca alternans in voltage-clamped cells, nmol/L) substantially increased LCR number and size (confocal line-scan im- demonstrating that suppression of Ca alternans was primarily caused by the aging) and reduced the LCR period (time from action potential-induced Ca2þ changes in AP morphology. Finally, activation of Kv11.1 channel significantly transient to subsequent LCR), which concurrently reduced the spontaneous reduced the degree of or even abolished pacing induced atrial T-wave alternans SANC cycle length and increased the average spontaneous SANC firing rate in isolated Langendorff perfused hearts, demonstrating that AP shortening is a po- (perforated patch) by30%. The CyA-induced increase in LCR parameters tential intervention to prevent alternans at whole heart level. was partially due to the increase in RyR phosphorylation at Ser2809 site, concur- In conclusion, alternation in AP morphology plays a significant role in the rent augmentation of L-type Ca2þ current amplitude and elevation of sarco- development and stabilization of atrial alternans. The demonstration that CaT plasmic reticulum Ca2þ load. To determine the role of PP2A activation in alternans can be controlled or even be prevented by modulating AP CyA-dependent acceleration of spontaneous SANC firing rate we employed se- morphology has important ramifications for arrhythmia prevention and therapy. lective PP2A inhibitor okadaic acid (100 nmol/L), which had no significant ef- fect on spontaneous SANC firing, LCR parameters or phospholamban 1458-Pos Board B367 phosphorylation. Further, in permeabilized SANC CyA markedly increased Connexin-43-Hemichannel-Mediated ATP Efflux Triggers Arrhythmo- D LCR number and size and purified PP1 suppressed LCRs. In contrast, neither genic CA2 Waves via P2X Purinoceptor Current in Atrial Myocytes okadaic acid nor purified PP2A affected LCR characteristics in permeabilized Joon-Chul Kim, Min-Jeong Son, Qui Anh Le, Sun-Hee Woo. SANC. Thus, net basal PP1 activity reduces basal PKA- and CaMKII- College of Pharmacy, Chungnam National University, Daejeon, Republic of dependent phosphorylation, limiting phosphorylation-dependent effects on Korea. LCRs and spontaneous SANC cycle length. Myocardium is subjected to mechanical stresses and adapts to them. Atrial my- ocytes are exposed to high shear stress during hemodynamic overload and 1456-Pos Board B365 blood regurgitation. We have previously shown evidence that shear stress Species Differences of Calcium Clock Functions in Human, Rabbit and (16 dyn/cm2)-induced atrial global Ca2þ waves are abolished by the block- Mouse Pacemaker Cells Recapitulate Species Differences in Heart Rate ades of ATP release, gap junction hemichannel or P2 purinergic signaling. In Syevda Sirenko, Kenta Tsutsui, Bruce D. Ziman, Oliver J. Monfredi, this study, we assessed activation of gap junction hemichannels by shear stress, Victor A. Maltsev, Edward G. Lakatta. and its role in induction of arrhythmogenic currents and in proarrhythmic Ca2þ National Institute on Aging, NIH, Baltimore, MD, USA. waves in rat atrial myocytes. Calcein dye efflux, but not oregon flux, was accel- Our prior studies in non-human sinoatrial nodal cells (SANC) revealed that erated by shear application. The shear-induced calcein efflux was enhanced by coupling of spontaneous local calcium releases (LCRs), generated by a Ca2þ zero external Ca2þ, and was suppressed by La3þ, but not by probenecid, sug- clock (sarcoplasmic reticulum-SR), with a membrane clock (sarcolemmal elec- gesting activation of connexins by shear stress. Shear stress produced inward trogenic molecules), regulates the spontaneous SANC action potential (AP) cation (Csþ) currents at resting potentials, and this current was completely sup- firing: the larger and earlier the ensemble of the LCR Ca2þ signal (a crucial pressed by La3þ or carbenoxolone, and was enhanced by quinine. The current LCR parameter), the earlier and larger NCX inward current, the more rapid was also partly suppressed by P2X receptor inhibition (by 50%) or by the basal AP firing rate. It’s well-know that the species differences in basal blockade of P2Y1 receptor/transient receptor potential melastatin subfamily 4 heart rate (HR) depends on body mass, and varies from 60-100beats/min in hu- (by 30%). Shear-induced NMDGþ current was one-fifth of the Csþ current, mans to 140-180beats/min in rabbit and 650-750beats/min in mice. We hypoth- showed linear voltage-dependence with a reversal at 0 mV and was eliminated esized that species differences in HR are linked to species differences in the by introduction of anti-connexin-43 antibodies. Shear-induced ATP release SANC Ca2þclock ticking rate, i.e. timing and magnitude of the LCR ensemble. from a monolayer of atrial cells, assessed by chemiluninescence, was abolished We isolated SANC from the adult human hearts rejected for transplantation, by either connexin-43 hemichannel inhibitor Gap 19 or connexin-43 knock- and from the hearts of other mammalian species. To have a direct access to down. Simultaneous measurement of ATP release and Ca2þ images using a the Ca2þclock without interference from the membrane clock, we permeabi- sniffer patch clamp and two-dimensional confocal microscopy, respectively, lized SANC (saponin) and recorded LCRs via line-scan confocal Ca2þ imaging further revealed that ATP releases occurred at 200-300-ms prior to the onset (Fluo-4salt, 30mM) when cells were bathed in 50nM free [Ca2þ]. SANC of all of the Ca2þ waves under shear stress and were sustained under prolonged shear species produced LCRs. In human (n=18), rabbit (n=16) and mouse (n=14), stimulus. Our data suggest that shear stress induces connexin-43-hemichannel- LCRs frequency (normalized per 100mmx1sec) was 13.352.24*#, mediated ATP release, thereby initiating P2X purinoceptor-dependent trig- 27.653.34*and 40.253.28, respectively (*P<0.05 human vs mouse, gered Ca2þ waves in atrial myocytes.

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1459-Pos Board B368 of the electrocardiogram (pQT), associated with conditions such as heart failure Intercellular Ultrafast Calcium Wave Velocity and Propagation of Sponta- and long-QT syndromes, is linked to increased vulnerability to arrhythmia. neous Electrical Activity in A7r5 Cells at Physiological Temperature Pharmacological management of arrhythmia associated with pQT has been Jairo C. Quijano1,2, Teddy Grand3, Stephan Rohr3, Jean Jacques Meister2. demonstrated to have limited effectiveness; Understanding the impact of phar- 1Politecnico Colombiano JIC, Medellı´n, Colombia, 2E´cole Polytechnique macological modulation on the complex interaction between trigger and sub- Federale de Lausanne, Lausanne, Switzerland, 3University of Bern, Bern, strate remains a significant yet important challenge in order to improve Switzerland. intervention efficacy. Temperature has pervasive effects on the functional properties of biological sys- Methods: We examined the efficacy of a hERG activator (MC-II-157c) to tems. In particular, mechanical performance and membrane conductance of reduce the manifestation of cell- and tissue-scale triggers and its concomitant muscles are greatly influenced by temperature. Experiments using an aligned mi- effects on the tissue substrate using a multi-scale modelling approach: a model cropatterned configuration of smooth muscle cells (A7r5) were performed at of the human ventricular action potential was integrated with a model of sto- 37C. Fluorescence microscopy revealed that both, mechanical and KCl stimu- chastic 3D spatiotemporal Ca2þ dynamics, which was then coarse-grained þ lation, evoked an ultrafast Ca2 wave with a velocity magnitude of almost two for suitability for 1D-3D tissue simulations. Parameters were modified to times the one obtained previously at room temperature and that is in agreement mimic pQT and MC-II-157c conditions. with the velocity reported previously by theoretical simulations (333 cells/s). Results: pQT conditions promoted the development of spontaneous release On the other hand, cultured A7r5 cells showed spontaneous electrical activity ex- events underlying afterdepolarisations during rapid pacing. MC-II-157c hibiting similarities with action potentials. In order to investigate the direction applied to pQT conditions shortened the action potential duration, inhibited and velocity of propagation of this spontaneous activity, we used optical map- the development of afterdepolarisations and reduced the probability of afterde- ping of transmembrane voltage that allowed the characterization of patterns of polarisations manifesting as triggered activity in single cells and ectopic activ- electrical activity in A7r5 cultures at 37C, at which temperature preparation ity in tissue. However, it could also increase transmural dispersion of had a high probability to exhibit spontaneous electrical activity. The optically repolarisation, which manifested as an increased vulnerable window for unidi- measured spatial and temporal characteristics of propagating ‘‘action poten- rectional conduction block. tials’’ revealed, that in patterned strands of A7r5 cells, conduction velocity Conclusions: The combination of stochastic release event modulation and þ was not statistically different from the velocities of the ultrafast Ca2 wave at transmural dispersion of repolarisation modulation by MC-II-157c resulted in 37C(29 mm/s). Together, our results suggest a direct correlation between an integrative behaviour wherein the arrhythmia trigger is reduced but the sub- þ the speed of ultrafast Ca2 wave/spreading velocity of the membrane depolari- strate is increased. Such variable overall vulnerability to arrhythmia cannot be zation with the speed of the phenomena known as electrical slow waves and also predicted from single-cell studies alone. validate the numerical data reported with experiments at physiological temper- atures. The ultrafast Ca2þ wave and the spontaneous electrical activity may 1462-Pos Board B371 trigger slower Ca2þ waves observed ex vivo and in vivo in smooth muscle cells. CellSpecks: A Software for Automated Detection and Analysis for Calcium Channels in Live Cells 1460-Pos Board B369 Syed Islamuddin Shah1, Martin Smith2, Ian Parker3, Ghanim Ullah1, Excitation-Contraction Coupling in HFpEF Angelo Demuro3. Peter Kilfoil, Xin Yue, Rui Zhang, Ryan Solymani, Daniel Soetkamp, 1Department of Physics, University of South Florida, Tampa, FL, USA, Eduardo Marba´n, Joshua Goldhaber. 2Pacific Biosciences, Menlo Park, CA, USA, 3Department of Neurobiology Heart Institute, Cedars-Sinai, Los Angeles, CA, USA. and Behavior, University of California Irvine, Irvine, CA, USA. Although the signature hemodynamic characteristic of heart failure with pre- With the intent to create a new approach to single channel recording that served ejection fraction (HFpEF) is diastolic dysfunction, contractile function could couple the fidelity of patch-clamp recording with a more high- may also be impaired. To investigate this possibility, we examined excitation- throughput screening capability, we have pioneered the development of a contraction (EC) coupling in cardiomyocytes isolated from Dahl salt-sensitive novel approach for single channel recording, we named Optical patch clamp rats with and without HFpEF. HFpEF was generated by feeding the rats a high recording. By combining the use of highly-sensitive fluorescent Ca2þ indica- salt diet for 11 weeks. We confirmed impaired diastolic relaxation using mitral tor dyes in conjunction with total internal fluorescence microscopy (TIRFM) inflow and tissue doppler echocardiography. We also documented clinical signs techniques, we monitor the flux of Ca2þ ions passing through the pores of a of heart failure consistent with HFpEF. In patch clamped myocytes, we charac- single open ion channels. This new approach provide channel gating informa- terized the voltage-dependence of ICa,L using 200 ms clamps from test potentials tion with time resolution of < 2 ms , with the great advantage of being ranging from 50 mV to þ50 mV; we found that ICa,L density was increased by massively parallel providing simultaneous and independent recording from 23.7% @0 mV in HFpEF (p<0.01) with a leftward shift in peak voltage. Steady- thousands of ion channels. However, manual analyses of these data by visual state activation of ICa,L was left-shifted in HFpEF (V1/2-act =-14.95 0.3 mV in inspection, presents severe challenges as each video recording can include control vs. 19.1 5 0.4 mV in HFpEF), whereas steady-state inactivation 15,000 or more images. To overcome this major drawback, we develop a was right-shifted (V1/2, inact=-37.9 5 0.2 mV in control vs. 34.3 5 0.1 mV computational image processing and analyses framework, we named Cell- in HFpEF), resulting in a doubling of window current. Voltage clamp-evoked Specks, capable of detecting and fully analyzing kinetics of ion channel con- þ Ca2 sparks measured using ultra-high speed laser scanning confocal imaging tained in a video sequence. By using a randomly generated synthetic data, we þ of fluo-4AM loaded myocytes demonstrated that Ca2 spark recruitment was tested the ability of CellSpecks to rapidly and efficiently detect and analyze maintained in HFpEF but spark latency was shortened (6.0750.05 ms) the activity of thousands of ion channels. Here, we report the use of compared to control (7.1450.09 ms, p<0.01). The variability of spark latency CellSpecks for the analyses of experimental data acquired by imaging muscle was also decreased in HFpEF (p<0.01). Since SR Ca content was unchanged nicotinic acetylcholine receptors (nAChRs), and Alzheimers disease in HFpEF, our findings suggest enhanced phosphorylation of Cav 1.2, RYR2, associated/multi-conductance levels Amyloid beta pore (Ab42) inserted in and/or modulating proteins. Stimulation with isoproterenol had a blunted effect the plasma membrane of Xenopus laevis oocytes. CellSpecks rapidly and effi- on ICa amplitude in HFpEF, with post-stimulation current densities virtually ciently, was able to generated maps of their locations, create raw and pro- identical between groups, suggesting elevated baseline phosphorylation of cessed time-dependent fluorescence traces, histograms distributions of open ICa,L in HFpEF. We conclude that EC coupling efficiency in HFpEF is maxi- and close dwell-times, and open probability. While, we process and analyze 2þ mized, at least in part due to increased phosphorylation of ICa. Enhanced window fluorescence data from Ca channels, CellSpecks is equally applicable to current density could also increase arrhythmia susceptibility. fluorescence imaging of other channels and molecules. 1461-Pos Board B370 1463-Pos Board B372 Trigger versus Substrate: Multi-Scale Considerations for Arrhythmia Relevance of InsP3 Receptor ROS Regulation in Atrial Myocytes Modulation by Pharmacological Action Jaime Desantiago, Kathrin Banach. Michael A. Colman1, Erick A. Perez Alday2, Arun V. Holden1, Internal Medicine/Cardiology, Rush University Medical Center, Chicago, IL, Al P. Benson1. USA. 1 2 University of Leeds, Leeds, United Kingdom, Oregon Health and Science In atrial tissue InsP3 induced InsP3 receptor type 2 (InsP3R2) mediated Ca release University, Portland, OR, USA. (IICR) has been linked to positive inotropy as well as to increased arrhythmic Ca- Background: Cardiac arrhythmias such as ventricular fibrillation are intrinsi- dependent after-depolarizations. InsP3 production in atrial myocytes is regulated 2þ cally tissue-scale phenomena, wherein single-cell triggers (e.g. Ca -induced through Gq protein coupled receptors like a1-adrenergic, endothelin-1 or Angio- spontaneous depolarisation) must interact with tissue-level substrate (e.g. het- tensin II (AngII) receptors and concomitant stimulation of PLC. Here we tested erogeneous repolarisation) to result in complex and irregular organ excitation the hypothesis that post-translational modifications of InsP3Rs in cardiomyocytes (e.g. self-perpetuating re-entrant excitation). Prolongation of the QT interval have significant consequences for cardiac excitation-contraction.

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Superfusion of field stimulated mouse atrial myocytes with AngII (1mM) creases in a time- and dose-dependent manner. Electrophysiological charac- induced a significant increase in diastolic [Ca], the Ca transient amplitude as terization of the cell line showed a corresponding dependence of hERG well as arrhythmic after-depolarizations. This increase in [Ca]i was suppressed current on [DOX] and time of treatment with the drug. As expected from pre- by treatment of the cells with the InsP3R2 blocker 2-Aminoethoxydiphenyl vious studies (Sale et al., 2008), currents exhibited an increase in deactiva- borate (2APB) as well as by the phopholipase C inhibitor U73122 (10mM) sup- tion time course and less rectification, resulting in larger steady-state porting the involvement of IICR. Simultaneously to changes in [Ca]i, AngII currents. Expression is tightly controlled with no evidence of hERG 1b promotes the stimulation of NADPH oxidase 2 (NOX2) dependent ROS pro- expression on western blots or in current kinetics prior to DOX application. duction. To determine if ROS contributes to AngII induced changes in [Ca]i Because hERG 1a and 1a/1b channels differ with respect to drug sensitivity the experiments were repeated in mice deficient for the expression of NOX2 (Abi-Gerges et al., 2011), this new cell line represents a useful reagent for phox-/- phox-/- (gp91 , p47 ). In NOX2 KO myocyes, AngII remained without effect more accurate predictions of off-target effects on IKr, particularly in compu- on [Ca]i supporting the notion that IICR mediated increase in [Ca]i depends on tational models relying on accurate IC50 measurements. Additionally, it will the presence of NOX2/ROS. facilitate studies of heteromeric assembly of hERG 1a and 1b subunits during ROS-dependent S-glutathionylation of InsP3R2 increases their affinity to InsP3 biogenesis. Supported by UW-Madison/WARF D2P Program and NINDS whereas it increases the ryanodine receptors (RYR) open probability. To deter- NS081320. mine the role of S-Glutathionlylation, atrial myocytes were superfused with Diamide (100mM). Diamide, comparable to AngII, induced an increase in basal 1466-Pos Board B375 and Ca transient amplitude leading to arrhythmic events. The change was IKR Enhancement in Stem CELL-Derived Cardiomyocytes by Morpholino reversible upon superfusion with the antioxidant DTT (1 mM) and preventable Anti-Sense Oligonucleotides by pre-treatment with 2APB. Mark W. Nowak1, Brian K. Panama1, Qiuming Gong2, Sanjot Singh1, The data support that ROS-dependent regulation of IICR plays a prominent role Randall Rasmusson3, Zhengfeng Zhou2, Glenna C.L. Bett4. 1 2 in the regulation atrial [Ca]i handling properties and is a sensitive readout for Cytocybernetics, Buffalo, NY, USA, Knight Cardiovascular Institute, NOX2/ROS production. Oregon Health & Science University, Portland, OR, USA, 3Physiology and Biophysics, SUNY, Buffalo, NY, USA, 4SUNY, Buffalo, NY, USA. Posters: Voltage-gated K Channels and We have previously shown that morpholino anti-sense (AS) oligonucleotides Mechanisms of Voltage Sensing and Gating II increases functional hERG expression in HEK293 cells (Gong et al, 2014). Here, we determined if morpholino AS oligonucleotides increase IKr expression 1464-Pos Board B373 in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Following 48 hr treatment with morpholino AS oligonucleotides, we observed Dynamic Rearrangement of the Intrinsic Ligand Regulates Gating of < KCNH Potassium Channels a significant increase (p 0.05) in IKr expression compared with inverse mor- pholino AS oligonucleotide control (Morpholino AS oligonucleotide (n=28): Gucan Dai, Zachary M. James, William N. Zagotta. 5 5 < Physiology and Biophysics, University of Washington, Seattle, WA, USA. 0.29 0.04 pA/pF, INV control (n=7) 0.11 0.08 pA/pF, (p 0.05). The KCNH voltage-gated potassium channels (EAG, ERG and ELK) play significant increase in current density was a result of a shift in the distribution of cells roles in neuronal and cardiac excitability. They contain cyclic nucleotide-binding expressing larger IKr current densities in the morpholino AS oligonucleotide- treated cells. In naı¨ve/vehicle treated cells, we observed a nearly equal fre- homology domains (CNBHD), but are not directly regulated by cyclic nucleo- % > tides. Instead, the ligand-binding cavity of the CNBHD is occupied by an intrinsic quency of cells expressing current densities 0.2 pA/pF and 0.2 pA/pF. Following morpholino AS oligonucleotide treatment, the percentage of cells ligand. The intrinsic ligand also resides at the interface between the amino- > < terminal eag domain and the carboxyl-terminal CNBHD. Here we show that, expressing current densities 0.2 pA/pF increased to 71% (p 0.05). We in D. rerio ELK (zELK) channels, deleting the intrinsic ligand produced opposite have demonstrated for the first time that morpholino AS oligonucleotide effects on the voltage-dependent gating in different patch-clamp configurations. treatment increases IKr expression in hiPSC-CMs, resulting in a higher fre- The intrinsic ligand inhibits the channel in the cell-attached configuration while quency of cells expressing larger IKr current densities. Increased IKr expression potentiates the channel after the run-up caused by patch excision. Using steady- in hiPSC-CMs will allow for more accurate screening of IKr-induced drug state and stopped-flow fluorescence anisotropy as well as electrophysiology, we effects on action potentials in both manual and multi-planar patch electrophys- demonstrate that an exogenous intrinsic-ligand peptide can bind to and regulate iology systems. zELK channels. Furthermore, the intrinsic ligand is critical for voltage-dependent potentiation (VDP), a process in which channel opening is stabilized by prior 1467-Pos Board B376 depolarization. The intrinsic ligand inhibits the channels before VDP and poten- Models of hERG Block Brandon Franks1, Glenna C.L. Bett2, Randall Rasmusson2. tiates the channels after VDP. Using patch-clamp fluorometry with a noncanon- 1 2 ical amino acid, L-Anap, combined with transition metal-ion FRET, we observed Cytocybernetics, Pendleton, NY, USA, Jacobs School of Medicine and that, during VDP, there is a rearrangement of the intrinsic ligand which was elim- Biomedical Sciences, SUNY, Buffalo, NY, USA. inated by deleting the eag domain. In addition, voltage-clamp fluorometry using Measurement of hERG block is crucial for assessment of a drug’s pro- L-Anap confirmed this movement of the intrinsic ligand with the time course of arrhythmic risk, and an in-Silico reconstruction of human cardiac cellular elec- VDP. We propose that the intrinsic ligand (1) can act as an allosteric modulator of trophysiology is a component of the CiPA initiative. Here, we compare several zELK channel gating, (2) switches from an antagonist to an agonist during the different hERG models to assess their similarities and applicability to experi- mental data.These four Markov models had explicit physical differences: 1) VDP or the run-up after patch excision, and (3) moves dynamically as a result 4 state linear model (C-C-I-O) of ferret IKr in atrial cells (20 C). 2) 5 state linear of the rearrangement of the eag domain/CNBHD interaction during the process of VDP. model (C-C-C-I-O) of HERG expressed in Xenopus Oocytes (20 C), 3) 5 state linear model (C-C-C-I-O) of HERG in CHO Cells (37C), 4) 6 state model (IC- 1465-Pos Board B374 IC-IO-C-C-O) of HERG in CHO cells (12 - 37C). These models differ in quan- A Novel hERG 1a/1b Stable Cell Line for Drug Screening and Research titative and qualitative aspects, reflecting different experimental preparations, Applications conditions, and experimental protocols used to define them. We examined these Erick B. Rı´os Perez, Fang Liu, Whitney Stevens-Sostre, Gail A. Robertson. models with respect to their performance during real-time simulations using Neuroscience, UW-Madison, Madison, WI, USA. dynamic clamp. The importance of having experimentally-constrained model New therapies for a wide range of diseases are counter-screened against parameters became immediately apparent. When we used quantitative analysis hERG potassium channels to reduce the risk of acquired long QT syndrome of the most complex models based on limited data sets, we observed some rate and life-threatening arrhythmias. Despite evidence that cardiac IKr is pro- parameters within certain models reached biophysically unrealistically fast duced by heteromeric hERG 1a/1b channels (Jones et al., 2004, Sale et al., values, effectively reducing two states to one state at some potentials. This sug- 2008, Jones et al., 2014), rather than the homomeric hERG 1a typically uti- gests that either the model structure was inappropriate or states were uncon- lized in screening, implementation of the more native-like channel has been strained during model development. Strategies for making use of these hampered by the silencing of hERG 1b expression in ‘‘stable’’ cell lines and models for integrated into a real-time dynamic clamp system, included limiting reversion of the channels to 1a homomers (Abi-Gerges et al., 2011). We have voltage dependent growth to prevent total numerical failure. These corrected developed a HEK293 cell line stably expressing heteromeric hERG1a/1b models showed dramatically different performances. This was evident during channels. Using the pTet-One technology (Clontech, USA) we stably trans- the period of deactivation between pulses, leading to different frequency depen- duced a well-characterized hERG 1a stable cell line with a hERG 1b expres- dencies among models. Since such models are critical in the FDA’s CiPA sion construct, allowing us to control 1b levels using different concentrations initiative, we demonstrate that additional model refinements using data from of doxycycline (DOX). Our results show that hERG1b protein expression in- protocols, such as the envelope of tails and re-activation currents, are critical

BPJ 8628_8631 Monday, February 19, 2018 293a for producing an accurate model structure and parameters for in-silico drug respectively, which suggested equal potency. The block of hERG currents by safety evaluations. norquetiapine was voltage-dependent with a steep increase over a range of volt- ages for channel activation. However, at more depolarized potentials where the 1468-Pos Board B377 channels were fully activated, the block by norquetiapine was voltage- External Protons Accelerate Deactivation of hERG Channels by Destabi- independent. The steady-state inactivation curve of the hERG currents was lizing the Relaxed State of the Voltage-Sensor shifted to the hyperpolarizing direction in the presence of norquetiapine. Nor- Yu Shi, Samrat Thouta, Tom Claydon. quetiapine did not produce a use-dependent block. A fast application of norque- Department of Biomedical Physiology and Kinesiology, Simon Fraser tiapine inhibited the hERG current elicited by a 5 s depolarizing pulse to þ60 University, Burnaby, BC, Canada. mV, which fully inactivated the hERG currents, suggesting an inactivated-state External protons accelerate the deactivation rate of cardiac hERG channels by an block. During a repolarizing pulse wherein the hERG current was slowly deac- unknown mechanism. Recently, we showed that the hERG voltage sensor dis- tivated, albeit remaining in an open state, the fast application of norquetiapine plays pronounced mode-shift behaviour due to relaxation, which stabilizes the 1 rapidly and reversibly inhibited the open state of the hERG current. Our results activated voltage sensor position and limits its return upon repolarization .We indicated that quetiapine and norquetiapine had equal potency in inhibiting hypothesized that protons accelerate deactivation by destabilizing the relaxed hERG tail currents. Norquetiapine inhibited the hERG current by preferentially state of the voltage sensor. To test this, we used cut-open voltage clamp and interacting with the activated channel in open and/or inactivated states. voltage clamp fluorimetry in Xenopus oocytes to measure voltage sensor move- ment, in addition to measurements of pore gating using two-electrode voltage 1471-Pos Board B380 clamp. Using durations that capture the majority of charge movement, the Molecular Mechanisms of hERG Potassium Channel Interactions with voltage-dependence of off-gating charge movement at pH 7.4 (V1/2 = 75.5 Ivabradine: Importance of the Lipophilic Route 5 2.3 mV, n=4) was found to be 34 mV left-shifted from that of on-gating Sergei Noskov, Henry Duff, Laura Perissinotti, Jiqing Guo, charge (V1/2 = 41.4 5 3.5 mV, n=5) indicative of relaxation-induced stabiliza- Meruyert Kudaibergenova. tion of the activated voltage sensor. At pH 6.5 this mode-shift was reduced to Sciences, University of Calgary, Calgary, AB, Canada. 19 mV (p<0.002; off-gating charge V1/2 = 51.8 5 3.3 mV, n=5; on-gating Human-ether-a-go-go-related channel (hERG) is a voltage gated potassium charge V1/2=-32.7 5 4.2 mV, n=5). This finding was corroborated by fluores- channel expressed in heart and brain. hERG is notorious for its interaction cence reports of voltage sensor movement, which showed that the mode-shift with various medications blocking Kþ transport, thereby prolonging action po- was reduced from 43 mV at pH 7.4 (n=5) to 23 mV at pH 6.5 (n=5). In tentials and resulting in an arrhythmias. Even recently released antiarrhythmic both of these reports of voltage sensor mode-shift, acidic pH induced a large de- agents such as ivabradine have been found to block hERG via unknown mech- polarizing shift in the voltage-dependence of voltage sensor return upon hyperpo- anisms. Common mechanisms of intracellular blockade of hERG imply access larization. These data suggest that the relaxed state of the voltage sensor is to major intra-cavitary binding site from the cytosol, while the second path in- destabilized by external protons and that this contributes to faster deactivation volves the ligand passing through the membrane and interacting with the trans- gating in hERG channels. 1Thouta et al., Biophys J.112:300-312.2017 membrane channel via a lipophilic pathway. The recent solution of the high- resolution Cryo-EM structure for open-state of the hERG1 channel (PDB:5VA1) 1469-Pos Board B378 D has provided an enhanced ability to resolve molecular details. Previous experi- Divalent Ions and H Block the Cardiac Potassium Channel HERG at an mental and computational efforts conducted by our lab have demonstrated the Outer Pore Site feasibility of the lipophilic route for the ivabradine mediated block of hERG cur- Gagandeep Singh, Kavaldeep Singh, Souad Hamade, Alan Miller. rents. This work used ivabradine as a molecular probe to map the lipophilic path Basic Sciences, Touro University - California, Vallejo, CA, USA. of the drug binding to the open and closed states of hERG channel. We developed Reduction of the current carried by the cardiac potassium channel HERG can biomolecular CHARMM force field compatible parameters for a different ioni- lead to Long QT syndrome, an arrhythmia characterized by a rapid heart rate zation states of ivabradine and used all-atom molecular dynamics simulations to and reduced cardiac output, which can, in certain situations, be fatal. The ef- compute kinetics and thermodynamics of their partitioning through hydrated fects of extracellular electrolytes on the biophysical properties of the HERG lipid membranes for different drug ionization states. The data combined from channel have been studied in some detail. In particular, increases in extracel- molecular docking and MD simulations of WT and mutated forms of hERG1 lular divalent ions such as calcium, magnesium, and zinc as well as increases enabled mapping key residues interacting with ivabradine and drug localization in extracellular hydrogen have been shown to slow channel activation, increase in the membrane. The residues essential for lipophilic blockade were further channel deactivation, and shift the G-V curve to more positive voltages. A tested by the combination of the site-directed mutagenesis and electrophysiolog- number of reports have also shown that hydrogen can reduce HERG current ical recordings. Our work established that ivabradine is interacting in the state- by a mechanism that does not involve an effect on channel deactivation and depending manner with the hydrophobic pocket formed by lipid-facing residues which likely involves pore block. We have previously described experiments from S5-S6 helices of the channel, the feature that may explain blockers risk pro- which suggest that there are two separate binding sites on HERG for calcium: pensities for arrhythmogenesis. one site that interacts with the voltage sensor and one site that may interact with the outer pore of the channel. Here we show, consistent with previously 1472-Pos Board B381 described experiments by others, that Hþ and Zn2þ also block HERG in a Sinusoidal Voltage Protocols for Rapid Characterisation of Ion Channel voltage dependent manner. We found that block of the double mutant Kinetics G628S631C by Hþ is reduced compared to block WT HERG by Hþ.We Kylie A. Beattie1, Adam P. Hill2, Remi Bardenet3, Yi Cui4, also found that arsenic trioxide blocks HERG at negative voltages, although Jamie I. Vandenberg2, David J. Gavaghan5, Teun P. de Boer6, at higher concentrations than previously tested by others. Combined, these Gary R. Mirams7. 1 data suggest that divalent ions, Hþ, and possibly arsenic, all block the pore School of Mathematical Sciences, FDA, White Oak, MD, USA, 2 of HERG, possibly at the same or nearby site in the outer mouth of the channel. Department of Molecular Cardiology and Biophysics, Victor Chang Cardiac Research Institute, Sydney, Australia, 3CNRS & CHRIStAL, University of 1470-Pos Board B379 Lille, Lille, France, 4Global Clinical Safety and Pharmacovigilance, Norquetiapine, the Active Metabolite of Quetiapine, Inhibits Cloned GlaxoSmithKline, Stevenage, United Kingdom, 5Department of Computer hERG Potassium Channels Science, University of Oxford, Oxford, United Kingdom, 6Department of Hong Joon Lee1, Jin-Sung Choi2, Sang June Hahn1. Medical Physiology, University Medical Center Utrecht, Utrecht, 1Department of Physiology, College of Medicine, The Catholic University of Netherlands, 7School of Mathematical Sciences, University of Nottingham, Korea, Seoul, Republic of Korea, 2College of Pharmacy, Catholic University Nottingham, United Kingdom. of Korea, Bucheon, Gyeonggi-do, Republic of Korea. Rationale: Understanding the roles of ion currents is crucial to predict the ac- Quetiapine is an atypical antipsychotic drug that is widely used for the treat- tion of pharmaceuticals and mutations in different scenarios, and thereby to ment of various psychotic and mood disorders. It is mainly metabolized by a guide clinical interventions in the heart, brain and other electrophysiological cytochrome P450 system in the liver. Norquetiapine is a major active metabo- systems. Our ability to predict how ion currents contribute to cellular electro- lite in humans with a pharmacological profile that differs distinctly from that of physiology is in turn critically dependent on our characterization of ion channel quetiapine. Many antipsychotic drugs, including quetiapine, cause QT interval kinetics - the voltage-dependent rates of transition between open, closed and prolongation commonly associated with blockade of hERG potassium chan- inactivated channel states. nels. Using the whole-cell patch-clamp technique, we investigated the effects Objective: The hERG potassium channel plays a fundamental role in controlling of norquetiapine on hERG channels that are stably expressed in HEK cells. electrical activity in the heart and many other tissues. There is a well-established Quetiapine and norquetiapine inhibited the hERG tail currents at 50 mV link between hERG mutations, or block by pharmaceuticals, and increased in a concentration-dependent manner with IC50 values of 8.3 and 10.8 mM, arrhythmic risk. We present a new method for rapidly exploring and characterizing

BPJ 8628_8631 294a Monday, February 19, 2018 ion channel kinetics, applying it to the hERG channel as an example, with the aim result in functional channels. The voltage dependence of these channels was of generating a quantitatively predictive representation of the ion current. comparable to control, indicating incorporation of L-ANAP and introduction Methods & Results: We fit a mathematical model to currents evoked by a novel of histidines did not measurably alter channel gating. We then performed PCF 8 second sinusoidal voltage clamp in CHO cells over-expressing hERG1a. The recordings of excised patches with L-ANAP incorporated hERG channels and model is then used to predict over 5 minutes of recordings in the same cell in found that hERG channels are stable in excised patches and that hERG response to further protocols: a series of traditional square step voltage clamps, E50X_S855H channels showed robust L-ANAP and Citrine fluorescence. The and also a novel voltage clamp comprised of a collection of physiologically- above techniques will determine how the eag-CNBHD interaction dynamically relevant action potentials. We demonstrate that we can make predictive cell- regulates conformational changes and ionic current in hERG. specfic models that outperform the use of averaged data from a number of different cells, and thereby examine which changes in gating are responsible 1475-Pos Board B384 for cell-cell variability in current kinetics. Calcium-Calmodulin Regulation of hEAG1 Channel Gating is also Impor- Conclusions: Our technique allows rapid collection of consistent and high qual- tant for the Enhanced Proliferation of hEAG1 Expressing cells ity data, from single cells, and produces more predictive mathematical ion Alina Finch, Raj Patel, Fred W. Muskett, John S. Mitcheson. channel models than traditional approaches. The approach will be widely appli- Molecular and Cell Biology, University of Leicester, Leicester, United Kingdom. cable to other voltage-gated ion currents both in the heart and other electro- þ physiological systems. The human ether-a-go-go 1 (hEAG1) voltage-gated K channel is a rapidly activating, non-inactivating channel that’s profoundly inhibited by elevated 1473-Pos Board B382 intracellular Ca2þ. This Ca2þ-sensitivity is mediated by calmodulin (CaM) Comparison of hERG and zERG Potassium Channel Function and binding to sites on the N (CaM-BD-N) and C-termini (CaM-BD-C). hEAG1 Pharmacology expression is normally restricted to neurons of the brain. However, it’s overex- Christina M. Hull1, Christine E. Genge2, Glen F. Tibbits1, pressed in >70% of human tumors and contributes to tumor progression. Thomas W. Claydon1. Numerous studies demonstrate hEAG1 increases proliferation and confers a 1Biomedical Phsyiology, Simon Fraser University, Burnaby, BC, Canada, transformed phenotype, but the underlying mechanisms remain elusive. In 2Stemcell Technologies, Vancouver, BC, Canada. this study we addressed the question does Ca2þ-CaM dependent regulation There is significant interest in the utility of hERG channel activator compounds of hEAG1 channel conduction also contribute to hEAG1 dependent increases to mitigate loss-of-function. Zebrafish (Daniorerio) hearts have been proposed of cell proliferation? CHO cell proliferation was measured with a 5-bromo- as a whole organ drug screening model. Here, we have investigated the biophys- 2-deoxyuridine incorporation assay 48 hours after transfection (n=4). Expres- ical and pharmacological properties of the zebrafish ortholog of hERG (zERG) to sion of WT hEAG1 increased proliferation by 64% (p<0.0005) compared to validate the zebrafish whole heart model for activator compound screening. We vector-controls. Mutation of CaM-BD-N and CaM-BD-C abolished the effect expressed zERG channels (zKCNH6a) in Xenopus oocytes and characterized of elevated Ca2þ on hEAG1 current, but only CaM-BD-C mutations signifi- channel gating properties using two-electrode voltage clamp. We show that cantly inhibited proliferation (p<0.001). Interactions of the eag domain zKCNH6a channel gating is similar to that of hERG. The voltage-dependence (eagD) and cyclic-nucleotide binding homology domain (cNBHD) are known of activation of zKCNH6a channels was 8.851.1 mV (n=7) compared with to be important for transducing Ca2þ-CaM binding to effects on hEAG1 cur- 29.052.4 mV (n=6) in hERG channels measured from 2 s depolarizing rent. DeagD and DcNBHD hEAG1 currents were insensitive to Ca2þ-CaM voltage steps (P<0.05). Deactivation gating was accelerated in zKCNH6a chan- and these mutants also reduced cell proliferation. However, there was no direct t 5 2þ nels: deact-110mV was 42.7 5.7 ms (n=7) in zKCNH6a compared with correlation with effects of Ca -CaM on conduction and cell proliferation. 147.0511.4 ms (n=5) in hERG (P<0.05). The voltage-dependence of inactiva- Deletion of the PAS-cap (D2-26) on the N-terminus and the cNBHD mutation tion gating in zKCNH6a channels was 62.050.9 mV (n=6) compared with E600R both increased hEAG1 currents in response to elevated Ca2þ (>12 42.151.8 mV (n=6) in hERG (P<0.05). Despite these differences, zKCNH6a fold), but only E600R attenuated proliferation (p<0.001). Overall the results channels produced a characteristic resurgent current in response to an action po- suggest that while eagD and cNBHD interactions regulate current responses tential voltage waveform that was similar to that observed from hERG channels. to Ca2þ-CaM, proliferation is mainly regulated by the C-terminal domains. zKCNH6a channels displayed a sensitivity to blocker compounds that was These are the first findings suggesting that the cNBHD and adjacent CaM bind- similar to hERG channels: dofetilide (5 mM) and terfenadine (5 mM) produced ing site are important for hEAG1-mediated effects on cell proliferation. 8352% (n=5) and 7452% (n=5) block of zKCNH6a channels, respectively, 5 5 1476-Pos Board B385 and 89 4% (n=5) and 82 4% (n=5) block of hERG channels, respectively. D Activator compounds NS-1643 (30 mM), PD-118057 (10 mM) and RPR- Protein Degradation Mechanism of Eag1 K Channel 260243 (10 mM) increased maximal relative conductance 1.5-, 2.4- and 2.3- Chih-Yung Tang1, Po-Hao Hsu1,2, Ya-Ching Fang1,2, Chung-Jiuan Jeng2. fold, respectively, in zKCNH6a (n=6), and 1.6-, 1.4- and 2.3-fold in hERG 1Department of Physiology, College of Medicine, National Taiwan 2 (n=4-6). These data represent, to our knowledge, the first characterization of University, Taipei, Taiwan, Institute of Anatomy and Cell Biology, School zKCNH6a channels and suggest that the zebrafish whole heart model may serve of Medicine, National Yang-Ming University, Taipei, Taiwan. as a valuable tool in the screening of hERG-modifying compounds. Mammalian ether-a`-go-go (Eag; Kv10) protein is neuron-specific, voltage-gated Kþ channel. Mutations in the gene encoding human Eag1 (Kv10.1) Kþ channel 1474-Pos Board B383 have been associated with congenital neurodevelopmental diseases. Little is Dynamics of the EAG Domain and Cyclic Nucleotide-Binding Homology known about the protein degradation mechanism of Eag Kþ channel. By perform- Domain Interaction Probed with a Fluorescent Noncanonical Amino ing yeast two-hybrid screening of a rat brain cDNA library, we identified specific Acid (L-ANAP) in hERG Potassium Channels RING E3 ubiquitin ligase as rat Eag1 binding partners. The E3 ubiquitin ligase and Ashley A. Johnson, Matthew C. Trudeau. rat Eag1 co-exist in the same protein complex in the brain, as well as substantially Physiology, University of Maryland, Baltimore, Baltimore, MD, USA. co-localizing at synaptic regions in neurons. The E3 ubiquitin ligase promotes pro- The voltage-gated potassium channel hERG (KCNH2, Kv11.1) plays a critical tein degradation of both endoplasmic reticulum- and plasma membrane-localized role in cardiac repolarization and is characterized by unusually slow deactivation rat Eag1 to the proteasome and the lysosome, respectively. Interestingly, the E3 kinetics. Slow deactivation in hERG channels is regulated by a direct interaction ubiquitin ligase also contributes to reduced protein expression of a disease- between the N-terminal eag domain and the C-terminal CNBHD. The eag- associated rat Eag1 mutant. Moreover, over-expression of the E3 ubiquitin ligase CNBHD interaction is sensitive to point mutations at the domain interface in significantly reduces the protein level of rat Eag2 (Kv10.2) channel. Overall, these hERG but less is known about potential dynamic rearrangements of the eag observations may shed new light on the endoplasmic reticulum and the peripheral domain relative to the CNBHD and the rest of the channel. Here, we report protein quality control mechanisms of Eag Kþ channels. that hERG channels formed from eag-CFP domains and Deag-Citrine channels showed FRET (Fo¨rster resonance energy transfer) that is sensitive to potassium- 1477-Pos Board B386 induced cell depolarization, consistent with an eag-CNBHD rearrangement. To Biophysical Characterization of a Predicted Shaker Splice Variant with an examine this in more detail, we took advantage of a fluorescent noncanonical Unconventional Initiation Codon amino acid (L-ANAP) and a metal ion bound to a dishistidine motif that can Hans J. Moldenhauer, Scarlett E. Delgado, Nieves Navarro, David Naranjo. be used to monitor small structural rearrangements within ion channels using Facultad de ciencias, Universidad de Valparaiso CINV, Valparaiso, Chile. patch-clamp fluorometry (PCF) and transition metal FRET. We found that amber Voltage gated potassium channels (VGKC) are important entities in the phys- stop codon-containing mutants N33X, E50X, and C64X in the eag domain incor- iology of the excitable cells. These channels are responsible of the action po- porate L-ANAPs as shown by robust currents measured with two-electrode tential frequency, the delay in initiation of action potentials burst and the voltage-clamp. Likewise, the addition of dihistidine mutations in the CNBHD postsynaptic modulation of synaptic potential amplitude. Shaker is the Rosetta (N33X_E788H,L790H, E50X_S855H,H851 and C64X_V796H,I798H) also stone of the VGKC. Cloned from Drosophila melanogaster the Shaker locus is

BPJ 8628_8631 Monday, February 19, 2018 295a a complex transcription unit which counts with 15 splice variant isoforms. region-specific differences of Kv2.1 phosphorylation may play a critical role in Some of those isoforms has been characterized in their biophysical properties, determining its functional properties in different brain regions. tissue localization and physiological role in Drosophila but we still do not have clues about an important number of them. Here we show the biophysical prop- 1480-Pos Board B389 erties of the uncharacterized splice variant, shaker S in Xenopus laevis oocytes. The Kv2.1 Potassium Channel Forms Endoplasmic Reticulum/Plasma From the tetramerization domain towards the C-terminus, this splice variant has Membrane Junctions via Interaction with VAP-A and VAP-B identical the same amino acid sequence to Shaker B, but the presence of the Benjamin T. Johnson1, Ashley Leek1, Michael Kirmiz2, Emily Maverick1, James Trimmer2. exon 22 confer to the 5’-untranslated region a remarkable sequence which gives 1 2 way to an unconventional isoleucine start codon followed by a unique amino Colorado State University, Fort Collins, CO, USA, University of acidic Ball domain sequence. To test the functionality of the unconventional California, Davis, Davis, CA, USA. initiation codon, we created a cDNA encoding Shaker S, synthetize in vitro Kv2.1 exhibits two distinct forms of localization on the neuronal plasma mem- RNA and injected into Xenopus oocytes. This transcript expressed healthy cur- brane: one population is freely diffusive and regulates electrical activity via rents with huge differences in the inactivation properties to all previously voltage-dependent potassium conductance while a second localizes to known Shaker splice variants. Inactivation time constant was 200 ms at micron-sized clusters that contain densely-packed, but non-conducting, chan- zero mV and recovery was two orders of magnitude slower. A Shaker S variant, nels. We and others have demonstrated that these Kv2.1 clusters are associated having an introduced conventional initiation codon to elucidate if the first with endo and exocytosis. We have also established that clusters represent amino acid in the protein was a Met or Ile, showed almost identical kinetics endoplasmic reticulum/plasma membrane (ER/PM) junctions and that Kv2.1 as Shaker S. In summary, the first biophysical characterization of Shaker S sug- is instrumental in forming these membrane contact sites. Clustering and the for- gests a very distinctive potential role in fly neuronal excitability. mation of ER/PM contacts is regulated by phosphorylation within the channel C-terminus, offering cells fast, dynamic control over the physical relationship 1478-Pos Board B387 between cortical ER and the PM. Changes in clustering, and thus ER remodel- The Mono-ADP-Ribosyltransferase ARTD10 Regulates KV1.1 through ing, occurs following repetitive-firing and ischemia. Given this regulation and PKCD the membrane trafficking functions associated with the Kv2.1-induced ER/PM Yuemin Tian1, Patricia Verheugd2, Priyanka Goswami3, Anand Goswami3, junctions it is paramount to understand the mechanisms by which Kv2.1 inter- Daniel Komnig4, Bernhard Luscher€ 2, Stefan Grunder€ 1. acts with the ER membrane. Using both proximity-based biotinylation 1Physiology, Aachen, Germany, 2Biochemistry and Molecular Biology, techniques in transfected HEK cells and chemical crosslinking/affinity purifica- Aachen, Germany, 3Neurophathology, Aachen, Germany, 4Neurology, tion approaches in mouse brain tissue we identified ER VAPs as potential Aachen, Germany. Kv2.1 interactors. VAPs were first identified as VAMP Associated Proteins Voltage-gated Kþ channels are integral transmembrane proteins found in all þ required for neurotransmitter release in Aplysia and are now known to be abun- excitable cells. In Kv1.1 K channels, fast inactivation occurs when the cyto- dant scaffolding proteins involved in membrane contact site formation plasmic N-terminus of an associated Kvb1 subunit enters the channel cavity throughout the ER. Confirmation that VAP-A and VAP-B bind Kv2.1 via a and blocks the pore of the Kva1.1 subunit. Modulation of Kva1.1 by Kvb1 non-canonical FFAT motif within the channel C-terminus employed FRAP-, is itself subject to several regulatory mechanisms. For example, phosphoryla- paGFP-, FRET-based assays, colocalization/redistribution analysis, and CD4 tion of Kva1.1 at serine 446 increases peak current amplitude, and increases chimeras. The VAP interactome includes not only membrane trafficking ma- the extent of inactivation. It has been shown that activation of protein kinase chinery but also proteins regulating non-vesicular lipid transport from the ER C (PKC) indirectly leads to serine 446 de-phosphorylation of Kva1.1, and to PM. Thus the Kv2.1-induced VAP concentration at ER/PM contact sites is thereby reduces the extent of inactivation of Kv1.1. predicted to have additional wide ranging effects on neuronal cell biology. ARTD10 is an enzyme mediating mono-ADP-ribosylation of substrate proteins. This involves the transfer of ADP-ribose from NADþ onto substrates. Both Kvb1 1481-Pos Board B390 and PKCd have been identified as potential substrates of ARTD10 through pro- Voltage-Gated Potassium Channel Kv3.1b is Regulated by the Sodium tein microarray analysis. In HeLa cells stably expressing ARTD10, we observed Channel Beta3 Subunit that ARTD10 reduced Kvb1 protein abundance and PKCd kinase activity. Since Jiseon Shim. PKCd indirectly leads to de-phosphorylation of Kva1.1, a larger fraction of Physiology, KyungHee University, Seoul, Republic of Korea. Kva1.1 was phosphorylated in these cells, and patch clamp indicated that Voltage-gated Kþ Kv3.1b channel has a crucial role in regulating the highfre- Kv1.1 current amplitude was indeed increased and inactivation enhanced. These quency firing properties of neurons and is abundantly expressed in the proximal results indicate that Kva1.1 is regulated by ARTD10 in vitro. dendrites, the somatodendritic region, and the axonal nodes of Ranvier. Here To assess the significance of this regulation in neurons, we isolated primary hip- we unexpectedly identified Navb3, an auxiliary subunit of Nav channels, as pocampal neurons, in which Kva1.1 is the predominant voltage gated Kþ chan- an integral part of the Kv3.1b channel complex in rat brain using mass spec- nel. A potent and selective inhibitor of ARTD10, OUL35, decreased peak current trometry. Navb3 reduces the stability of the heterologously expressed amplitude and decreased extent of inactivation of voltage-gated Kþ currents, sug- Kv3.1b proteins. Interestingly, we found the differences of Kv3.1b and gestingthatARTD10regulatedKva1.1 also in hippocampal neurons. At the Navb3 expression between distinct regions of the brain. Kv3.1b protein is high- same time, OUL35 increased excitability of hippocampal neurons. ly expressed in cerebellum and brainstem, whereas Navb3 is expressed in ce- Our work demonstrates that Kv1.1 is indirectly regulated by ARTD10 through rebrum and brain stem. This shows an age-dependent pattern in rat brain. PKC-dependent de-phosphorylation. Our work identifies a completely new Navb3 significantly decreases the current densities of Kv3.1b in a voltage- regulator of Kv1.1 that may have an important role in the regulation of neuronal dependent manner and alters in the voltage dependence of its steady-state inac- excitability. tivation. In conclusion, our data indicate Navb3 as a component of neuronal Kv3.1b channel complexes and a functional modulator for Kv3.1b. 1479-Pos Board B388 Region-Specific Phosphorylation of Voltage-Gated Potassium Channel 1482-Pos Board B391 Kv2.1 in Mammalian Brain The Functional Modulation of Sodium-Activated Potassium (Slack) Chan- Jiyeon Hwang. nels by Phosphatase and Actin Regulator 1 (Phactr1) Physiology, KyungHee University, Seoul, Republic of Korea. Syed R. Ali, Leonard K. Kaczmarek. The voltage-gated potassium channel Kv2.1 is highly phosphorylated in mamma- Pharmacology, Yale University, New Haven, CT, USA. lian brain, and its variable phosphorylation sites modulate the activity-dependent The Slack gene encodes sodium-activated potassium channels that are abun- regulation of the channel functions. Previous our studies reported that the differ- dantly expressed in the central nervous system. Human mutations alter the ential phosphorylation subsets of Kv2.1 affect its physiological properties in function of Slack channels, resulting in epilepsy and intellectual disability. different cell types under basal conditions. Here we found the differences of Most of the disease-causing mutations are located in the extended cytoplasmic Kv2.1 phosphorylation status and sets between distinct regions of the brain. C-terminus of Slack channels and most result in increased Slack current. Pre- Mass spectrometry was used to determine level of phosphorylation at fourteen vious experiment using a yeast two-hybrid system indicate that the C-terminus identified sites on Kv2.1 purified from different regions of brain. Three sites of Slack channels binds a number of cytoplasmic signaling proteins. One of were found only in cerebellum, on the other hands, three sites were identified these is Phactr1, a protein that is believed to target protein phosphatase 1 only in cerebrum. Biochemical analysis of Kv2.1 by phospho-specific antibodies (PP1) to its phosphoprotein substrates. Phactr1 is also known to be an actin- showed different molecular weight shifts of Kv2.1 as well as its distinct phos- binding protein. We have now found by co-immunoprecipitation that all three phorylation states in cerebrum, cerebellum, and brainstem. In the kainite model components, Phactr1, PP1 and actin exist in a complex with Slack channels. We of continuous seizures in rat, we found the site-specific differences of then investigated the role of disease-causing Slack mutations in the modulation Kv2.1 phosphorylation in distinct brain regions. Our findings demonstrate that of the Slack-Phactr1 complex and found that R455 and R1085 modulates the

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Slack-Phactr1 complex formation. In parallel experiments using whole-cell chronic nicotine treatment, total cell lysates displayed a shift in stoichiometry patch clamp recordings of Slack currents, we found that co-expression of towards (a3)2(b4)3 from (a3)3(b4)2. a3b4 nAChRs are highly expressed in Phactr1 produced a suppression of Slack current. Furthermore, phosphorylation the medial habenula and interpeduncular nuclei, which are involved in reward of Slack channels by TPA dissociate phactr1. Our findings are consistent with processing, and possibly nicotine addiction and withdrawal. We studied effects the hypo-thesis that Phactr1 recruitment of PP1 to the channel results in following chronic treatment (at least 24 hours) of 500 nM menthol at a3b4 dephosphorylation and loss of current amplitude. Our findings suggest that tar- nAChR using Fo¨rster resonance energy transfer (FRET), total internal reflec- geting Slack-Phactr1 interactions may be helpful in developing novel therapies tion fluorescence microscopy (TIRFM), and whole-cell patch-clamp electro- for brain disorders associated with the malfunction of Slack channels. physiology of Neuro-2a cells transiently expressing fluorescently labeled subunits. FRET experiments indicated a shift in stoichiometry toward (a3) 1483-Pos Board B392 3(b4)2 from (a3)2(b4)3. TIRFM experiments revealed a3 subunit up- Development of an Automated Electrophysiology Assay for Kv1.3 using regulation in the endoplasmic reticulum and a3b4 nAChR reduction in the Nanion Syncropatch 384PE plasma membrane. Our FRET experiments, however, include contributions Kaylee Choi, Joe McGivern. from intracellular a3b4 nAChR stoichiometry. In contrast, patch clamp exper- Amgen, South San Francisco, CA, USA. iments measuring Zn2þ inhibition of acetylcholine-evoked currents indicate Recently, a couple of giga-seal automated patch clamp (APC) platforms in a exclusively the functional cell surface a3b4 nAChR stoichiometry. Neither standard 384-well plate format have been introduced. These APC instruments chronic menthol, chronic nicotine, nor combined chronic menthol and nicotine can deliver higher throughput measurements with high-quality data for ion detectably alters Zn2þ inhibition of acetylcholine, showing that neither alters channel drug discovery. In this study, we developed an automated electrophys- functional plasma membrane a3b4 nAChR stoichiometry. Furthermore, iology assay for the voltage-gated potassium channel, Kv1.3 as a test case on chronic menthol treatment shifts by < 1.5-fold the EC50 of acetylcholine at the Nanion SyncroPatch 384PE, one of the latest APC platforms. We achieved a3b4 nAChRs. These findings are consistent with our fluorescence-based ex- > a high cell-catch rate (98% wells/plate, defined by membrane resistance 16 periments showing a reduction in endoplasmic reticulum exit sites following U M /cell) by screening plate types (number of holes and resistance), and ob- chronic menthol, which consequently reduces a3b4 nAChR delivery to the tained a stable seal (87% wells/plate, defined by initial/final seal resistance plasma membrane. Therefore, despite their intracellular effects, neither > U 500 M /cell) by optimizing recording solutions. We generated data at an menthol nor nicotine influences cell surface a3b4 nAChR stoichiometry. Sup- > overall success rate (defined by initial peak current 300 pA/cell, initial/final port: DA037743, DA036061, DA40047. seal resistance >500 MU/cell, and current stability <57% change/min) of 79% on average. Using the same platform, we validated a dose-response assay 1486-Pos Board B395 using clotrimazole, a potent Kv1.3 inhibitor. The assay was robust with Z’ fac- Ancestral Reconstruction Approach to Acetylcholine Receptor Structure tor 0.52 and the success rate 75%. Altogether, our results demonstrated that the and Function SyncroPatch represents a reliable platform for voltage-gated potassium channel Corrie J.B. daCosta, Jethro E. Prinston, Johnathon R. Emlaw, assays. Mathieu F. Dextraze, Christian J.G. Tessier, F. Javier Perez-Areales, Melissa S. McNulty. Posters: Ligand-gated Channels II Chemistry and Biomol. Sci., University of Ottawa, Ottawa, ON, Canada. Acetylcholine receptors (AChRs) are members of a superfamily of proteins 1484-Pos Board B393 called pentameric ligand-gated ion channels, which are found in almost all Agonist-Specific Pharmacological Effects of CMPI and NS9283 at (a4) forms of life, and thus have a rich evolutionary history. Muscle-type AChRs 3(b2)2 Neuronal Nicotinic. Acetylcholine Receptors are heteropentameric complexes assembled from four related subunits (a, b, Kemburli Munoz, Farah Deba, Ayman K. Hamouda. d, and ε). Here we reconstruct the amino acid sequence of a b-subunit ancestor Pharmaceutical Sciences, Texas A&M Health Science Center, Kingsville, shared by humans and cartilaginous fishes (i.e. Torpedo). Then, by resurrecting TX, USA. this ancestral b subunit and co-expressing it with human a-, d-, and ε-subunits, Positive allosteric modulators (PAMs) of neuronal nicotinic acetylcholine re- we show that despite 132 substitutions, the ancestral subunit is capable of form- ceptors (nAChRs) have potential clinical applications in neuropsychiatric dis- ing human/ancestral hybrid AChRs. Whole cell currents demonstrate that the orders where specific subsets of nAChR are involved. They potentiate agonist- agonist acetylcholine has reduced potency for hybrid receptors, while single induced responses via binding at site(s) distinct from the agonist binding sites channel recordings reveal that hybrid receptors display reduced conductance and may provide the required specificity by binding to unique site(s) present and open probability. Our results outline a promising strategy for studies of only in a nAChR subpopulation. Previous studies have established CMPI and AChR evolution aimed at identifying the amino acid origins of AChR structure NS9283 as potent nAChR PAMs with high selectivity for the a4-containing and function. nAChRs. Furthermore, CMPI and NS9283 have been shown to potentiate re- sponses of (a4)3(b2)2 but not (a4)2(b2)3 nAChR isoforms. Mutational and 1487-Pos Board B396 computational analyses established that NS9283 and CMPI potentiate (a4) Allosteric Activation of Unliganded AChRs: Multiple, Bumpy Pathways 3(b2)2 nAChR by binding to pockets at the ‘‘non-canonical’’ a4:a4 subunit Tapan K. Nayak, Anthony Auerbach. interface, which is present only in the (a4)3(b2)2 nAChR. NS9283 and Physiology and Biophysics, SUNY, University at Buffalo, NY, Buffalo, NY, CMPI potentiation was characterized using ACh, a full agonist of (a4)3(b2)2 USA. nAChR that binds with higher affinity at a4:b2 agonist binding sites and Acetylcholine receptors (AChRs) are allosteric proteins that isomerize (‘gates’) with lower affinity at the a4:a4 subunit interface. Because ACh binds at the between resting and active conformations. We used single-channel electro- a4:a4 subunit interface to a site that completely or partially overlaps physiology to study gating of mouse adult-type endplate AChRs in the absence NS9283 and CMPI binding sites, respectively, here we extend these studies of agonists (free energy change, DG ). Many different point mutations in to characterize the pharmacology of NS9283 and CMPI potentiation of (a4) 0 AChRs change DG . For pairs of mutations, the net change in DG was about 3(b2)2 nAChR using agonists that bind at a4:b2 but not a4:a4 agonist binding 0 0 the same as the sum of the individual changes, indicating that mutations were site. Our preliminary results indicate that NS9283 and CMPI effect on the po- independent and had mostly local effects. By correlating changes in DG with tency and/or efficacy of agonist depends on the agonist’s ability to bind at 0 side chain partition coefficients, we could infer the hydrophobicity and volume a4:a4 subunit interface. of each residue’s local environment. The kinetics of unliganded gating is com- 1485-Pos Board B394 plex, but was explained by schemes having 3-shut and 2-open states that all D Effects of Chronic Menthol at Alpha3Beta4 (a3b4)-Containing Nicotinic produced the same G0. The longest-lived open component was eliminated Acetylcholine Receptors by some mutations (aromatic residues at the agonist-binding site and in M1) Selvan Bavan1, Suparna Patowary1, Charlene H. Kim1, and a-conotoxin (a specific blocker of the a-d binding site), without affecting D Brandon J. Henderson2, Henry A. Lester1. G0. This suggests that long-lived unliganded openings are outside of the main 1California Institute of Technology, Pasadena, CA, USA, 2Marshall activation sequence. In diliganded gating, in the a subunit there is a coarse- University, Huntington, WV, USA. grained gradient in gating f values (a conformational ‘wave’) that is less Some heteropentameric nicotinic acetylcholine receptors (nAChRs) are up- distinct in unliganded gating. Furthermore, without agonists the relative posi- regulated by chronic nicotine. Menthol is present in 30% of tobacco ciga- tion of the gating transition state was more malleable compared to with agonists f D rettes sold in the United States. Compared with non-mentholated cigarettes, (larger Hammond shifts). The degree of shift (d /d G0) was a function of the menthol-containing cigarettes are associated with reduced smoking cessation. residue f-value, and was largest at f0.5. We hypothesize that without ago- Chronic menthol favors the lower sensitivity (a4)3(b2)2, whereas chronic nists, there are multiple activation pathways that traverse a rugged potential nicotine favors the higher sensitivity (a4)2(b2)3 stoichiometry. Following energy surface.

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1488-Pos Board B397 tions of a, b, g, d, and ε subunits, resulting in distinct receptors with diverse Alternative Binding Mode of Full and Partial Agonists in a Pentameric ion selectivity, physiological function and pharmacology. Drugs targeting Ligand-Gated Ion Channel Stabilises Loop C in an Open Conformation nAChRs are commonly used in the treatment of neurological disorders and Marc A. Damgen€ 1, Timo Greiner2, Remigijus Lape2, Lucia G. Sivilotti2, as muscle relaxants. Using azobenzene as basic molecular skeleton, we have Philip C. Biggin1. designed a series of novel drugs that differentially effect nAChR activity as a 1Department of Biochemistry, University of Oxford, Oxford, United function of their isomeric state; isomerization was reversibly induced by light Kingdom, 2Department of Neuroscience, Physiology and Pharmacology, irradiation. Two compounds (CH168 and CH376) behaved as potent ligands University College London, London, United Kingdom. with a clear selectivity (up to 60-fold) for the muscular nAChRs (Ki = 35-42 Pentameric ligand-gated ion channels are crucial for fast synaptic transmission and nM), compared to neuronal a7 and a4b2 nAChRs. The trans-isomer of both important drug targets. Upon agonist binding the transmembrane pore opens, al- compounds displayed higher activity against nAChR. CH168 behaved as an in- lowing ions to flow into the cell. While agonists with varying ability to open the hibitor of muscular nAChR expressed in Xenopus oocytes. At 10 mM, CH168 ion channel are known, an explanation of partial agonism at atomistic resolution completely inhibited the activation by acetylcholine (ACh, 50 mM). Cis- remains an unsolved problem. An understanding of this could be exploited for isomerization of CH168 by irradiation with UV light (LED, peak at 340 nm) drug design, as the ideal therapeutic drug should elicit a fine-tuned ion flow that reverted about 20% of inhibition at the same concentration. Our observations is, depending on the clinical situation, at a specific position of the spectrum be- suggest that the cis-isomer of CH168 remained bound to nAChR after isomer- tween a full agonist and a silent antagonist. We have performed molecular dy- ization, but it was less effective in inhibiting activation by ACh than the trans- namics simulations of the glycine receptor with full and partial agonists in the isomer. The trans-isomer of CH376 behaved as an agonist, activating nAChR at orthosteric binding site. The observed principal binding mode of glycine is in concentrations of 500 nM and below. In contrast, the cis-isomer of CH376 was excellent agreement with a recent crystal structure where density of the agonist unable to render the channel open. Intriguingly, CH168 and CH376 are struc- with a stable water molecule in the binding pocket is discernible. Here, we report turally similar, differing in a single carbon atom in their structure. Thus, we analternativebinding mode, located slightly further awayfromthe transmembrane have identified a key element in the structure of these compounds that will domain, at the subunit interface that stabilises loop C in an open conformation. The be exploited to develop analogues that we can use as either inhibitors or ago- stability of this alternative pose varies for full and partial agonists, but the results nists of muscular nAChR. Funding: MINECO (SAF2015-64948-C2-1-R), are consistent with the view that the conformation of loop C is associated with the CSIC (PIE-201580E109), Nutting Foundation Grant (CAVG). efficiency of different agonists. The differences between the principal and this alternative binding mode for a range of different agonists will be discussed in 1491-Pos Board B400 the context of free energy calculations. The results suggest the dynamics of Endocytosis Participation in Nicotine-Induced Upregulation of Alpha 7 ligand-binding in these receptors is even more complex than originally supposed. (A7) Nicotinic Acetylcholine Receptors (nAChRs) in Xenopus Oocytes Joseph Farley, Jayharsh Panchal, Kristi DeBoeuf, Mohammad Islam, 1489-Pos Board B398 Jonathan Blake Anderson, Vasu Sheel, Josh Hoffer. Pharmacological Properties and Evaluations of a Novel Positive Allosteric Neuroscience, Indiana University, Bloomington, IN, USA. Modulator of a7 nAChR for Attenuation of Schizophrenia-Like Behavior The a7 nAChR is widely expressed throughout peripheral and central nervous in Mice systems and is implicated in many neuropathologies/syndromes (e.g., Alz- Lilan Sun1, Taoyi Yang1, Wenxuan Jiao2, Qi Sun3, Kewei Wang1. heimer’s Disease, inflammation, chronic pain, stroke, cancer, schizophrenia) 1Qingdao University, Qingdao, China, 2Peking University, Peking, China, and nicotine addiction. Functional/numerical upregulation of a7Rsbymany 3Peking University, Beijing, China. compounds has been reported and several different mechanisms have been sug- The alpha-7 neuronal nicotinic acetylcholine receptor (a7 nAChR), consisting of gested to participate. Prolonged nicotine exposure may upregulate a7(similarto homomeric a7 subunits, is a ligand-gated Ca2þ-permeable ion channel critical for other nAChRs) and contribute to nicotine addiction. We’ve previously shown cognition, sensory processing, attention, and working memory. Dysfunctional a7 2-fold functional and numerical upregulation of a7 Rs in oocytes following nAChR is implicated in cognition and neuropsychiatric disorders. Therefore, 12 hr of 100 mM nicotine and extensive washout. Nic-upregulation was depen- þ enhancement of a7 nAChR function is considered to be therapeutic strategy dent upon intracellular Ca2 (being blocked by BAPTA-AM), but independent þ þ aimed at ameliorating cognitive deficits of neuropsychiatric disorders such as of Ca2 - influx from extracellular media. We further identified several Ca2 - Alzheimer’s disease (AD) and schizophrenia. We recently synthesized a novel signaling pathways involved in nic-upregulation. Because a7 R (like another series of 6-(2-Chloro-6-methylphenyl)-2-((3-fluoro-4-methylphenyl)amino)thia- Cys-loop LGIC, Gly R1) contains a canonical G-protein binding cluster zolo[4,5-d]pyrimidin-7(6H)-ones as positive allosteric modulators (PAM) of a7 (GPBC) in the M3-M4 intracellular loop, whose mutation abolishes GPCR- þ nAChR. In this study we evaluated the effect of a representative compound Ca2 -signaling of a7 Rs (King et al., 2015), we tested the involvement of this JWX-A0108 on activation of a7 and auditory gating or cognitive deficits in cluster in nic-upregulation. Mutation of GPBC (RMKR to AAAA), preventing þ mice using a combination of assays including two-electrode voltage clamp interaction of Gaq/ Gbg with GPBC and disrupting Gaq-PLC-IP3-Ca2 release, (TEVC) recordings of Xenopus oocytes expressing human a7 nAChR, brain slice abolished nic-upregulation of a7 Rs. We also found that the calcineurin inhibitor recordings, pre-pulse inhibition (PPI) for startle response and Y-maze sponta- cyclosporine A (CsA) produced 2X-upregulation of wt a7 Rs, similar to nicotine, neous alternation test. Compound JWX-A0108 exhibits as type I PAM that spe- and occluded nic-upregulation. A dynamin inhibitor (Dynasore), produced cifically activates a7 current in the presence of 100 mM ACh in dose-dependent 2X upregulation of both wt and mutant a7 Rs and occluded nic- manner with EC50 at 5.7 5 0.7 mM and 6-fold increase of the current. Applica- upregulation. Brefeldin A (inhibitor of protein transport from ER to Golgi) failed tion of JWX-A0108 (1-10 mg/kg, i.p.) dose-dependently reversed the auditory to affect nic-upregulation. Our results suggest that nic-upregulation of a7arises þ gating deficit induced by NMDA antagonist MK-801 in prepulse inhibition assay. from sustained GPCR-Ca2 -signaling by a7 Rs, inhibiting basal/constitutive þ In Y-maze, JWX-A0108 (0.03-0.3 mg/kg, i.p.) significantly increased the time calcineurin-dynamin mediated endocytosis of a7 Rs. Although GPCR-Ca2 spent in novel arm from 45% to 63% in mice treated with MK-801. We also eval- signaling is blocked in mutant a7 Rs channels, the downstream effects of calci- uated the effect of JWX-A0108 on locomotor activity, and fount that JWX- neurin- and dynamin-inhibition are still produced pharmacologically in these A0108 (0.03 mg/kg, i.p.) significantly reduced MK-801-induced hyperactivity. mutant channels, reproducing the 2X nic-upregulation. The effects of JWX-A0108 on selectivity, pERK signaling and synaptic transmis- sions are currently being investigated. 1492-Pos Board B401 Agonist Binding to Endplate AChRs: MWC Is A-Ok 1490-Pos Board B399 Anthony Auerbach, Tapan K. Nayak. Functional Characterization of Novel Photo-Switchable Neuromuscular Physiol/Biophys, SUNY Buffalo, Buffalo, NY, USA. Blockers Agonists turn on receptors because they bind with a higher affinity to active Clara Herrera-Arozamena1, Olaia Martı´-Marı´1, Martı´n Estrada1, versus resting conformations of their target sites. Receptor activation can occur Mario Dela Fuente-Revenga2, Carlos A. Villalba-Galea3, by either of two pathways that connect to form a cycle (sometimes called Marı´a Isabel Rodrı´guez-Franco1. MWC): 1) an agonist binds to a resting receptor that then activates, or 2) a 1Medicinal Chemistry Institute, Spanish National Research Council, Madrid, resting receptor first activates and then binds an agonist. We used mutations Spain, 2Department of Physiology and Biophysics, Virginia Commonwealth to construct mouse endplate acetylcholine receptors (AChRs) having only 1 University, Richmond, VA, USA, 3Department of Physiology and functional target site, and single-channel electrophysiology to measure inde- Pharmacology, Thomas J. Long School of Pharmacy & Health Sciences, pendently equilibrium dissociation constants for 4 different agonists to both University of the Pacific, Stockton, CA, USA. resting and active conformations of each site. For all agonists and sites, the total Nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels found free energy change in each pathway was the same, confirming an activation cy- in the nervous system and skeletal muscles. They are composed by combina- cle without external energy. Other results show that i) there is no cooperativity

BPJ 8628_8631 298a Monday, February 19, 2018 between sites, ii) agonist association is slower than diffusion in resting recep- mutations is the major cause hyperekplexia, a rare neuromotor disorder tors but nearly diffusional in active receptors, iii) resting affinity is determined that is characterized by exaggerated startle reflexes. The V280M mutation mainly by agonist association, iv) active affinity is determined mainly by in the a1 subunit causes heperekplexia but, surprisingly, results in a gain agonist dissociation, and v) at each site and for all agonists, receptor activation of function of the receptor. The mutant receptor shows spontaneous activa- approximately doubles the agonist binding free energy. Agonist binding to tion and prolonged deactivation. It has been suggested that V280M might AChRs is a 3-step process that involves diffusion (‘dock’), a local conforma- change GlyRs oligomerization and the formation of clusters. We tested tional change that is modulated by receptor activation (‘catch’), and a local this idea by using Number and Brightness (N&B) analysis to determine gating rearrangement that switches the binding site from low-to-high affinity GlyR oligomerization. A SNAP tag was fused to the N-terminal of wt (‘hold’). The ‘catch’ (that contributes to affinity) and ‘hold’ (that contributes and V280M human a1 GlyR. Receptors were expressed in HEK-293 cells. to efficacy) structural changes are obligatorily-linked parts of a single, global Cells were incubated with the Atto-488 fluorescent probe that covalently la- allosteric transition. We hypothesize that ‘catch’ ensures proper signal recogni- bels SNAP tag-containing receptors. N&B analysis measures the variance tion in complex chemical environments, and that binding-site compaction is a in fluorescence as receptors diffuse on the surface of the basolateral cell determinant of both resting and active binding energies. membrane. Larger variance corresponds to larger diffusing entities - multi- mers of GlyRs. Our tentative stoichiometry assignment is monomer, dimer, 1493-Pos Board B402 4-mer and 8-mer. Using outside-out patch clamp recordings of macroscopic Dissecting Kinetic Differences in Ancestral/Extant Hybrid Acetylcholine currents, we found that the SNAP-tagged wt and V280M GlyRs had similar Receptors glycine-sensitivity and kinetics compared to the corresponding untagged re- Christian Tessier, Corrie daCosta. ceptors. Using fluorescence measurements, we found that SNAP-tagged wt University of Ottawa, University of Ottawa, Ottawa, ON, Canada. receptors tended to form dimers and 4-mers as the dominant species. In the Muscle type nicotinic acetylcholine receptors (nAChRs) are heteropentameric presence of 3 mM glycine ,monomers were predominant. Addition of 1 mM a b d ligand-gated ion channels composed of four homologous subunits (2 ,1 ,1 , strychnine antagonized the effect of glycine. SNAP-tagged V280M recep- e b and 1 ). Previously, we resurrected an ancestral -subunit shared between hu- tors exhibit a higher level of oligomerization than wt receptors. In prelim- b mans and cartilaginous fishes. Despite 132 substitutions, this ancestral -sub- inary data from 12 cells, the 8-mer species was dominant. Further unit was able to replace its human counterpart and form hybrid ancestral/ experiments are needed to determine whether changes in receptor clustering extant nAChRs. Macroscopic currents showed that hybrid nAChRs containing underlie the hyperekplexia phenotype of V280M a1GlyRs. the ancestral b-subunit displayed reduced acetylcholine sensitivity. To deter- mine the basis for reduced agonist sensitivity, we have acquired single channel 1496-Pos Board B405 recordings spanning a full acetylcholine concentration range. The sequence of Effects of 5-HT3A Intracellular Domain Modifications on Oligomerization open and closed dwell times were globally fit to a modified del Castillo-Katz Antonia Stuebler1, Michaela Jansen2. mechanism to determine the underlying rates of acetylcholine binding and 1Pharmacology and Neuroscience, Center for Membrane Protein Research, nAChR apparent gating. Texas Tech University Health Science Center, Lubbock, TX, USA, 2Cell 1494-Pos Board B403 Physiology and Molecular Biophysics, Center for Membrane Protein Allosteric Modulation of the Pentameric Ligand-Gated Ion Channel ELIC Research, Texas Tech University Health Science Center, Lubbock, TX, USA. by Functionally Active Nanobodies The 5-Hydroxytryptamine type 3 (5-HT3) receptor is a cation-selective pen- Marijke Brams1, Hannelore De Peuter1, Radovan Spurny2, Els Pardon3, tameric ligand-gated ion channel (pLGIC) that contributes to fast synaptic Daniel Bertrand4, Jan Steyaert3, Cedric Govaerts5, Chris Ulens1. neurotransmission in the central and peripheral nervous system. Targeting 1KU Leuven, Leuven, Belgium, 2FEI Thermo Fisher Scientific, Brno, Czech these receptors could be a potential treatment for anxiety, depression, and Republic, 3VUB-VIB, Brussels, Belgium, 4HiQScreen, Geneva, Switzerland, an alternative to antipsychotics drugs. Two of the three eukaryotic pentameric 5ULB, Brussels, Belgium. domains, the extracellular and transmembrane domain of the 5-HT3A,have Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are a been studied and fully crystallized. However, the intracellular domain class of ion channels involved in fast synaptic transmission in the central and (ICD), which is the most divergent domain of the pLGIC superfamily, has peripheral nervous systems. Members of this family include the nicotinic only been partially resolved, and its function remains mainly unknown. The ICD of homomeric 5-HT3A has been shown to form a pentameric quater- acetylcholine receptors, serotonin type-3 receptors, GABAA/C (gamma-amino- butyric acid) receptors and glycine receptors. These receptors are the target for nary structure in the absence of the two other domains. When mutations are a wide variety of therapeutics, including benzodiazepines such as diazepam introduced into this domain, alterations in receptor conductance have been observed. Based on this knowledge, we investigated if such alterations would (Valium), which act as positive allosteric modulators (PAMs) of the GABAA receptor and are prescribed as a sedative, anxiolytic or anti-epileptic. In this also alter the oligomeric state of the receptor, and ultimately, resolve its struc- study, we employed the prokaryote ligand-gated ion channel ELIC as a model ture. Chimeras of the ICD were created and a modified maltose-binding pro- to understand the structural mechanism of allosteric modulation. We raised tein (MBP) was attached to its N-terminus by a short alanine linker, expressed nanobodies, which are antibody fragments derived from camelids and have in Escherichia coli, and the resulting protein construct was purified using the potential to trap conformationally transient states, and used them as crystal- amylose column purification and size-exclusion chromatography (SEC). To lization chaperones for ELIC. Functional characterization of ELIC nanobodies further analyze the oligomeric state of the ICD, we employed SEC in line using electrophysiological techniques demonstrated that these nanobodies are with Multi-Angle Light Scattering (SEC-MALS) to measure the absolute mo- active as channel modulators. We demonstrate that nanobodies can either lecular weight. Additionally, the constructs are used for spectroscopic studies inhibit or potentiate agonist-evoked channel responses, suggesting that nano- of their stability and secondary structure content (DSC, CD), as well as for bodies can act as negative (NAMs) or positive allosteric modulators (PAMs), high-throughput crystallization trials. respectively. The X-ray crystal structure of ELIC in complex with a nanobody reveals that a nanobody binds at an allosteric binding site and near to a binding 1497-Pos Board B406 site previously known to mediate potentiation of ELIC by the benzodiazepine Interaction Sites of Serotonin Type 3A Intracellular Domain (5-HT3A- flurazepam. Given the structural conservation of allosteric binding sites be- ICD) with Chaperon Protein RIC-3 1 2 tween ELIC and human receptors our study reveals common mechanisms for Elham Pirayesh , Michaela Jansen . 1Cell Physiology and Molecular Biophysics, Texas Tech University HSC, allosteric modulation in this family of channels. Our expanded knowledge of 2 the structural determinants of allosteric modulation opens opportunities for Lubbock, TX, USA, Cell Physiology and Molecular Biophysics, Texas Tech the development of new therapeutics targeting pLGICs in ion channel-related University HSC, Lubbock, TX, USA. disorders. The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective member of the pentameric ligand-gated ion channel (pLGIC) superfamily. 1495-Pos Board B404 Members of this superfamily assemble from five subunits, each of which con- Glycine Receptor Oligomerization Characterized by Number and Bright- sists of three domains, extracellular (ECD), transmembrane (TMD), and intra- ness Analysis cellular domain (ICD). Recently, we have shown that 5-HT3A-ICD fused to Mohammed A. Shanawaz1, Sheena Mago2, Allen Stekol2, maltose binding protein (MBP) directly interacts with the chaperone protein Mario J. Rebecchi1,2, James P. Dilger1,2. resistance to inhibitors of choline esterase (RIC-3). Additionally, we have 1 Physiology and Biophysics, Stony Brook University, Stony Brook, NY, also demonstrated that 5-HT3A-ICD is required and sufficient for the interaction 2 USA, Anesthesiology, Stony Brook University, Stony Brook, NY, USA. between 5-HT3A and RIC-3. To elucidate the molecular determinants of this Glycine receptors (GlyR) are ligand-gated chloride channels that mediate interaction we developed different MBP-fused 5-HT3A-ICD constructs by dele- synaptic inhibition in the CNS. A loss of GlyR function by single site tion of large portions of its amino acid sequence. We have expressed two

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mutants (MBP-5-HT3A-ICD-DA and MBP-5-HT3A-ICD-DB) in Escherichia 1500-Pos Board B409 coli and purified them to homogeneity. Additionally, we have also purified Probing Conformational Motions Underlying Anesthetic Drug Actions in a RIC-3 to be utilized in protein-protein interaction experiments. Using RIC-3 af- Ligand-Gated Ion Channel 1 2 3 finity pull down, the interaction of MBP-5HT3A-ICD constructs and RIC-3 is Sritejasvinthi Karimikonda , Varun Tiwari , Candice S. Klug , 1 investigated. Furthermore, we co-expressed 5-HT3A and RIC-3 in Xenopus oo- Cynthia Czajkowski . cytes to study the interaction in-vivo by two electrode voltage clamp (TEVC) 1Neuroscience, University of Wisconsin - Madison, Madison, WI, USA, 2 recordings. Full-length 5-HT3A-mediated currents are significantly reduced Biophysics Training Program, University of Wisconsin - Madison, Madison, 3 when RIC-3 is co-expressed. Here, we study if individual MBP-5-HT3A-ICD WI, USA, Biophysics Training Program, Medical College of Wisconsin - constructs rescue 5-HT3A-mediated currents in the presence of RIC-3. Our re- Milwaukee, Milwaukee, WI, USA. sults support the hypothesis that interaction of 5-HT3A-ICD and RIC-3 is medi- General anesthetics and ethanol exert their functional effects on pentameric ated by specific segments of the 5-HT3A-ICD as opposed to the complete ligand-gated ion channels (pLGICs) by binding to distinct allosteric sites domain. Further studies are directed toward deciphering the molecular determi- that are far from the agonist binding sites. While recent high-resolution struc- nants of RIC-3 protein-protein interaction. tures of prokaryotic and eukaryotic pLGICs, solved in the presence of these drugs have illuminated their binding sites, the structural mechanisms under- 1498-Pos Board B407 lying how these drugs enhance or inhibit pLGIC function are largely un- Mapping Two Neursteroid Modulatory Sites in GLIC: A Prototypic Pen- known. Recently, a single residue variant in the bacterial pLGIC, GLIC, tameric Ligand Gated Ion Channel was identified that is potentiated by ethanol (PNAS 108:12149-12154, Wayland W.L. Cheng, Zi-Wei Chen, Bracamontes R. John, 2011). Taking advantage of this GLIC ethanol-sensitive mutant, we are using Melissa M. Budelier, Kathiresan Krishnan, Daniel J. Shin, Cunde Wang, site directed spin labeling electron paramagnetic resonance (SDSL EPR) Xin Jiang, Douglas F. Covey, Gustav Akk, Alex S. Evers. spectroscopy to study motions underlying ethanol potentiation of GLIC cur- Anesthesiology, Washington University in St. Louis, Saint Louis, MO, USA. rents. Previous studies have shown that agonist-mediated channel opening is Neurosteroids are endogenous sterols that modulate pentameric ligand gated associated with an inward tilting of the extracellular binding domain (ECD). ion channels (pLGICs), and can be anesthetics, analgesics, and anti- Here, we examined if, and how, the ECD moves in response to ethanol bind- epileptics. The complex effects of neurosteroids on pLGICs suggest the pres- ing in the transmembrane channel domain. Individual cysteine residues were ence of multiple binding sites in these receptors. However, the structural deter- introduced in the ECD of an ethanol-sensitive GLIC construct (F14’A) at the minants of these sites remain unknown. We used a novel series of neurosteroid top of b-strands (b1, 2, 5, 6, 8), labeled with the spin probe MTSL and photolabeling reagents combined with top-down and middle-down mass spec- ethanol-induced motions were measured using double electron-electron reso- trometry to determine the stoichiometry, sites and orientation of neurosteroid nance (DEER) spectroscopy and compared to agonist (proton)-induced gating binding in Gloeobacter ligand gated ion channel (GLIC), a prototypic pLGIC. motions. DEER distance distributions for spin labels attached to N19C (b1), Neurosteroids photolabel two sites per GLIC subunit, both within the trans- S93C (b5) and Q101C (b6) revealed that the attached probes moved closer membrane domain; one site is an intrasubunit site and the other is located in together at pH 3.0, indicating that the inner b-sheet of GLIC ECD tilts in- the interface between subunits. By using complementary photo-reactive groups wards upon agonist activation. Interestingly, DEER distance distributions positioned throughout the neurosteroid backbone, we precisely map the orien- for N19 (b1) and S93 (b5) showed no significant changes in the presence tation of neurosteroid binding within each site. Docking simulations are consis- of ethanol, indicating that structural mechanisms underlying ethanol allo- tent with both sites being neurosteroid binding sites. Mutations introduced to steric drug modulation of pLGIC function are different than agonist- either site alter neurosteroid modulation of GLIC channel activity demon- mediated motions that lead to channel activation. strating the functional role of both sites. These results provide a detailed molec- ular framework for understanding multi-site allosteric modulation of the family 1501-Pos Board B410 of pLGICs by neurosteroids. Expansion of a Transmembrane Cavity Facilitates Anesthetic Potentiation of a Pentameric Ligand Gated Ion Channel 1499-Pos Board B408 Marie Lycksell1, Stephanie A. Heusser1, Rebecca J. Howard1, 1,2 Structures and Functions of ELIC-GABAAR Chimeras Erik Lindahl . Qiang Chen1, Marta M. Wells1, Palaniappa Arjunan1, Tommy S. Tillman1, 1Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden, Devin Adell2, Aina E. Cohen3, Yan Xu1, Pei Tang1. 2Theoretical Physics, KTH Royal Institute of Technology, Stockholm, 1University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, Sweden. 2Appalachian State University, Boone, NC, USA, 3Stanford Synchrotron Ligand-gated ion channels are important molecular targets of general anes- Radiation Lightsource, Menlo Park, CA, USA. thetics,someofwhichhavebeeninusesincethemid19thcentury.However, g-Aminobutyric acid type A receptors (GABAARs) are the major inhibitory the molecular mechanism of these drug-receptor interactions is poorly under- neurotransmitter-gated receptors in the central nervous system. General an- stood, creating barriers to the development of new anesthetics. Biochemical esthetics target these pentameric ligand-gated ion channels (pLGICs), pri- and electrophysiological studies have shown that general anesthetics bind to marily to the transmembrane domains (TMDs). High-resolution structures small pockets in Cys-loop receptors, also known as pentameric ligand-gated of GABAARs can facilitate our understanding of the mechanism of action ion channels. In addition, recent X-ray structures of the Gloeobacter viola- of anesthetics and the discovery of new anesthetics, but thus far only one ceus ligand-gated ion channel show differential binding of the common anes- structure of a desensitized homopentameric b3GABAAR is available. To thetic propofol in the presence of sensitizing mutations. In this work, we used speed up structure determination, we took advantage of the more easily molecular dynamics simulations to investigate how anesthetic binding relates crystallized prokaryotic homologs, such as ELIC, to construct chimeras to receptor structure. We found a correlation between anesthetic occupancy that have the extracellular domain (ECD) of ELIC fused to the TMD of and binding site expansion. This correlation was removed by a mutation a1orb3GABAAR. The constructed heteromeric ELIC-a1b3GABAAR that increased functional sensitivity to anesthetic potentiation. Contraction and homomeric ELIC-b3GABAAR chimeras are activated by the agonists of the apo binding site from the expanded conformation was associated GABA or propylamine. Resembling wild-type (WT) GABAARs, ELIC- with a pinching motion of two transmembrane helices, this motion being a1b3GABAAR and ELIC-b3GABAAR are potentiated or even activated less pronounced in the mutant than in the wild-type channel. The sensitizing at a higher concentration by general anesthetics. WT a1GABAAR does mutation also influenced flexibility of the mutated amino acid side chain and not form a functional channel; however, the homomeric ELIC-a1GABAAR the bound anesthetic. These novel dynamic observations enabled us to refine chimera can be activated by GABA or propylamine, but with a smaller cur- an allosteric mechanism for bimodal anesthetic modulation in the Cys-loop rent than that observed in the ELIC-a1b3GABAAR and ELIC-b3GABAAR receptor family, and provided testable hypotheses for further structure- chimeras. Furthermore, we expressed, purified and crystallized the chimeras function experiments. in homomeric and heteromeric forms. The crystal structure of apo ELIC- a1GABAARshowsasimilarporeprofiletothatofb3GABAAR, but with 1502-Pos Board B411 0 a smaller pore radius except at the 2 position. The replacement of the The Pros of nACh and 5-HT3 Receptors TMD may have affected the ECD structure, where the loop F, loop D, Sarah C. Lummis1, Richard Mosesso2, Dennis A. Dougherty2. loop C, and loop A regions show notable deviations compared to the apo 1Dept of Biochemistry, University of Cambridge, Cambridge, United WT ELIC structure. This work establishes an alternative approach for Kingdom, 2Chemistry, California Institute of Technology, Pasadena, CA, high-resolution crystallographic study of GABAARs, for mechanistic under- USA. standing of anesthetic action, and for structure-based discovery of novel Pentameric ligand gated ion channels (pLGIC) mediate fast synaptic trans- GABAAR modulators. Research is supported by NIH. mission in animals, where they are known as Cys-loop receptors.

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Prokaryotic homologues of these proteins have been found in bacteria, 1504-Pos Board B413 although these do not possess the eponymous Cys-loop. Each receptor is Highly Pure Soluble Chimeras of the Intracellular Domain of Anionic Pen- constituted of five subunits which surround a central pore. A detailed anal- tameric Ligand-Gated Ion Channels ysis of sequences of all these subunits reveals that only the Pro in the Cys- Akash Pandhare1, Ali F. Ahmed2, Jackson V. Littlejohn2, Michaela Jansen1. loop is absolutely conserved; this has led to the proposal (which has not 1Cell Physiology and Molecular Biophysics, Center for Membrane Protein been widely adopted) that these receptors should be renamed ‘Pro-loop’ re- Research, Texas Tech University Health Sciences Center, Lubbock, TX, ceptors1. In addition to this Pro, there are 3 other Pros that are largely USA, 2Medical Student Summer Research Program, School of Medicine, conserved and have been shown to play important but different roles 2-4. Texas Tech University Health Sciences Center, Lubbock, TX, USA. Here we combine old and new data to compare the characteristics of these The past few years have seen remarkable advances in structural information Pro residues that allow them to have these distinct roles. Pro has a unique gleaned from atomic resolution X-ray crystallographs of important anionic chemistry in proteins because it lacks a backbone hydrogen bond, and has and cationic pentameric ligand-gated ion channels (pLGIC) from the a stronger cis-bias, and greater steric bulk at the amine than other amino Cys-loop superfamily. The highly conserved extracellular domain (ECD) and acids. Determining which of these properties is essential at which location transmembrane domain (TMD), and the diverse intracellular domain (ICD) in Cys-loop receptors can only be accomplished using proline analogues, constitute each subunit of eukaryotic pLGICs. The ECD and TMD of different i.e non-conventional amino acids. These were inserted using nonsense Cys-loop receptors are prime targets for several clinical drugs. These drugs are suppression into nACh and 5-HT3 receptors and receptor characteristics thus notorious for binding indiscriminately to undesired receptors within the su- were determined using two electrode voltage clamp in Xenopus oocytes. perfamily resulting in a wide range of unwanted side-effects. Intriguingly, the The data reveal some similarities but also some differences in the marked variability of ICDs between different subtypes can be exploited to aid roles of Pro at equivalent positions, and identify differences in the in the development of newer drugs with refined specific binding. However, the specific Pro properties that allow these two related Cys-loop receptors to ICD of any Cys-loop receptor has yet to be fully characterized. function. The two prominent anion-conducting pLGICs, glycine-a1 (Gly-a1) and g- 1. Jaiteh et al PLOS one 11(3) e0151934; 2. Rienzo et al. (2016) J Biol Chem amino butyric acid type A-r1 (GABAA-r1) receptors, of the Cys-loop super- 291 :6272-80; 3. Limapichat et al. (2010) J Biol Chem 285 :8976-84; 4. Dang family are critical in mediating postsynaptic inhibition in the central nervous et al. (2000) Mol. Pharmacol. 57(6):1114-22 system. Dysfunction of these pLGICs has been implicated in numerous serious neuropsychiatric and neurological diseases. 1503-Pos Board B412 The goal of our current project was to develop a strategy for protein expression Allosteric Modulation of the Pentameric Ligand-Gated Ion Channel ELIC and purification of the ICD of Gly-a1 as well as GABAA-r1 receptors. Here, we by Barbiturates generated chimeras containing maltose-binding protein (MBP) fused to the Hannelore De Peuter1, Marijke Brams1, Delphine Joseph2, N-terminus of the ICD of either Gly-a1 receptor or that of GABAA-r1 receptor Daniel Bertrand3, Chris Ulens1. and separated by a short alanine linker. We expressed these chimeras in E. coli 1KU Leuven, Leuven, Belgium, 2Universite Paris Sud, Chaˆtenay-Malabry, 3 and were able to purify both proteins of interest to homogeneity by employing France, HiQScreen, Geneva, Switzerland. 3-step purification. Further characterization to determine size using size exclu- The family of pentameric ligand-gated ion channels or Cys-loop receptors sion chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) is a class of ion channels involved in synaptic transmission in the peripheral revealed the oligomeric state. Experiments are underway to gain detailed struc- and central nervous systems. Among the different family members, the hu- tural insights. man GABAA receptors are an important target for a wide range of frequently prescribed therapeutics, including benzodiazepines, which act 1505-Pos Board B414 as hypnotics, anxiolytics and anti-epileptics, as well as barbiturates, which High Precision in Fluidic Control Provides Needed and Reliable Accuracy are mainly used as anti-epileptics. Among the barbiturates, an example in Ligand Induced Current Responses of Acetylcholine and GABA Recep- molecule is pentobarbital, which displays a complex mode of action. In tors this study, we employed the prokaryote ligand-gated ion channel ELIC as Ali Yehia1, Haiyang Wei2. a model to understand mechanistic effects of barbiturates. First, we func- 1Fluxion Biosciences, Alameda, CA, USA, 2Eurofins Pharma Discovery tionally characterized the effects of a series of barbiturate analogs on Services, St. Charles, MO, USA. ELIC channels expressed in Xenopus oocytes. Using the two-electrode Comprehensive studies of allosteric modulators depend on reliable, accurate voltage clamp technique we observed that all tested barbiturates, namely and repetitive responses of receptors to the co-applications of agonists. If liquid pentobarbital, brallobarbital, thiopental and selenobarbital, act as positive exchange is not precise and complete, receptor desensitization would ulti- allosteric modulators (PAMs), with levels of potentiation up to 100% at mately act as the hindering step in an assay. Although automated patch clamp an EC10-20 concentration of the agonist GABA. This result suggests that systems have evolved markedly in the last 11 years, in most cases the control barbiturates may potentiate ELIC through a binding site that is common over fluidic exchange did not advance beyond simple liquid addition to a well. to or resembling a modulatory site in the human GABAA receptors. Even with the implementation of microfluidics, precise control of fluidic ex- Next, our goal is to gain structural insight into the molecular determinants change while applying continuous flow of solutions have been elusive to of barbiturate recognition in ELIC. To accomplish this, we will pursue most setups. Here we present a new technique that enhances an already estab- X-ray structures of ELIC in complex with heavy atom barbiturate analogs, lished microfluidics based automated patch clamp system by allowing precise such as the bromo-derivative brallobarbital, which is clinically available, addition of ligand at the exact interval facilitating downstream receptor modu- and the seleno-derivative selenobarbital. Together, this study offers func- lation studies. We demonstrate the effect on recovery from desensitization of tional and structural insight into the mechanism of allosteric modulation nAChR alpha 7 receptors, repetitive and serial activation of GABA receptors, of ELIC by the therapeutically important class of barbiturates. as well as precise parallel addition of agonist to all receptors.

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Posters: Ion Channel Regulatory Mechanisms II 1508-Pos Board B417 Temperature Conditioning Increases CA2D Sensitivity and Activation 1506-Pos Board B415 Kinetics of ANO6 Variants PKC Activation Induces Ubiquitination-Dependent KV1.3 Endocytosis Joo Hyun Nam1, Yung Kyu Kim1, Sung Joon Kim2. 1Physiology, Dongguk University, College of Medicine, Gyeongju-si, Mediated by Nedd4-2 Ubiquitin Ligase 2 Ramo´n Martı´nez-Ma´rmol1, Katarzyna Styrczewska2, Republic of Korea, Physiology, Seoul National University College of 2 2 3 Medicine, Seoul, Republic of Korea. Mireia Perez-Verdaguer , Albert Vallejo-Gracia ,Nu´ria Comes , 2þ Alexander Sorkin4, Antonio Felipe2. ANO6 belongs to a family of Ca -activated Cl channel (CaCC) pro- 1Clem Jones Centre for Ageing Dementia Research, Queensland Brain teins and has four splice variants. Unlike classic CaCCs, ANO6 activa- 2þ 2þ > Institute, The University of Queensland, Brisbane, Australia, 2Biochemistry tion requires a high concentration of free intracellular Ca ([Ca ]i 1 m and Molecular Biomedicine, University of Barcelona, Barcelona, Spain, M) and is often delayed; thus, ANO6 needs several minutes to reach a 3Laboratory of Neurophysiology, University of Barcelona, Barcelona, Spain, fully activated state. These properties are evident in three ANO6 4 variants 1, 2 and 5 (V1, V2, and V5). In this study, we used the Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. whole-cell patch-clamp technique to identify a temperature (37 C) The voltage-dependent potassium channel Kv1.3 participates in the that increases ANO6 calcium sensitivity and activation kinetics. At 37 m 2þ immune system physiology. Kv1.3 regulates the membrane potential C, all ANO6 variants are activated by 1 M[Ca ]i. Additionally, triggering downstream Ca2þ-dependent pathways. This channel concentrates this temperature shortened the ANO6 latency time (the time taken to in specific membrane microdomains serving as signaling platforms. Altered reach the maximal current, after performing the whole-cell configura- expression of Kv1.3 is observed at the onset of several autoimmune tion). Moreover, V2 and V5 produced larger peak currents than those diseases. We show that adenosine (ADO), acting as a potent endogenous by V1, even though V1 showed the highest membrane expression level, modulator, stimulates PKC, thereby causing immunosuppression. PKC as confirmed by membrane protein biotinylation. Interestingly, the delayed activation disappeared in all isoforms. ANO6 also functions as activation down-regulates Kv1.3 triggering a clathrin-mediated internaliza- tion that targets the channel to lysosomes. Thus, the amount of Kv1.3 a scramblase; however, the temperature of 37 C did not induce at the plasma membrane decreases, which is clearly compatible with phosphatidylserine exposure (PS) exposure at a sub-micromolar an effective anti-inflammatory response. This mechanism involves ubiquiti- calcium concentration (300 nM), contrary to the results of patch nation of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. Postsynaptic recordings. All three of the functional ANO6 isoforms showed a similar PS exposure level. Together, these results suggest that density protein 95 (PSD-95), member of the MAGUK family, 2þ situates Kv1.3 into lipid-raft microdomains impairing the ubiquitination all functional ANO6 isoforms are activated by relatively low [Ca ]i % m and endocytosis of the channel. Therefore, the association of PSD-95 ( 1 M), when conditioned at a physiological temperature, although with Kv1.3 would modulate the anti-inflammatory response in leukocytes. the isoforms showed different calcium sensitivities and activation This work elucidates the PKC-dependent molecular mechanisms that kinetics. Furthermore, we concluded that there is no correlation between target Kv1.3 during immunomodulation in leukocytes. Supported scramblase and channel activity. This study may provide a basis for by MINECO and FEDER (BFU2014-54928-R and BFU2017-87104-R), ANO6 channel studies and the possibility for ANO6 to function as Spain. an ion channel in various tissues and cells depending on the variant form. 1507-Pos Board B416 Revealing Molecular Coupling between Anion Selectivity and Structural 1509-Pos Board B418 Transitions in Voltage-Dependent Anion Channel (VDAC) Effects and Activation Mechanisms of Ivermectin on G-Protein-Gated Van Ngo1,2, Tatiana K. Rostovtseva3, Sergey M. Bezrukov3, Inwardly Rectifying Potassium Channels Sergei Y. Noskov4. I-Shan Chen1,2, Michihiro Tateyama1,2, Yuko Fukata2,3, Motonari Uesugi4,5, 1 2 Yoshihiro Kubo1,2. Physics, University of Calgary, Calgary, AB, Canada, Los Alamos National 1 Lab, Los Alamos, NM, USA, 3Section on Molecular Transport, NIH, Division of Biophysics and Neurobiology, Department of Molecular and 4 Cellular Physiology, National Institute for Physiological Sciences, Okazaki, Bethesda, MD, USA, Biological Sciences, University of Calgary, Calgary, 2 AB, Canada. Japan, Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Hayama, VDAC regulates the fluxes of metabolites (ATP/ADP) between the 3 mitochondria and other cellular compartments by switching between Japan, Division of Membrane Physiology, Department of Molecular and uniquely high-conducting ‘‘open state’’ and low conducting ‘‘closed states’’. Cellular Physiology, National Institute for Physiological Sciences, Okazaki, Japan, 4Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto These transitions involve conformational rearrangements that both alter 5 ion selectivity and hinder the passage of metabolites. Although the x-ray University, Uji, Japan, Institute for Chemical Research, Kyoto University, structure of its open-state displays a cylindrical beta-barrel pore with Uji, Japan. the N-terminus tail leaning on the inside wall, a closed structure of Ivermectin (IVM) is a worldwide used antiparasitic drug, which was VDAC remains unsolved. To reveal the molecular basis of the transitions, discovered by Dr. Omura and Dr. Campbell to whom the 2015 Nobel Prize 5 in Physiology or Medicine was awarded. IVM kills nematode by activating we performed all-atom Molecular Dynamics simulations at 150 mV - with ionization states corresponding to pH=7 and 4 for enhancing the glutamate-gated Cl channel, and it also targets several ligand-gated voltage sensitivity. Over 70-ms atomistic simulations reveal gating transi- channels, such as GABAAR, GlyR, nAChR and P2X4. It is known that tions between open and closed states. We built the Markov State Model IVM binds to the transmembrane domains (TMs) of these channels. We found that IVM also activates a novel target, the G-protein-gated inwardly correlating the structural changes of the beta-barrel and movements of þ key residues with ionic permeation and ion-occupancy configurations. We rectifying K (GIRK) channel. Electrophysiological recordings in found that the beta-barrel oscillates between stable states at an order of Xenopus oocytes revealed that IVM activates GIRK1/GIRK2 remarkably magnitude faster than the N-terminus tail, which remains inside the pore and GIRK1/GIRK4 weakly in a phosphatidylinositol-4,5-biphosphate during the entire simulations. The charged residues of the N-terminus tail (PIP2)-dependent manner, and that the activation is independent of Gbg re-orient differently in the opposite electrical fields, and creating a subunits. In cultured rat hippocampal neurons, we also observed that IVM gating-charge transfer of 1.5 to 1.8|e|. The gating of VDAC depends on activates native GIRK current. By chimeric and mutagenesis analyses, we the protonation state with apparent impact on the stability of open and identified an amino acid residue critical for the activation, Ile82, which is closed states. The fluctuations of the pore diameter at pH=4 appears to be unique to GIRK2 among the GIRK family and located at the slide helix be- much larger than at pH=7. The acidic pH leads to rapid conformational tween the TM1 and the N-terminal cytoplasmic tail domain (CTD). Trp91 changes of VDAC, 50-percent reduction of ionic currents, and a clear and Ile195 located at the cytoplasmic ends of TM1 and TM2 also modulate enhancement of the ion selectivity. The proposed gating mechanism based the IVM-dependent activation. The results demonstrate that the TM-CTD on the coupled dynamics and the gating-charge transfer differs from the interface in GIRK channels, rather than the TMs, governs IVM-mediated voltage-sensing mechanisms common for Kþ or Naþ channels, but reminis- activation. These findings provide us with novel insights on the mode of ac- cent of the ‘‘squeezing’’ barrel model explaining the complexity of gating tion of IVM in ion channels that could lead to identification of new pharma- dynamics in VDAC. cophores which activate the GIRK channel.

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1510-Pos Board B419 1512-Pos Board B421 Gaussian-Accelerated Molecular Dynamics Modeling Leads to Identifica- Human Sperm Rotation is Regulated by Asymmetrically Positioned tion of SLAC1 Anion Channel Residues for CO2 Signaling in Arabidopsis Flagellar Control Units Guard Cell Nadja Mannowetz1, Melissa R. Miller1, Samuel J. Kenny2, Jingbo Zhang1, Nuo Wang2, Yinglong Miao3, Felix Hauser1, Steven A. Mansell1, Michal Wojcik2, Robert S. Zucker1,KeXu2, Wouter-Jan Rappel4, J. Andrew McCammon2, Julian Schroeder1. Polina V. Lishko1. 1Division of Biological Sciences, UC San Diego, La Jolla, CA, USA, 1Department of Molecular and Cell Biology, University of California, 2Department of Chemistry and Biochemistry, UC San Diego, La Jolla, CA, Berkeley, Berkeley, CA, USA, 2Department of Chemistry, University of USA, 3Center for Computational Biology and Department of Molecular California, Berkeley, Berkeley, CA, USA. Biosciences, University of Kansas, Lawrence, KS, USA, 4Physics To fertilize an egg, spermatozoa have to overcome the tenacious egg’s Department, UC San Diego, La Jolla, CA, USA. protective vestment. CatSper, the principal calcium channel of human Plants control CO2 exchange and water loss to the atmosphere in sperm is indispensable for this process since it initiates the asymmetrical response to endogenous and environmental stimuli via stomatal pores. bending of the sperm tail via calcium influx termed hyperactivation. - Atmospheric [CO2] elevation triggers stomatal closure by Cl efflux Apart from hyperactivation, human sperm also rotate, which enables mediated via the SLAC1 anion channel in the plasma membrane of them to swim against the liquid flow in the fallopian tube and reach - guard cells. Previous research showed that bicarbonate (HCO3 )can the egg. However, the mechanistic details how the influx of calcium directly up-regulate the SLAC1 channel activity and the transmembrane ions triggers hyperactivation and rotation have not been identified yet. - domain of SLAC1 is involved in the stomatal CO2/HCO3 response. CatSper is pH-dependent and for its full activation the intracellular pH - The mechanism by which HCO3 regulates the SLAC1 anion channel must be alkaline. Alkalinization can be accomplished by either proton activity is unknown. Here, we predicted candidate bicarbonate binding transporters or a faster mechanism, such as the voltage-gated proton sites within the SLAC1 anion channel through long-timescale channel Hv1. CatSper, Hv1 and regulatory proteins must be spatially Gaussian accelerated molecular dynamics (GaMD) simulations. compartmentalized to guarantee effective regulation of calcium influx - Mutations of two putative HCO3 -interacting residues abolished the into the sperm tail via CatSper. With this work we characterize - enhancement of the SLAC1 anion channel activity by CO2/HCO3 , while nanodomains along the tail of human sperm, which consist of Hv1, SLAC1 activation by other stimuli remained intact. Furthermore, gas CatSper and its regulatory protein ABHD2. With super-resolution exchange experiments with complemented slac1 mutant plants microscopy we show that CatSper and ABHD2 form four symmetrically expressing mutated SLAC1 proteins revealed that one of these SLAC1 positioned longitudinal lines spanning the sperm tail, whereas Hv1 forms residues is required for the stomatal CO2 response. Patch clamp only two asymmetrical longitudinal lines. Inhibition of Hv1 leads to a analyses of guard cells showed that activation of the S-type decrease in sperm rotation underpinning its role and the specific - anion channel activity by CO2/HCO3 was impaired, being consistent structural arrangement in this motility patterns. In conclusion, Hv1, with the above analyses. Results from this study provide important CatSper, and ABHD2 are organized in distinct regulatory nanodomains, insights into how plants respond to the continuing atmospheric rise of which control hyperactivated motility and rotation thus ensuring [CO2]. fertilization.

1511-Pos Board B420 1513-Pos Board B422 Chimeric Kv7.4 Channel with Amino-Terminus of Kv7.5 Has Putative Modulation of KCNQ Channels by Intracellular Zinc Protein Kinase a Phosphorylation Site and is Sufficient to Confer Partial Haixia Gao1,2, Aurelien Boillat1, Dongyang Huang2, Ce Liang2, Chris Peers3, Sensitivity to Cyclic Adenosine Monophosphate/Protein Kinase a Nikita Gamper1,2. (cAMP/PKA) Pathway 1Faculty of Biological Sciences, University of Leeds, Leeds, United Lyubov I. Brueggemann1, Leanne L. Cribbs2, Kenneth L. Byron1. Kingdom, 2Department of Pharmacology, Hebei Medical University, 1Molecular Pharmacology and Therapeutics, Loyola University Chicago, Shijiazhuang, China, 3Faculty of Medicine and Health, University of Leeds, Maywood, IL, USA, 2Cell and Molecular Physiology, Loyola University Leeds, United Kingdom. Chicago, Maywood, IL, USA. KCNQ (Kv7) potassium channels are important regulators of excitability We previously found that enhancement of Kv7.5 current by activation of in neurons and muscle cells. Suppression of KCNQ channel activity or cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway expression underscores many physiological and pathological mechanisms is associated with phosphorylation of Kv7.5 channels, whereas Kv7.4 of excitation. On the other hand, pharmacological augmentation of lacks PKA-dependent regulation. We mutated several putative phosphory- KCNQ channel activity is a validated strategy for treatment of lation sites that differ between Kv7.4 and Kv7.5. Serine residues S406 hyperexcitability disorders such as epilepsy and pain. Yet, known and S412 and threonine T32 were mutated to alanines, but none of these physiological mechanisms resulting in acute potentiation of KCNQ chan- mutations prevented cAMP/PKA-dependent enhancement of Kv7.5 cur- nel activity are rare. We report that intracellular free zinc directly and rents by forskolin/IBMX. In contrast, S53A mutation did significantly reversibly augments activity of recombinant KCNQ2, KCNQ2/3 and reduce forskolin/IBMX-induced enhancement from 2.8-fold to 1.3-fold. KCNQ4 channels as well as native M current in cultured sensory neurons The S53A mutation was also associated with a negative shift of the acti- and hippocampal slices. This effect is mechanistically distinct from the vation curve (V0.5= 44 mV for WT Kv7.5 and 56 mV for redox-dependent KCNQ channel potentiation as it does not require the Kv7.5S53A). Since Kv7.4 channels lack the putative amino-terminal redox-sensitive cysteine cluster in the S2-S3 linker. Interestingly, the ef- S53 PKA phosphorylation site, we replaced the amino-terminus of fect of zinc could not be attributed to a single histidine- or cysteine- Kv7.4 with the amino-terminus of Kv7.5 (NKv7.5). The NKv7.5-Kv7.4 containing zinc binding site within KCNQ channels. Instead, zinc dramat- channels had voltage dependence similar to Kv7.4 (V0.5=-31 5 3mV ically reduced KCNQ channel dependence on its obligatory physiological for NKv7.5-Kv7.4 and 34 5 2 mV for Kv7.4). Current through activator, phosphatidylinositol 4,5-bisphosphate (PIP2). Thus, intracellular chimeric NKv7.5-Kv7.4 channel was significantly enhanced by forskolin zinc activated single KCNQ2/3 channels in the excised patch recordings in (1 mM); the 29% enhancement was significantly less than 61% enhance- the absence of added PIP2. Moreover, zinc abolished KCNQ channel ment of WT Kv7.5 current, but greater than 8% enhancement of WT inhibition by voltage-sensitive phosphatase ciVSP and by muscarinic M1 Kv7.4. Further modification of Kv7.4 by replacing its carboxy-terminal receptor triggering. In addition, KCNQ3 subunit, which has intrinsically PIP2 binding domain with the corresponding Kv7.5 PIP2 binding domain high apparent PIP2 affinity, is insensitive to zinc modulation, whereas (DCKv7.5) resulted in 6 mV positive shift of voltage dependence relative KCNQ3 mutants with reduced PIP2 affinity were potentiated. We hypoth- to WT Kv7.4. Forskolin (1 mM) enhancement of NKv7.5-Kv7.4- DCKv7.5 esize that zinc facilitates interactions of the lipid-facing interface of a currents was not significantly different from NKv7.5-Kv7.4, but was KCNQ protein with the inner leaflet of the plasma membrane in a way significantly less than its enhancement of WT Kv7.5 channel. Our findings similar to that promoted by PIP2. Zinc is increasingly recognized as ubiq- suggest that PKA-dependent phosphorylation of amino-terminal S53 of uitous intracellular second messenger, therefore, this discovery may repre- Kv7.5 channels contributes to the enhancement of channel activity, but sent a hitherto unknown pathway of physiological M channel modulation. additional sites are required to confer full responsiveness to PKA Additionally, our data may inform the development of novel KCNQ activators. openers for therapeutic purposes.

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1514-Pos Board B423 by Gq-coupled-M1AChR stimulation via PIP2 depletion by phospholipase Assigning Function to the D and E Helices of HCN CNBD C(PLC), and other GqPCRs by Ca2þ signals. Ca2þ rises are suggested to 1 1 1 2 Andrea Saponaro , Alessandro Porro , Chiara Donadoni , Bina Santoro , stimulate synthesis of PIP2. In hippocampus, M/KCNQ-channels and 3 1 Gerhard Thiel , Anna Moroni . M1AChRs are widely expressed, and cholinergic innervation strongly af- 1Biosciences, University of Milan, Milan, Italy, 2Neuroscience, Columbia fects circuit excitability. We investigated mAChR neuromodulation of University, New York City, NY, USA, 3Biology, TU-Darmstadt, Darmstadt, hippocampal neurons using brain-slice electrophysiology and Ca2þ imag- Germany. ing. Using whole-cell voltage-clamp, we observed fraction of deactivating HCN (hyperpolarization-activated cyclic-nucleotide sensitive cation nonselec- relaxation at 60 mV due to IM/ERG current by kinetics and highly- tive) channels are activated by voltage and modulated by the direct binding of specific blockers (XE991,E-4031). In dentate gyrus granule cells(DGGCs), cAMP to their cytoplasmic C-terminal region named CNBD (cyclic nucleotide >95% of current was from M-channels, whereas in CA1 neurons, M/ERG binding domain) (Zagotta et al., 2003). HCN channels are further regulated by current ratio was 60/40. We stimulated mAChRs with oxotremorine-M(1- TRIP8b, a brain-specific auxiliary subunit which controls channel trafficking 10mM) or M1R-selective agonist 77LH281(3-10mM). Unexpectedly, both and gating. In particular, TRIP8b interacts with the HCN channel CNBD and agonists enhanced IM in DGGCs (maximum 2.4-fold), whereas IM was antagonizes the facilitatory effect of cAMP on channel opening (Hu et al., suppressed in CA1 neurons (78% block). M1R-induced enhancement of 2013 ). Recently, the cryo-EM structure of human HCN1 was solved in both IM was abolished by edelfosine blockade of PLC. Testing M1R-induced the cAMP-free and cAMP-bound states (Lee and Mackinnon, 2017). A increase of PIP2 abundance in DGGCs as underlying increases in IM, comparison of the two structures shows that, in the full-length protein, we quantified commensurate increase of GIRK current during muscarinic cAMP binding induces all the same changes previously highlighted in the stimulation, thus acting as a PIP2 biosensor. Blockade of PI(4)P5-kinase isolated CNBD fragment using NMR (Saponaro et al., 2014), plus the by UNC3230(3mM) caused muscarinic stimulation to instead suppress 2þ 2þ unexpected folding of two additional helices at the C-terminus of the CNBD. IM in DGGCs by 60%. Ca imaging revealed robust [Ca ]i increase These helices were not included in any of the previous experiments conducted in DGGCs by muscarinic stimulation. Despite pronounced increase in using the isolated C-linker/CNBD fragment. To understand whether these two IM by mAChR stimulation in DGGCs, it was accompanied by robust in- dynamic helices play a role in regulating both cAMP and TRIP8b binding, we crease in excitability (action potential frequency), leading us to have compared, using an in vitro binding assay, two CNBD constructs with or hypothesize PLC-induced activation of other channels. When the TRPC- without the newly discovered helices. Surprisingly, their presence increases the blocker, ML204 (10mM), was applied concurrently with muscarinic 2þ affinity for cAMP without affecting TRIP8b binding. This result assigns to the agonist, neither excitability nor [Ca ]i were significantly changed. new C-terminal helices of the HCN channel CNBD a specific role in controlling Thus, DGGC excitability is regulated by muscarinic signals in concert cyclic nucleotide affinity. with M-type/TRPC channels, whereas CA1 neurons are regulated in a distinct fashion. 1515-Pos Board B424 Caveolae-Mediated Activation of Mechanosensitive I Channels CL,SWELL 1517-Pos Board B426 Disrupts Conduction and Promotes Arrhythmogenesis in Pulmonary The Unfolded Protein Response Contributes to Electrical Remodeling in Veins and Superior Vena Cava Human Cardiomyocytes Di Lang1, Yuri V. Egorov2, Rose Wang1, Leonid V. Rosenshtraukh2, Man Liu1, Guangbin Shi2, Anyu Zhou2, Samuel C. Dudley Jr.1 Alexey V. Glukhov1. 1Lillihei Heart Institute, University of Minnesota, Minneapolis, MN, USA, 1Medicine, University of Wisconsin Madison, Madison, WI, USA, 2Cardiovascular Research Center, Brown University and Lifespan Rhode 2Laboratory of Heart Electrophysiology, Cardiology Research Center, Island Hospital, Providence, RI, USA. Moscow, Russian Federation. Background: The unfolded protein response (UPR) is active in cardiomyopa- Atrial fibrillation is the most common heart rhythm disorder, often associated thy and decreases protein levels by increasing mRNA decay, accelerating with hypertension, heart failure and other cardiovascular conditions, where protein degradation, and inhibiting protein translation. myocardium suffers elevated mechanical stretch. Caveolae, small (50-100 Objective: We investigated whether the UPR regulated cardiac ion channels in nm) invaginations of the plasma membrane, have been recently reported to human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). act as mechanosensors by buffering mechanical forces and contributing to cell Methods: We measured changes of cardiac action potentials (AP) and ion volume regulation via modulation of activity of volume-activated Cl channels channels at the mRNA, protein, current levels. Tunicamycin (TM, 5 mg/mL), I . We hypothesize that atrial stretch disrupts caveolae structures and Cl,swell GSK2606414 (GSK, 300 nM) and 4m8C (5 mM) was utilized to activate the enables activation of I that disrupts conduction and promotes arrhythmo- Cl,swell UPR and inhibit PERK and IRE1, respectively. All treatments were 20-24 h genesis in pulmonary veins (PV) and superior vena cava (SVC). High resolution at 37 C in a 95%CO /5%O incubator. optical mapping and simultaneous recordings from two glass-microelectrodes 2 2 Results: TM-induced UPR activation caused prolongation of the AP duration were employed to study action potential propagation in rat and mouse PV and (APD : 15755154 vs. 477523 ms in controls) and a reduction of the SVC. Stretch (tension applied from 1 to 100mN) force-dependently depolarized 90 maximum upstroke velocity (dV/dt :6651 vs. 7754 V/s in controls). These resting potential (RP) and decreased action potential amplitude (APA) exclu- max changes were explained by reductions in all major currents: I ,I ,I and sively in the distal PV/SVC, but not in the PV/SVC ostium, which led to the Na CaL to I þI .Na1.5, Ca 1.2, K 4.3, and K LQT1 channels showed concomitant development of intra-vein block (25% at 25 mN vs. 75% at 100 mN, P<0.01) Kr Ks v v v v reductions in mRNA and protein levels. Protein levels of hERG, KChIP2, and both triggered and reentrant activities. Conduction block was abolished and K 1.4 were elevated with decreased or unaltered mRNA levels. Inhibition by the application of I channel blockers 100mM DIDS and 100mM v Cl,swell of PERK or IRE1 shortened the APD partially and restored dV/dt 9-AC by restoring APA (from 1554mVto7154 mV, P<0.01 in PV; and max completely. PERK downregulated the mRNA and current of Na 1.5, K 4.3, 652mVto6659 mV, P<0.01 in SVC) and RP (from 6150.5 mV to v v hERG, and K LQT1. The IRE1 branch downregulated mRNA, protein, and 8251mV,P<0.01 in PV; and from 5352mVto7555 mV, P<0.01 v current levels of Na 1.5, hERG, and K LQT1, and Ca 1.2 protein and current in SVC). Immunofluorescent imaging revealed co-localization of caveolae v v v levels. scaffolding protein, caveolin-3, and I channels, CLC-2, in PV. Stretch Cl,swell Conclusions: Activated UPR downregulates all major cardiac ion channels and decreased caveolin-3 expression level which likely disrupted caveolae structures results in electrical remodeling in hiPSC-CMs, which can be partially reversed and unmasked I channels promoting their activation and the development Cl,swell by inhibition of either the PERK or IRE1 branch. Therefore, targeting the UPR of conduction block. Our findings demonstrate that caveolae-mediated could be a novel antiarrhythmic strategy. activation of mechanosensitive ICl,swell appears to be essential component of atrial mechano-electrical transduction and arrhythmogenesis. 1518-Pos Board B427 1516-Pos Board B425 K2P2.1 (TREK-1)-Activator Complexes Reveal a Cryptic Selectivity Filter Muscarinic Receptor Neuromodulation of KCNQ M-Type KD, and Binding Site Other, Channels in Hippocampal Principal Neurons Involves Striking Marco Lolicato1, Cristina Arrigoni1, Takahiro Mori2, Yoko Sekioka2, Cell-Specific Regulation Controlling Excitability Clifford Bryant3, Kimberly A. Clark1, Daniel L. Minor1. Chase M. Carver, Mark S. Shapiro. 1CVRI, UCSF, San Francisco, CA, USA, 2Ono Pharmaceutical Co. Ltd, Physiology, UT Health San Antonio, San Antonio, TX, USA. Osaka, Japan, 3Departments of Biochemistry and Biophysics, UCSF, Voltage-gated ‘‘M-type’’ channels are important for neuronal discharge San Francisco, CA, USA. properties and require interactions with phosphatidylinositol 4,5-bisphos- Polymodal thermo- and mechanosensitive two-pore domain potassium phate(PIP2). In sympathetic ganglia, M-current(IM) is strongly depressed (K2P) channels of the TREK subfamily generate ‘leak’ currents that regulate

BPJ 8632_8635 304a Monday, February 19, 2018 neuronal excitability, respond to lipids, temperature and mechanical stretch, measured in Langendorff-perfused hearts, which may due to decreases and influence pain, temperature perception and anaesthetic responses. These in L-type-Ca2þ-currents with CL316243 pre-treatment. We conclude dimeric voltage-gated ion channel (VGIC) superfamily members have a that sustained exposure to b3-ARs stimulation regulates cardiac ionic unique topology comprising two pore-forming regions per subunit. In channel currents, providing an impression of its direct ionic current contrast to other potassium channels, K2P channels use a selectivity filter remodeling effects at cellular level, which seems not enough to compan- ‘C-type’ gate as the principal gating site. Despite recent advances, poor sate the depressive effect of b3-ARs stimulation on mechanical activity pharmacological profiles of K2P channels limit mechanistic and biological of heart in overweight insulin resistant rats. Importantly, these arrhyth- studies. Here we describe a class of small-molecule TREK activators that mogenic ionic remodeling with b3-ARs stimulation in MetS rats, in directly stimulate the C-type gate by acting as molecular wedges that restrict part, can be important to prevent apperance of arrhythmia in further interdomain interface movement behind the selectivity filter. Structures of stage of MetS such as diabetes. K2P2.1 (also known as TREK-1) alone and with two selective K2P2.1 (Supported through TUBITAK for grant SBAG-214S254). (TREK-1) and K2P10.1 (TREK-2) activators-an N-aryl-sulfonamide, ML335, and a thiophene-carboxamide, ML402-define a cryptic binding 1521-Pos Board B430 Modeling Mechanisms of Cardiac L-Type Ca2D Channel Regulation: pocket unlike other ion channel small-molecule binding sites and, together 2D with functional studies, identify a cation-p interaction that controls selec- Interactions of Voltage, Ca , and Isoflurane Neeraj Manhas1, Amadou K.S. Camara2, Ranjan K. Dash3. tivity. Together, our data reveal a druggable K2P site that stabilizes the C- 1Biomedical Engineering, Medical College of Wisconsin, Milwaukee, WI, type gate ‘leak mode’ and provide direct evidence for K2P selectivity filter 2 gating. USA, Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA, 3Biomedical Engineering and Physiology, Medical College of 1519-Pos Board B428 Wisconsin, Milwaukee, WI, USA. Structural and Functional Characterization of Bestrophin Channel Calcium influx via L-type Ca2þ channel (LCC) in cardiomyocytes (CM) is Inactivation regulated by both voltage-dependent and Ca2þ-dependent inactivation George Vaisey, Stephen B. Long. (VDI, CDI) mechanisms, which are not well understood. Also, how isoflur- Structural Biology Dpt, MSKCC, New York, NY, USA. ane (ISO), a volatile anesthetic cardioprotective agent, exerts negative The bestrophin anion channel is a calcium-activated chloride channel: inotropic effect on CM by modulating LCC, possibly VDI and/or CDI, is cytoplasmic calcium (Ca2þ) activates the channel, allowing chloride lacking. We extended a previously developed Markov model of LCC to ions to flow down their electrochemical gradient. Mutations in bestro- investigate these different regulation mechanisms, based on diverse pub- phin 1 (BEST1) cause macular degenerative disorders. Previous whole- lished kinetic data on whole-cell patch-clamp of ventricular myocytes, cell recordings have shown that BEST1 currents decrease over time at with different charge carriers, ISO concentrations, and voltage-clamp proto- higher Ca2þ concentrations. Here, we present electrophysiological cols. It is understood experimentally that the accumulated Ca2þ in the vicin- studies of purified wild-type and mutant BEST1 channels which, in ity (nanodomain) of LCC Ca2þ sensors, as Ca2þ passes through the channel, combination with single particle cryo-electron microscopy (cryo-EM) is responsible for most of CDI, independent of Ca2þ in the dyadic space (mi- analysis, enable a better understanding of this behavior. We show that crodomain) due to Ca2þ induced Ca2þ release (CICR). The previous LCC currents of purified chicken BEST1 decrease over time in a Ca2þ-depen- model, which includes distinct voltage and Ca2þ modes of LCC, cannot dent manner. Because this current rundown is exhibited by the purified fully describe CDI mechanism. Interestingly, it is also observed that ISO channel, and can be altered by mutagenesis, we refer to it as Ca2þ- dose-dependently inhibits LCC and its steady state inactivation/availability. dependent inactivation. Truncation or mutation of a C-terminal ‘‘tail’’, To quantify these mechanisms, we extended the previous LCC model to which wraps around the periphery of the BEST1 channel, abolishes or incorporate possible interactions of ISO with the two LCC modes. We reduces inactivation. Alanine substitutions within a hydrophobic also incorporated a Ca2þ nanodomain in the vicinity of LCC Ca2þ sensors ‘‘neck’’ of the pore cause the channel to be non-inactivating. Previously and simulated Ca2þ dynamics in this region to quantify the native role of we had shown the neck to be a Ca2þ-dependent gate for activation of Ca2þ entry on CDI of LCC, independent of CICR. The extended LCC the channel. These experiments suggest the neck is also a seal that model systematically reproduces various aspects of LCC regulation closes during inactivation. Ongoing cryo-EM studies of non- underlying VDI and CDI, and their regulation by ISO, in addition to inactivating BEST1 mutants support the functional data and provide providing contributions of Ca2þ nanodomain for CDI, independent of new insight into the mechanism of Ca2þ-dependent inactivation. CICR. The LCC model also predicts that the depressant action of ISO on LCC is dependent on ISO interactions with the open and inactivated states 1520-Pos Board B429 of LCC. b3-Adrenergic Receptor Regulation of Cardiac Ion Channels in Overweight Insulin Resistant Rats 1522-Pos Board B431 Aysegul Durak, Yusuf Olgar, Erkan Tuncay, Belma Turan. Viscosity and Conductivity Tunable Diode-Like Behavior for MESO- and Biophysics, Ankara University School of Medicine, Ankara, Turkey. Micropores Beta3-adrenergic receptors (b3-ARs) are expressed in mammalian heart Rachel A. Lucas, Yinghua Qiu, Zuzanna S. Siwy. and their stimulation generally exert negative inotropic effect through Physics and Astronomy, University of California, Irvine, CA, USA. activation of eNOS. Animal studies showed their anti-obesity and anti- Rectifying pores, which transport ions mainly in one direction, blocking the diabetic effects due to rapid lipid oxidation, afffecting insulin signaling ionic flow in the other, were shown to be important in the preparation of and exerting glucose improvements. However, studies also demonstrated chemical sensors, components of ionic circuits, and mimics of biological that their expression level are increased markedly under pathological channels. Ionic rectiflcation has been shown with various engineered sys- conditions. Therefore, we aimed to examine the role of b3-ARs stimula- tems, but pores with similar opening diameters often rectify to a various un- tion with an selective agonist CL316243 on cardiac ionic currents in controlled extent. In this work we present a system of single meso-pores, high carbohydrate diet induced overweight insulin resistant rats (rats whose currentvoltage curves and rectiflcation can be tuned with great pre- with metabolic syndrome, MetS) under in vitro conditions. Markedly cision via viscosity and conductivity gradients of solutions placed on both prolonged action potentials, due to mostly depressed Kþ-channel cur- sides of the membrane. The mechanism of rectification is based on electro- rents recorded in freshly isolated cardiomyocytes from MetS rats, were osmotically induced fiow, which fills the entire volume of the pore with a normalized pre-treatment of cardiomyocytes with CL316243 (1 mMfor single solution from either side of the membrane. How the pore surface 20 min incubation) while L-type-Ca2þ-currents were depreesed, signifi- gets wetted by the solutions in contact with the membrane is investigated catly. In addition, markedly increased both TTX-sensitive Naþ-channel here. This understanding is especially important if different solvents are and Naþ/Kþ-pump currents in MetS rats were further increased with present on both sides of the membrane, which leads to a complex depen- CL316243 pre-treatment while these further increases could not further dence of the solvent composition along the pore axis. We also examine affect the amplitude of action potentials. Although we have some impor- the role of pore surface charge properties in inducing electoosmotic flow tant benefits on cardiac ionic currents with b3-ARs stimulation at in more complex solutions, especially with water/organic solvent mixtures. cellular level in MetS rats, however, this stimulation exerted further In the presence of media with difierent dielectric constants, local surface negative effect on depressed left ventricular developed pressure charge density, medium conductivity, ionic mobility, and electrostatic

BPJ 8632_8635 Monday, February 19, 2018 305a interactions are altered compared to purely aqueous solutions. These effects PMA-induced ROS production. In osteoclasts, ROS are generated during are expected to influence electroosmotic flow and consequently ion current RANK-RANKL-mediated signalling cascade, and are essential for differ- rectification governed by electroosmosis, as reported here. This highly entiation, activation, and survival. During later culturing passages of predictable rectifying system can find various applications, including RAW cells, where osteoclast-forming potency declined, Pi-induced in- measuring viscosity of unknown media and tuning electrokinetic passage creases in Hþ channel activities and ROS production were both modest. of particles. The present study suggests that Pi works as a common endogenous signal to enhance Hþ channels and ROS production in osteoclasts, which 1523-Pos Board B432 may support osteoclastogenesis. PKA-Dependent Phosphorylation Underlies Functional Upregulation of SK Channels in Ventricular Myocytes From Hypertrophic Hearts 1525-Pos Board B434 Iuliia Polina, Shanna Hamilton, Radmila Terentyeva, Karim Roder, The Sigma-1 Receptor Modulates K 1.2 Channels in the Absence of the Gideon Koren, Dmitry Terentyev. V KVb2 Subunit Cardiovascular Research Center, Rhode Island Hospital, Providence, RI, Madelyn J. Abraham1, Adrian Y.C. Wong2, Richard Bergeron2. USA. 1 þ Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON, Canada, Small conductance calcium-activated potassium (K ) channels (SK) expressed 2Neuroscience, Ottawa Hospital Research Institute, Ottawa, ON, Canada. in ventricular myocytes (VMs) are dormant in health but become functional in Sigma-1 receptors (s-1R) are interorganelle signalling molecules which have cardiac disease, leading to partial offset of disease-related reduction in been shown to modulate various ion channels, including Shaker-type repolarization reserve. However, the mechanism of functional upregulation voltage-gated potassium channels (Kv1.x). Previous studies have shown that of SKs remains unknown. We aimed to test the hypothesis that posttranslational pharmacological activation of s-1R decreases Kv1.x channel current amplitude modifications of SKs in conditions accompanied by enhanced catecholamin- and increases channel inactivation, leading to reduced voltage-sensitivity. ergic drive play a central role in disease-related activation of SKs using the While this is likely due to a direct interaction between the s-1R and the rat model of hypertrophy induced by thoracic aortic banding (TAB). pore-forming Kv1a subunit, the molecular locus of this interaction remains Western blot analysis using anti-pan-Ser/Thr antibodies demonstrated to be elucidated. In vivo, the Kv1a subunit exists as a hetero-oligomer with enhanced phosphorylation of immuno-precipitated SK channels (SK2) in auxiliary Kvb subunits. Curiously, expression of the Kvb subunit modulates VMs from TAB rats vs Shams, which was fully reversible by incubation of cells Kva channel inactivation in a qualitatively analogous fashion to an activated m with PKA inhibitor H89 (1 M, 30 min) but not CaMKII inhibitor KN93 (500 s-1R, suggesting that both modulators may share a common binding site on nM, 30 min, n=4). Patch-clamped VMs from TABs but not Shams exhibited the Kva subunit. Thus, we hypothesized that overexpression of Kvb-subunits outward current sensitive to specific SK inhibitor Apamin (APA, 100 nM). may occlude the regulatory effects of the ligand-activated s-1R. To test this, In current-clamp experiments, APA prolonged action potential (AP) in TAB we transiently transfected HEK293 cells with a the Kv1.2 channel subunit m VMs but not in Shams. Introduction of specific PKA inhibitor, PKI (10 M) together with s-1R-YFP alone, or alongside Kvb2. Electrophysiological b þ into the patch-pipette alleviated ISK in TAB VMs. Conversely, -adrenergic recording of whole cell K currents from these cells show that ligand activation agonist Isoproterenol (ISO, 100 nM, 3 min) evoked ISK in Shams. Furthermore, of the s-1R results in a 30% decrease in current amplitude and a small in current-clamp experiments APA prolonged APD in ISO-treated Sham VMs, inhibitory shift in voltage-dependence of inactivation of Kv1.2. Interestingly, rendering them similar to TABs. Using site-directed mutagenesis we tested expression of Kvb2 abolishes the effect of s-1R ligand activation on Kv1.2, which of 5 known rat SK2-Ser, that can be phosphorylated by PKA, are suggesting either a common locus of binding or physical occlusion of the s- involved in modulation of channel function. Adenoviral overexpression of 1R binding site by Kvb2. Our preliminary data provides further evidence of de-phosphomimetic mutant S136A in adult rat VMs maintained in culture a direct, functional interaction between the s-1R and Kv1.2. We also suggest for 48 hrs demonstrated 10-fold reduction in conductance and lack of that s-1R and Kvb2 modulation of Kv1.x channels are mutually exclusive response to ISO in comparison to rSK2-WT and other phospho- and events, and that s-1R modulation of Kv1.2 can only occur when Kvb subunits dephospho-mimetic mutants. In conclusion, disease-related upregulation of are not expressed. SKs in VMs is mediated by PKA-dependent phosphorylation conferred by single N-terminal S136. 1526-Pos Board B435 Evidence for Mechanosensitive Channel Activity of Tentonin 3/TMEM150C 1524-Pos Board B433 Gyu-Sang Hong. Extracellular Phosphate Is an Endogenous Regulator for Voltage-Gated Neuroscience, Korea Institute of Science & Technology, Seoul, Republic of Proton Channels and Production of Reactive Oxygen Species in Korea. Osteoclasts Mechanosensation is essential for various physiological processes, and it is Guangshuai Li1, Katsuuki Miura2, Yoshiko Hino1, Yoshie Moriura1, mediated by mechanotransduction channels. Recently, we reported that Junko Kawawaki3, Hiromu Sakai1, Miyuki Kuno1. TMEM150C/Tentonin 3 (TTN3) confers mechanically activated currents 1 Physiology, Osaka City University Graduate School of Medicine, Osaka, with slow inactivation kinetics in several cell types, including dorsal root gan- 2 Japan, Applied Pharmacology and Therapeutics, Osaka City University glion neurons (Hong et al., 2016). The accompanying Matters Arising by Du- 3 Graduate School of Medicine, Osaka, Japan, Research Support Platform, bin, Murthy, and colleagues confirms that naive heterologous cells demonstrate Osaka City University Graduate School of Medicine, Osaka, Japan. a mechanically activated current, but finds that this response is absent in Osteoclasts, bone-resorbing cells, express voltage-gated proton (Hþ) þ CRISPR-Cas9 Piezo1 knockout cell lines and suggests that TTN3 is a modu- channels in the plasma membrane. As in other cells expressing H chan- lator of Piezo1. We present and discuss evidence based on coexpression of nels, their activities are increased by addition of PMA, a protein kinase TTN3 and Peizo1 and mutant variants of the pore region of TTN3 to support C activator, but thier natural activating stimuli for osteoclasts have not that TTN3 is a pore-forming unit, not an amplifying adaptor for Piezo1 activity. been defined yet. In resorption pits, inorganic phosphates (Pi) are accu- This Matters Arising Response paper, along with Zhao et al. (2017), addresses mulated during degradation of hydroxyapatite, a major component of the Matters Arising from Dubin et al. (2017), published concurrently in this bone mineral. Consequently osteoclastsareexposedtohighconcentra- issue of Neuron. tions of Pi. Pi is known as an extracellular signal responsible for regu- þ lation of bone metabolism. Here, we examined effects of Pi on H 1527-Pos Board B436 channels and NADPH oxidase-mediated production of reactive oxygen Gramicidin Ion Binding and Conductance: New Insights from 17O Solid species (ROS) in RAW264-derived osteoclast-like cells. Pi enhanced þ State NMR Spectroscopy in a 1.5 GHZ Spectrometer H channels by increasing the maximal conductance, decreasing the Joana Paulino1, Ivan Hung1, Eduard Chekmenev2, Zhehong Gan1, activation time constant (enhanced gating), and shifting the activation Timothy A. Cross1. to more negative voltages. The enhanced gating was mainly caused by 1NHMFL, Tallahassee, FL, USA, 2Ingram Cancer Center, Vanderbilt, elevation of intracellular Pi levels, and the accompanying decrease in Nashville, TN, USA. intracellular pH increased the maximal conductance additionally. The Protein NMR is based on 1H, 13C and 15N nuclei, however limited chemistry enhanced gating was inhibited partially by GF109203X (PKC inhibitor) takes place at these atoms. Oxygen, on the other hand, is involved in many and staurosporine (general kinase inhibitor), but was not affected by different processes. Solid state (ss) NMR is an essential technique for the phosphatase activities. Pi and PMA upregulated ROS production. The study of functional biology of membrane proteins. Gramicidin A (gA), a ROS production was inhibited by GF109203X. Zinc decreased the peptide with alternating L and D amino acids, forms a dimeric Kþ channel

BPJ 8632_8635 306a Monday, February 19, 2018 with a 4 A˚ diameter pore lined by the carbonyl oxygens of the backbone. gA PM-ER junctions by co-expressing some of these channels with the two JPs, in ion conduction has similarities to the selectivity filter of Kþ channels. Cation tsA201 cells and using confocal microscopy and electrophysiology to assess their binding to gA has been studied previously by 15N and 17O ssNMR. 17O is a interaction. Our results show that JP3 and JP4 selectively recruited L-type, 5/2 quadrupolar nuclei with large CSA and MHz quadrupolar coupling. The P/Q-type and N-type, but not T-type, channels to PM-ER junctions and that spectroscopy of 17O at fields of 19 to 21T was shown to be promising, how- JP3, and to a greater extent JP4, substantially slowed inactivation of P/Q and ever it still suffers from low sensitivity and poor resolution. At 35T the S/N of N-type channels. A similar effect on channel activity was observed using JPs gA 17O-Leu10 increased by an order of magnitude. Line narrowing due to the in which deletion of the C-terminal, ER-anchoring transmembrane domain increase in field revealed that the 30 ppm wide seemingly single peak of disrupts their ability to form clusters. This indicates that channel activity is Leu10 at 19T consist of 2 peaks with linewidths of about 5 ppm each. Ion regulated by direct interaction with JPs and not as a result of channel clustering binding to Leu10 follows previous reported trends Liþ>Kþ>Ba2þ.Also, in the PM-ER junction. ion binding was shown to happen with different affinities to each of the monomers Leu10. The presence of two NMR peaks in the absence of ions, however, could possibly be correlated to different monomer geometry and/ Posters: Ion Channels, Pharmacology, and or differences in water hydrogen bonding. This study aims to unravel the Disease I detailed chemistry of ion binding and hydrogen bonding to gA demonstrating the new frontiers that can be reached by the use of 17O NMR at very high 1530-Pos Board B439 fields. Istaroxime Accelerates Calcium Transient Decay in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes 1528-Pos Board B437 Beatrice Badone1, Roel Spatjens2, Cristina Altrocchi2, Paul Volders2, Model Development of SK Channel Gating Incorporating Calcium Sensi- Antonio Zaza1. 1 tivity and Drug Interaction Biotechnology and Biosciences, University Milan-Bicocca, Milan, Italy, 2 Ilse van Herck1, Bo H. Bentzen2,3, Vincent Seutin4, Hemenegild Arevalo1, Cardiology, University of Maastricht, Maastricht, Netherlands. 2þ Mary M. Maleckar1, Neil V. Marrion5, Andrew G. Edwards1,6. Altered cardiomyocyte Ca handling is involved in many pathological conditions. 1Simula Research Laboratory, Oslo, Norway, 2Acesion Pharma, Patients with acute heart failure (AHF) may require immediate hemodynamic sup- Copenhagen, Denmark, 3Biomedical Institute, University of Copenhagen, port by using positive inotropic/lusitropic agents. Istaroxime is a luso-inotropic 2þ Copenhagen, Denmark, 4Neurophysiology unit, GIGA Neurosciences, compound that stimulates SERCA (sarcoplasmic reticulum Ca -ATPase) by pre- 5 venting its interaction with phospholamban (PLN) and, as an ancillary property, University of Lie`ge, Lie`ge, Belgium, School of Physiology, Pharmacology þ þ and Neuroscience, University of Bristol, Bristol, United Kingdom, also inhibits the Na /K ATP-ase. Istaroxime has been extensively characterized 6Physiology and Cell Biology, University of Oslo, Oslo, Norway. on adult mammalian cardiomyocytes. Here we test whether istaroxime is able to Small conductance calcium activated potassium (SK) channels are stimulate SERCA function also in human induced pluripotent stem cell-derived predominantly expressed in heart atria and contribute to late repolarization of cardiomyocytes (hiPSC-CMs). This precedes the evaluation of istaroxime effects the action potential. Inhibition of SK current has been proposed as a therapy on disease-specific hiPSC-CMs. 2þ for atrial fibrillation (AF). We aim to develop an accurate model of SK channel Methods: we performed simultaneous voltage-clamp and intracellular Ca 2þ gating and drug interaction that can be used to probe the role of SK channels in recordings. Ca transients were recorded during 200 ms voltage steps from atrial health and disease. The model, based on Hirschberg et al’s (1998) SK2 40 mV to 0 mV applied at 3-second cycle length. Cells were superfused model, incorporates 4 closed and 2 open states. To generate a model with utility with Tyrode’s solution (control condition) and, subsequently, with istarox- 2þ at physiologic temperature, we performed inside-out macropatch voltage clamp ime (4 mM). Analysis was focused on Ca -transient amplitude and decay 2þ ramps at 23 and 37 C, and observed a pronounced leftward EC50 shift at 37 C rate, which mainly reflect the ability of SERCA to reuptake Ca into the (0.53 5 0.07 mM and 0.38 5 0.02 mM to 0.23 5 0.02 mM and 0.28 5 0.01 mM sarcoplasmic reticulum (SR). Results: we found that istaroxime accelerates 2þ for hSK3 and hSK2, respectively). Thus, taking calcium activation of the major Ca transient decay (n=10 cells; tdecay from 465579 ms to 313546 ms; hSK isoforms into the diastolic calcium range. To provide a first-principles ba- mean5SEM; p<0.005). In contrast to adult guinea pig cardiomyocytes, is- 2þ sis for constraining the kinetics of multi-step calcium-dependent activation, and taroxime did not change Ca -transient amplitude in hiPSC-CMs (from binding of known and novel pharmacologic inhibitors, we performed single- 1.3450.04 nM to 1.2950.05 nM). Conclusions: although hiPSC-CMs 2þ channel recordings of SK2 at 23 C. Opening events without inhibitors were differ from adult cardiomyocytes in several Ca handling properties, our 2þ best fit with two major open-state conductances (low: 10.9 5 1.6 pS, 60 5 data indicate that istaroxime modulates Ca dynamics in a way compatible 2þ 41% of events; high: 14.9 5 1.4 pS, 40 5 41% of events), justifying the mul- with SERCA stimulation. Lack of effect on the expected increase in Ca - 2þ tiple open states included in the model and to be further analysed regarding transient amplitude may be due to experimental run-down of L-type Ca state specific kinetics. We will also present single channel data on known inhib- current. itor apamin and novel modulator AP14145 constraining a pharmacological model. In conclusion, these experiments provided novel findings on tempera- 1531-Pos Board B440 ture sensitivity of SK channels. These will be incorporated into an improved Patient-Specific Mutations Impair Bestrophin1’s Essential Role in Medi- Markov model of SK channel dynamics to elucidate the roles of SK channels ating Ca2D-Dependent CL- Currents in Human RPE in health and disease. Yao Li1, Yu Zhang2,YuXu3, Alec Kittredge2, Nancy Ward2, Shoudeng Chen4, Stephen Tsang1, Tingting Yang2. 1529-Pos Board B438 1Columbia University, New York, NY, USA, 2University of Rochester, Role of Neuronal Junctophilins in Recruitment and Modulation of Rochester, NY, USA, 3Xinhua Hospital affiliated to Shanghai Jiao Tong Voltage-Gated Calcium Channels in PM-ER Junctions University, Shanghai, China, 4The Fifth Affiliated Hospital of Sun Yat-sen Stefano Perni, Kurt G. Beam. University, Zhuhai, China. Physiology and Biophysics, University of Colorado, Aurora, CO, USA. Mutations in the human BEST1 gene lead to retinal degenerative diseases Junctions between the plasma membrane (PM) and the endoplasmic reticulum displaying progressive vision loss and even blindness. BESTROPHIN1, (ER), which have been observed in many cell types, are subcellular encoded by BEST1, is predominantly expressed in retinal pigment epithe- compartments in which multiple channels, both in the PM (ligand or lium (RPE), but its physiological role has been a mystery for the last two voltage-gated channels) and in the ER (ryanodine receptors and IP3 receptors) decades. Using a patient-specific iPSC-based disease model and interdisci- cross-talk with one other and regulate local calcium dynamics. Junctophilins plinary approaches, we comprehensively analyzed two distinct BEST1 pa- (JPs) were among the first identified proteins to be involved in the formation tient mutations, and discovered mechanistic correlations between patient and stabilization of PM-ER junctions, principally in muscle and neurons. While clinical phenotypes, electrophysiology in their RPEs, and the structure and the crucial role in cardiac and skeletal muscle contractility of the two muscle function of BESTROPHIN1 mutant channels. Our results revealed that the isoforms JP1 and JP2 is demonstrated by the severity of the KO phenotypes disease-causing mechanism of BEST1 mutations is centered on the (perinatal and embryonic lethality respectively), the roles of the neuronal indispensable role of BESTROPHIN1 in mediating the long speculated junctophilins (JP3 and JP4) appear to be more subtle and less well understood. Ca2þ-dependent Cl- current in RPE, and demonstrate that the pathological While knock-outs for JP3 or JP4 exhibit only mild or no phenotype, double potential of BEST1 mutations can be evaluated and predicted with our knock-outs show motor discoordination, impaired motor learning and impaired iPSC-based ‘‘disease-in-a-dish’’ approach. Moreover, we demonstrated memory, suggesting multiple roles of JPs in the nervous system. Here we tried that patient RPE is rescuable with viral gene supplementation, providing a to clarify how voltage-gated calcium channels localize and function in neuronal proof-of-concept for curing BEST1-associated diseases.

BPJ 8632_8635 Monday, February 19, 2018 307a

1532-Pos Board B441 (polyQ) tract within the N-terminal region of the huntingtin (HTT) gene. The D242N, a KV7.1 LQTS Mutation Uncovers a KEY Residue for IKS Voltage disease affects 1 in 10000 individuals and the main symptoms are cognitive Dependence dysfunction and motor disorders. Cristina Moreno, Anna Oliveras, Chiara Bartolucci, Carmen Mun˜oz, Ryanodine receptors (RyRs) are calcium (Ca2þ) release channels on the Alicia de la Cruz, Diego A. Peraza, Juan R. Gimeno, endo/sarcoplasmic reticulum (ER/SR). RyR is normally closed at low cytosolic Mercedes Martin-Martinez, Stefano Severi, Antonio Felipe, [Ca2þ] (100-200nM). The closed state of RyR is stabilized by calstabin Pier D. Lambiase, Teresa Gonzalez, Carmen Valenzuela. proteins, which prevent pathological intracellular Ca2þ leak. Institute of Biomedical Research CSIC-UAM, Madrid, Spain. Excessive basal Ca2þ release (Ca2þ leak) through RyR leading to depletion of KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most internal Ca2þ store has been reported in HD. However, the mechanisms by important repolarizing currents of the cardiac action potential. Its relevance is which neuronal RyR2 channel dysfunction contributes to HD remain to be underscored by the identification of more than 500 mutations in KV7.1 and, at fully elucidated. In this study we show that RyR2 are leaky in post least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this mortem brains of HD patients using single channel recordings. Via biochemical study was to characterize the biophysical and cellular consequences of the analysis we report that RyR2 undergoes post-translational modifications D242N KV7.1 mutation associated with the LQTS. The mutation is located (phosphorylation, oxidation and nitrosylation) in brains of 2 murine models of in the S4 transmembrane segment, within the voltage sensor of the KV7.1 chan- HD (Q111, Q175). Moreover, RyR2 is depleted of the stabilizing nel, disrupting the conserved charge balance of this region. Perforated patch- subunit calstabin2 (FKBP 12.6) from the channel macromolecular complex lead- clamp experiments show that, unexpectedly, the mutation did not disrupt the ing to ER Ca2þ leak as determined using a microsomal Ca2þ leak assay. Rycal voltage-dependent activation but it removed the inactivation and slowed the (S107), a RyR stabilizing drug, repairs the leak in RyR2 channels by inhibiting activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface pro- oxidation-induced dissociation of calstabin2 from the RyR2 channels. tein and co-immunoprecipitation experiments revealed that neither plasma These results provide evidence suggesting that ER Ca2þ leak via neuronal membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered RyR2 channels contributes to HD neuropathology. by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized po- 1535-Pos Board B444 Structural Modeling of Full-Length KCa Channels using Rosetta tentials (þ50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical Heesung Shim, Heike Wulff, Kevin DeMarco, Vladimir Yarov-yarovoy. phenotype of the LQTS patients carrying the D242N mutation is due to UC Davis, Davis, CA, USA. impaired action potential adaptation to exercise and, in particular, to increase Small-conductance (KCa2.1-2.3) and intermediate-conductance (KCa3.1) in heart rate. Moreover, our data identify D242 aminoacidic position as a po- calcium-activated potassium channels are crucial modulators of neuronal tential residue involved in the KCNE1-mediated regulation of the voltage excitability and calcium signaling throughout the body. While KCa2 chan- nels are widely expressed in the central nervous system, where they underlie dependence of activation of the KV7.1 channel. Funded by SAF2013-45800- R, SAF2016-75021-R, BFU2014-54928-R, RIC RD12/0042/0019 and CIBER the neuronal after hyperpolarization, KCa3.1 is predominantly found in pe- CB/11/00222. ripheral tissues such as the vascular endothelium and white and red blood cells. KCa channels have, therefore, been suggested as drug targets for epi- 1533-Pos Board B442 lepsy, ataxia, hypertension and immunsuppression. However, there currently is no available crystal structure of these medically relevant channels that Normethsuximide and Ethosuximide Potentiate a1b3g2 GABAA Recep- tors and Alleviate Pentylenetetrazol-Mediated Inhibition in Cultured could be used for structure-assisted drug design. In this work, we used Roset- HEK293 Cells taCM computational modeling software to generate a full-length open-state Brendan Ito, Yongli Chen. model of KCa3.1. The KCa3.1 S1-S4 region was modeled using the crystal Hawai’i Pacific University, Kaneohe, HI, USA. structure of KCNQ1 (PDB ID: 5VMS) as a template. The pore region was The succinimides methsuximide (MS) and ethosuximide (ES) are first-line generated based on the pore-forming domain structure of the Kv1.2-Kv2.1 treatments for absence seizures, and much of the work over the past two de- chimeric channel (PDB ID:2R9R) and the calmodulin binding domain cades has focused on their action as open-channel blockers of the T-type (Cam-BD) was modeled using the KCa2.2 Cam-BD crystal structure (PDB voltage-dependent calcium channel (VDCCs). However, it is questionable ID:4J9Z). Furthermore, we modeled the pore region of KCa2.2 using the as to whether the blockade of T-type VDCCs fully explains the therapeutic KCa3.1 pore homology model as a template. We generated 10,000 KCa3.1 actions of succinimides, and there are mixed reports regarding the affinity of and KCa2.2 models and evaluated them using the membrane environment specific energy function and clustering approach. We are currently probing these compounds to GABAA receptors (GABAARs), suggesting ES and MS may have novel anti-absence seizure actions that are mediated via specific both the full-length KCa3.1 and the KCa2.2 pore models by docking KCa channel inhibitors and gating modulators using RosettaLigand. Our struc- subtypes of GABAARs. Here, we examine the impact of ES, MS, and the primary metabolite of MS, a-methyl-a-succinimide (normethsuximide/ tural modeling using Rosetta may help elucidate the molecular mechanism of KCa channel modulators and could be useful for structure-based drug NMS), on GABAAR currents using in vitro whole-cell patch-clamp electro- physiology. Specifically, we compare the effects of these compounds on design. HEK293 cells expressing specific subtypes of GABAARs, the WSS-1 cell 1536-Pos Board B445 line stably expressing a1b3g2GABAARs and HEK293 cells transiently ex- a b g Dual Effect of Amiodarone on the Oncogenic Kv10.1 Channel pressing 1 3(G32R) 2GABAARs containing a single amino acid residue Froylan Gomez-Lagunas, Carolina Barriga-Montoya, mutation. In addition, we use real-time quantitative PCR (RT-qPCR) to Areli Huanosta-Gutierrez. verify the expression of GABAAR subunit mRNA transcripts in these cell Physiology, UNAM, Mexico City, Mexico. lines. Our preliminary results show that ES and NMS, but not MS, þ a b g Ectopic expression of the voltage-dependent K channel Eag1 (or Kv10.1) is potentiates 1 3 2GABAARs and alleviates pentylenetetrazol (PTZ)- associated with 70% of human cancers and, reciprocally, Kv10.1 channel inhi- mediated inhibition of the receptor. We also discuss the implications bition hinders tumors growth. In agreement with its particular role in cancer, of our results within the context of generalized absence seizures, and recent reports have shown that Kv10.1 presents structural, and functional, þ consider how changes in the expression of GABAAR subtypes as a conse- characteristics that depart from those common to canonical, Shaker-related, K quence of absence seizures may contribute to the efficacy of these channels. Amiodarone is an anti-arrhythmic drug thought to bind within the compounds. pore of canonical Kþ-channels. In this work we studied the interaction of amio- darone with Kv10.1. The drug affects these channels at nanomollar concentra- 1534-Pos Board B443 tions, and that amiodarone exerts a dual effect on Kv10.1, depending on the Exploring the Role of Ryanodine Receptors in Huntington’s Disease time-window of channel activity and on the resting membrane potential from Pathophysiology which channels are activated. On a time scale of hundreds of milliseconds to sec- 1 1 1 Panagiota Apostolou , Steven Reiken , Qi Yuan , Kaylee Wedderburn- onds amiodarone induces a slow channel inhibition (or apparent inactivation) that 1 2 1 1 1 Pugh , Felicia Benoit , Ari Moscona , Kavin Chada , Andrew Marks . reduces the Kþ conductance. On the other hand, looking at the first milliseconds 1Physiology and Cellular Biophysics, Columbia University Medical Center, New 2 of channel activity, we observed that, surprisingly, amiodarone accelerates the ki- York, NY, USA, Psychology, New York University, New York, NY, USA. netics of Kþ current activation. As a result of this, the initial Kþ current surge of Huntington’s disease (HD) is an autosomal dominant progressive Ad-modified channels may transiently surpass IK of unmodified channels, depend- neurodegenerative disorder caused by an abnormal expansion of polyglutamine ing on the resting membrane potential from which the currents are activated. The

BPJ 8632_8635 308a Monday, February 19, 2018 latter is observed in external solutions containing either zero or 2 mM response identical to that of cbv1 alone: ethanol inhibition was blunted. external Mg2þ ions. These observations suggest that amiodarone binds to the Collectively, our data suggest that hydrogen bonding between Ser160 voltage-sensor module, and hence modifies conformational changes of Kv10.1 and ethanol’s hydroxyl group is essential for ethanol-mediated inhibition channels. of slo1þbeta1 current. Consistent with in vitro electrophysiology find- ings, KCNMB1 K/O mouse arteries electroporated with beta1S160A 1537-Pos Board B446 cDNA failed to constrict in response to ethanol whereas electroporation Polypeptide Toxins: Two New Inhibitors of the Oncogenic Potassium with wt beta1 cDNA evoked ethanol-induced vasoconstriction. In Channel Kv10.1 conclusion, Ser160 in the beta1-TM2 region is required for ethanol- 1 2 2 Enoch Luis , Erika Monserrat Torres-Moales , Arlet Loza-Huerta , induced inhibition of beta1-containing BK channels and the resulting ce- 3 2 Sergio Roma´n-Gonza´lez , Roberto Arreguin-Espinosa , Cesar Oliver Lara- rebral artery constriction. Support: R37-AA11560 (AMD) and AHA Pre- 2 2 4 2 Figueroa , Arturo Herna´ndez-Cruz , Lourival D. Possani , Arturo Picones . doctoral Fellowship (GK). 1CONACyT - Instituto de Fisiologı´a Celular, UNAM, Mexico City, Mexico, 2National Laboratory of Channelopathies. Universidad Nacional Auto´noma 1539-Pos Board B448 3 de Mexico, Mexico City, Mexico, Instituto de Quı´mica. Universidad Introducing Simulated IK1 into Human iPSC-Cardiomyocytes using Nacional Autonoma de Mexico, Mexico City, Mexico, 4Instituto de Dynamic Clamp on an Automated Patch Clamp Platform Biotecnologı´a. Universidad Nacional Auto´noma de Mexico, Cuernavaca, Corina Bot1, Nadine Becker2, Birgit Goversen3, Sonja Stoelzle-Feix2, Morelos, Mexico. Alison Obergrussberger2, Toon A.B. van Veen3, Niels Fertig2, In recent years, different studies have shown that the expression of the Teun P. de Boer3. voltage-gated potassium channel ether a` go-go 1 (eag1, Kv10.1) induces 1Nanion Technologies Inc, Livingston, NJ, USA, 2Nanion Technologies, proliferation in several cancer cell lines and in vivo tumor models, while Munich, Germany, 3Department of Medical Physiology, Division of Heart & blockage or silencing of the channel inhibits proliferation. Due to these find- Lungs, University Medical Center Utrecht, Utrecht, Netherlands. ings, Kv10.1 has been proposed as an early cancer target. The goal of this Dynamic clamp is a powerful tool involving injection of real-time work is to identify new and potent Kv10.1 channel antagonists. Using simulated membrane currents into patch clamped cells. This technique HEK293 cells stably expressing Kv10.1 (HEK-Kv10.1), we initially has been employed in conventional patch clamp electrophysiology to screened 40 different new potential modulatory polypeptides by measuring introduce inward rectifier IK1 current into human induced pluripotent stem þ Tl inflow through Kv10.1 by a fluorescence-based assay (FLIPRÒ Potas- cell-derived cardiomyocytes (hiPSC-CMs). IK1 is expressed at low levels in sium Assay Kit, Molecular Devices). We found two new polypeptide toxins, these cells. For this reason, hiPSC-CMs display a more depolarized one isolated from the marine cone snail Conus regularis (RsXXVIIA) and membrane potential than adult cardiomyocytes, limiting their use in safety the other from the scorpion Centruroides noxius (CnMt9), exerting potent pharmacology. Therefore, introducing simulated IK1 into hiPSC-CMs may inhibitory effects on the Tlþ influx. Whole-cell patch-clamp recordings of improve maturity of these cells and ensure that they represent a viable Kþ currents recorded from HEK-Kv10.1, revealed that RsXXVIIA (1 alternative to the scarcely available dissociated adult human cardiomyocytes. nM) and CnMt9 (2 mg/ml) inhibit Kv10.1 currents measured at þ50 mV Indeed, they are attractive cells types because of their unlimited availability by 75% and 88%, respectively. Conductance-voltage relationship fitted and human origin. with Boltzmann equations showed that RsXXVIIA (1 nM) shifts to the right In this study, we combined dynamic clamp with an automated patch the half-activation voltage (V1/2) by 27.7 mV, without changing the slope clamp platform to demonstrate that IK1 conductance can be added to hiPSC- factor; CnMt9 shifts to the right the V1/2 by 19.1 mV and increased the slope CMs on this platform. Our results show that IK1 can be successfully added to factor from 15.5 to 33.0 (control versus CnMt9 at 2 mg/ml). RsXXVIIA and hiPSC-CMs to up to 4 cells simultaneously and that this results in a more sta- CnMt9 exerted a concentration-dependent inhibition of Kv10.1 bilized and hyperpolarized resting membrane potential. Action potential (AP) channels with an IC50 of 4.5 pM and 14.2 ng/ml, respectively. The potent shape also changes when IK1 is added. We have used with different amounts inhibitory effects of these two new polypeptide toxins on Kv10.1 activity of IK1 (100-2000 pS/pF) and show that increasing IK1 results in AP shortening make them attractive compounds for targeting Kv10.1 with therapeutic and an acceleration of the upstroke. We could measure native Ba2þ-sensitive potential. IK1 in voltage clamp mode in approximately 50% of these cells, but IK1 was Supported by grants 279820 (Laboratorio Nacional de Canalopatı´as) and CB small, on average 1.9850.42 pA/pF (mean 5 SEM). Adding a Ca2þ channel 240305 (CONACYT), and PAPIIT IN211616 (DGAPA-UNAM). activator (BayK 8644), or blocker (nifedipine) caused an increase and decrease of the AP duration, respectively. In conclusion, combining dynamic clamp with 1538-Pos Board B447 automated patch clamping results in an enhanced, medium-throughput platform Identification of an Ethanol Recognition Site in BK Beta1 Subunit that for safety pharmacology. Mediates Ethanol-Induced Cerebral Artery Myocyte BK Channel Inhibition and the Resulting Artery Constriction Guruprasad Kuntamallappanavar, Anna Bukiya, Alex Dopico. 1540-Pos Board B449 Pharmacology, The University of Tennessee Hlth. Sci. Ctr., Memphis, TN, Mechanism of Gating of the Intermediate-Conductance Calcium-Activated USA. Potassium Channel (KCa3.1) Ethanol at concentrations obtained in circulation during moderate-heavy Brandon M. Brown, Heesung Shim, Heike Wulff. episodic drinking (30-80 mM) decreases the activity of voltage/calcium- Pharmacology, UC Davis, Davis, CA, USA. gated, large conductance potassium (BK) channels in smooth muscle Small-conductance (KCa2) and intermediate-conductance (KCa3.1) calcium- (SM), which leads to cerebral artery constriction (Liu et al., 2004). activated Kþ channels are voltage-independent and share a common The SM-abundant BK beta1 subunit (encoded by KCNMB1) is necessary calcium/calmodulin mediated gating machinery. The existing KCa activators, for ethanol to inhibit cerebral artery SM BK channels under physiolog- such as SKA-31, NS309, and EBIO, activate both channel types with similar ical voltages and calcium (Bukiya et al., 2009, Kuntamallappanavar and potencies, though they are slightly (5-10 fold) selective for KCa3.1 over Dopico, 2016). Moreover, beta1 transmembrane domain 2 (TM2) is KCa2. Previously, we optimized the benzothiazole pharmacophore of required for such action of ethanol (Kuntamallappanavar and Dopico, SKA-31 toward KCa3.1 selectivity and identified SKA-121, which 2017). The precise ethanol recognition site(s) in beta1-TM2 that medi- exhibits 40-fold selectivity for KCa3.1 over KCa2.3. . We then tested why ates ethanol-induced BK channel inhibition and associated cerebral ar- introducing a single CH3 group in 5-position of the benzothiazole/oxazole tery constriction has remained unknown. Here, we address this system achieves such a gain in selectivity for KCa3.1 by generating models question by combining computational modeling, mutagenesis, patch- of the KCa3.1 and KCa2.3 CaM-BD/CaM complexes with SKA-121 using clamp electrophysiology, in vitro artery electroporation and pressuriza- Rosetta computational modeling software. We found that all benzothiazole/ tion. Computational dynamics revealed two potential ethanol- oxazole-type KCa activators bind relatively "deep" in the CaM-BD/CaM recognition residues in beta1-TM2: Ser160 and Thr165. Macroscopic interface and hydrogen bond with E54 on CaM. In KCa3.1, SKA-121 forms currents evoked from BK channel-forming cbv1 subunits co-expressed an additional hydrogen bond network with R362. In contrast, NS309 sits with beta1T165A displayed an ethanol response similar to that of more "forward" and directly hydrogen bonds with R362 in KCa3.1. Mutating cbv1þbeta1, allowing ethanol inhibition of current at physiological, in- R362 to serine, the corresponding residue in KCa2.3 reduces the potency of ternal calcium>2 microM. In contrast, currents from cbv1þbeta1- SKA-121 by 7-fold, suggesting that R362 is responsible for the S160A,T165A and cbv1þbeta1S160A channels displayed an ethanol generally greater potency of KCa activators on KCa3.1. The increase in

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SKA-121’s KCa3.1 selectivity compared with its parent, SKA-31, seems to be glutamate, receptor desensitization, probability of opening, receptor due to better overall shape complementarity and hydrophobic interactions with surface trafficking, and the effect of: pregn-5-ene-20-on-3b-yl-sulfate, S372 and M368 on KCa3.1 and M72 on CaM at the KCa3.1-CaM-BD/CaM 5b-pregnan-20-on-3b-yl-20-butyric acid, and 5b-androstan-3a-yl-hemi- interface. Investigations into SKA-121’s mechanism of action revealed that succinate (AhS) (steroids that exhibit a positive allosteric effect at both SKA-121 and NS309 not only shift the Ca2þ concentration-response NMDARs). The following human receptors were studied: GluN1/ curve of KCa3.1 to the left but also increase PO at saturating Ca2þ concentra- GluN2B (P553L; V558I; A590T; W607C; N615I; V618G; S628F; tions. We are currently investigating whether PIP2 is a cofactor for activation, E657G; Q662P; D668N; R682C; G799S; E807K; G820E; G820A; like it is for KCa2, and whether it is involved in the effects of SKA-121 and M824R; L825V). Our results indicate on multiple effects of mutations NS309. - 4 did not respond to agonist application while 9 responded with Supported by R21NS101876 and UL1 TR001860 (NIH). currents significantly diminished compared to the WT; 5 had reduced surface expression; 2 had reduced glutamate or glycine affinity; 1 had 1541-Pos Board B450 increased receptor desensitization, and 3 exhibited lower probability of Tetrahy- Development of KV1.3-Blocking Monoclonal Antibodies using channel opening. Steroidal compounds exhibited potentiating action mena thermophila that varied depending on both the type of mutation and the steroid. 1 2 3 4 Janna Bednenko , Rian Harriman , Lore Marie¨n , Hai M. Nguyen , The most prominent potentiation (1822%) was observed for 30 mM 1 1 1 1 Alka Agrawal , Ashot Papoyan , Yelena Bisharyan , Joanna Cardarelli , AhS at GluN1/GluN2B(L825V) receptors. These results suggest potential 1,5 5 3 3 Ted Clark , Donna Cassidy-Hanley , Bas van der Woning , Hans de Haard , therapeutic role neurosteroid analogsmayplayinpatientswithNMDAR 2 4 1 Ellen Collarini , Heike Wulff , Paul Colussi . channelopathies. 1TetraGenetics Inc, Arlington, MA, USA, 2Crystal Bioscience, Emeryville, CA, USA, 3argenx, Zwijnaarde, Belgium, 4Pharmacology, University of 1543-Pos Board B452 California, Davis, CA, USA, 5Immunology and Microbiology, Cornell Novel Drugs that Augment KCNQ (KV7, ‘‘M-Type’’) Potassium Channels University, Ithaca, NY, USA. as a Post-Event Treatment for Traumatic Brain Injury Expression of the voltage-gated Kv1.3 potassium ion channel, whose role Isamar Sanchez, Fabio Antonio, Borges Vigil, Eda Bozdemir, involves regulation of membrane potential and calcium signalling in both Rafael J. Veraza, Liliana Espinoza, Deborah M. Holstein, MaryAnn Hobbs, excitable and non-excitable cell types, is upregulated in activated immune Vladislav Bugay, James Lechleiter, Robert Brenner, Mark S. Shapiro. cells associated with inflammatory and autoimmune diseases such as Departments of Cell and Integrative Physiology, Cell & Structural Biology, ischemic stroke, Alzheimer’s disease, type 1 diabetes, multiple sclerosis, and Neurosurgery, The University of Texas Health Science Center at San and psoriasis. Many studies have shown that specific Kv1.3 blockers are Antonio, San Antonio, TX, USA. effective in a number of animal models of these inflammatory conditions Traumatic brain injury (TBI) is a risk factor for development of many demonstrating that Kv1.3 is an attractive target for therapeutic interven- cerebral disorders, i.e. epilepsy. We hypothesize that reducing neuronal tion. Despite its clinical relevance, however, current small molecule and excitability soon after TBI blunts epilepsy development by abrogation of peptide toxin drugs targeting Kv1.3 suffer a lack of selectivity, less neuronal hyperexcitability, inflammatory responses, and as yet unknown than optimal bioavailability and/or poor pharmacokinetics. Monoclonal an- cascades causing myriad behavioral, cognitive and psychiatric disorders. tibodies (mAbs) are widely considered superior to classical small mole- Since enhancing KCNQ (‘‘M-type’’) Kþ currents silences neurons and pre- cule and peptide drugs in these aspects but technical difficulties in vents seizures, such as via the M-channel ‘‘opener’’ and anti-convulsant producing sufficient recombinant immunizing channel proteins remain a drug, retigabine (RTG), we used a controlled closed-cortical impact serious challenge in their development. We discuss here a general strategy (CCCI) model of TBI to test whether RTG administration soon after TBI to overcome this critical hurdle by combining overexpression of recombi- prevents post-TBI dysfunction. Mice were subjected to CCCI TBI, followed nant Kv1.3 in Tetrahymenathermophila with immunization of phylogenet- by RTG (i.p. 1 mg/kg) or vehicle within 30 minutes post-TBI. After 24h, ically diverse species and unique screening tools that allow deep-mining mice were monitored continually by video/EEG for electrical activity and for antibodies that could potentially bind functionally important regions seizures. To assay seizure susceptibility post-TBI, mice were challenged of the channel protein. We show that the resulting recombinant Kv1.3 with pilocarpine (3X, 75 mg/kg, 6 days after TBI), and mouse cohorts traffics to the cell surface, is correctly folded, adopts a native tetrameric compared. Somatic areas were quantified in Nissl-stained brain slices to conformation and is readily purified from membranes. Recombinant Kv1.3 measure osmotic cell swelling, which was significantly reduced in was then used to generate and recover 70 full-length anti-Kv1.3 mAbs RTG-treated post-TBI mice. Inflammation and astrogliosis are hypothesized from immunized chickens and llamas, of which, 10 were able to inhibit to be critical mediators of post-TBI maladaptive responses, both brain Kv1.3 current. Select antibodies were shown to be potent (IC50<10nM) hemispheres were assayed by immunoblotting for expression of CD40L and specific for Kv1.3 over related Kv1 family members, hERG and and GFAP, two known markers of these processes, respectively, that are hNav1.5. correlated with TBI severity. We found that TBI caused spontaneous seizures in 33% of vehicle-only-treated mice. Of mice administered 1542-Pos Board B451 RTG after TBI, not a single mouse displayed spontaneous seizures. After Function, Expression, and Pharmacology of Disease-Associated Mutations pilocarpine challenge, about half of vehicle-treated mice displayed seizures of NMDA Receptors within 24h, whereas less than a third of RTG treated mice did so, and RTG 1 1 1 1 Vojtech Vyklilcky , Barbora Karusova , Bohdan Kysilov , Marek Ladislav , treatment significantly increased the latency to first seizure. Thus, KCNQ/M 1 1 1 2 Pavla Hubalkova , Tereza Smejkalova , Martin Horak , Hana Chodounska , channel-enhancing drugs may serve as a novel, effective treatment for Eva Kudova2, Jiri Cerny1, Ladislav Vyklicky1. 1 2 deleterious effects that follow TBI. Supported by DoD grants W81XWH- Institute of Physiology CAS, Prague, Czech Republic, Institute of Organic 13/15-1-0284. Chemistry and Biochemistry CAS, Prague, Czech Republic. N-methyl-D-aspartate receptors (NMDARs) mediate synaptic plasticity, 1544-Pos Board B453 and their dysfunction is implicated in multiple brain disorders. Inherited Disease-Associated Mutations Reveal a Conserved Glycine that Stabilizes or de novo mutations in human GRIN2B gene, encoding the GluN2B Opposing Channel Conformations in Ionotropic Glutamate Receptors subunit of the NMDAR, have been linked to a number of neurodevelop- Johansen Amin1, Xiaodong Pang2, Aaron Gochman3, Mark E. Bowen4, mental disorders. Functional analysis of these receptor variants is often Huan-Xiang Zhou2, Lonnie P. Wollmuth5. missing, thus the link between mutations in GluN2B subunit and patient 1Molecular and Cellular Pharmacology, Stony Brook University, Port phenotype as well as possible treatment strategies remain unclear. Here Jefferson, NY, USA, 2Department of Physics and Institute of Molecular wefocusedontheidentificationoffunctional, pharmacological and sur- Biophysics, Florida State University, Tallahassee, FL, USA, 3Neurobiology face trafficking abnormalities of NMDARs containing single point and Behavior, Stony Brook University, Stony Brook, NY, USA, 4Physiology missense mutations in GluN2B subunit that have been found in patients and Biophysics, Stony Brook University, Stony Brook, NY, USA, diagnosed with autism spectrum disorders and/or intellectual disability. 5Neurobiology and Behavior, Stony Brook University, Port Jefferson, NY, USA. Patch-clamp recordings and immunohistochemistry of human NMDARs A variety of de novo missense mutations associated with neurological heterologously expressed in HEK293 cells was used to look for disorders occur in ionotropic glutamate receptors (iGluRs) subunits. These differences between wild-type (WT) and mutated NMDAR. Particularly, disease mutations often appear in motifs critical to the process of we determined the GluN1/GluN2B receptor affinity for glycine and glutamate-induced channel opening or gating. Numerous missense

BPJ 8632_8635 310a Monday, February 19, 2018 mutations are found in the NMDA receptor M4 segments, a transmembrane identified in several genes encoding proteins that collectively participate segment peripheral to the pore domain in eukaryotic iGluRs. Subsets of in glycinergic neurotransmission. The major target of hyperekplexia mu- these missense mutations affect receptor gating but very dramatic effects, tations is the a1 subunit within the glycine receptor family, which con- including in one instance halting gating, occurred at a conserved glycine, sists of five homologous members, four a subunits (GLYRA1-4) and a positioned at the extracellular end of M4. Even alanine substitutions at single b subunit (GLYRB). Glycine receptors are pentameric ligand- this glycine, most notably in GluN1, severely restricted the stability of gated chloride channels that are predominantly expressed in inhibitory the open state. Molecular dynamics simulations suggest that this glycine interneurons of the spinal cord and brain stem, where they mediate post- in GluN1 permits unique backbone interactions as well as the extreme extra- synaptic hyperpolarization. The GLYRA1 can functionally assemble both cellular end of M4 to unravel in the open state. Surprisingly, in an AMPAR as a homopentamer and a GLYRA1/GLYRB heteropentamer. Here, we structure, reorientation of the extreme extracellular end of M4 centered on addressed the question whether a loss-of-function of the GLYRA1 may this conserved glycine occurs, but in contrast to NMDARs, in a closed state. result from a mutation-induced defect in the pentameric assembly. To Consistent with these structural differences, an alanine substitution at this this end, we individually expressed a total of 42 hyperekplexia-causing glycine in AMPARs destabilizes a closed state, in direct contrast to that GLYRA1 point mutants, each with a C-terminal hexahistidyl tag, in for NMDARs. Hence, structural features unique to glycine stabilize Xenopus laevis oocytes. We labeled the newly synthesized and the opposing conformations in iGluR subtypes. In NMDARs, missense plasma membrane-bound GLYRA1 mutants with [35S]methionine and mutations at this conserved glycine dramatically alter their function at an amine-reactive fluorescent dye, respectively. We purified the recom- synapses. These results have strong implications for how such disorders binant proteins by metal affinity chromatography, and analyzed their are caused at the ion channel level and highlight how structural features oligomeric and monomeric state by blue native polyacrylamide gel elec- in NMDAR and AMPAR have evolved to permit them to carry out different trophoresis (BN-PAGE) and SDS-PAGE, respectively. We also ex- functional roles at synapses. pressed the same constructs for two-electrode voltage clamp recordings. About 40% of the hyperekplexia mutations led to a signifi- 1545-Pos Board B454 cant reduction of the homopentameric assembly. The assembly- Development and Application of a Peptide Inhibitor-Bound Quantum Dot affecting mutations were predominantly located in the transmembrane Targeting the Voltage-Gated Potassium Channel KV1.3 in the Olfactory helices TM1, TM3 and TM4 that we have previously shown to be Bulb crucial for homopentamer formation (Haeger et al., NSMB 17, 90-98, 1 2 3 4 Austin B. Schwartz , Anshika Kapur , Zhenbo Huang , Raveendra Anangi , 2010). In contrast, mutations located in the pore-lining TM2 or the 4 3 2 4 Zoltan Dekan , Erminia Fardone , Goutam Palui , Glenn F. King , loop regions mostly affected glycine-gated channel function but not 2 5 Hedi Mattoussi , Debra A. Fadool . homopentamerization. 1The Molecular Biophysics Program, Florida State University, Tallahassee, FL, USA, 2Department of Chemistry and Biochemistry, Florida State 1547-Pos Board B456 University, Tallahassee, FL, USA, 3Department of Biological Science, Atomistic Simulation of Lipid Membrane Permeation for Cardiac Ion Program in Neuroscience, Florida State University, Tallahassee, FL, USA, Channel Blockers 4Institute for Molecular Bioscience, University of Queensland, Brisbane, Kevin R. DeMarco1,2, Slava Bekker3, Colleen E. Clancy2, Australia, 5Department of Biological Sciences, Program in Neuroscience and Sergei Y. Noskov4, Igor Vorobyov2,5. The Molecular Biophysics Program, Florida State University, Tallahassee, 1Biophysics Graduate Group, UC Davis, Davis, CA, USA, 2Physiology and FL, USA. Membrane Biology, UC Davis, Davis, CA, USA, 3Hartnell College, Salinas, The voltage-gated potassium channel Kv1.3 has a select distribution and CA, USA, 4Centre for Molecular Simulations, Biological Sciences, is well characterized for its role in immunity, glucose metabolism, and University of Calgary, Calgary, AB, Canada, 5Pharmacology, UC Davis, olfaction. Our interest lies in the channel’s ability to regulate excitability Davis, CA, USA. in mitral cells of the olfactory bulb (OB), where it has the potential to The inability to accurately predict the risk of drug cardiotoxicity is the primary serve as a metabolic target to balance body weight and enhance olfactory reason for drug candidates being abandoned during development, or removed ability. To aid in targeted delivery, we have conjugated the venom- from the market. Many drugs are known to interact with cardiac ion channels, derived Kv1.3 inhibitor margatoxin (MgTx) to luminescent quantum including the potassium-selective voltage-gated channel Kv11.1 encoded by the dots (QDs). Herein, we improved upon our previous conjugation strategy human ether-a-go-go gene (hERG). This channel is responsible for a major re- þ through insertion of a polyhistidine tag on the N-terminus of the peptide polarizing K current (IKr) during the cardiac action potential, and drug- (HisMgTx), allowing for a facile conjugation and controlled orientation of induced attenuation of this current can lead to acquired heart rhythm distur- MgTx on the QDs. Using recombinant expression and synthetic chemistry, bances, such as long QT syndrome (LQTS), a standard ECG-based clinical we produced three forms of HisMgTx. When measured by whole-cell, marker for increased risk of deadly arrhythmias. The blockade of hERG, and patch-clamp electrophysiology, the produced peptides inhibit 80% of the other cardiac ion channels, is mediated in large part by the propensity of a outward current in Kv1.3-transfected HEK-293 cells and have an IC50 drug to passively diffuse into the cell from the extracellular space, and therefore of 100 - 350 pM. When using His-conjugation, as many as 40 HisMgTx lipid membrane permeation of a drug is a critical factor in its pharmacokinetics. peptides can be self-assembled on an individual QD, as quantified by a However, at the molecular level, little is known about the specific ionization change in mobility shift in an agarose gel. Our electrophysiological states, spatial localization, or aggregation patterns of drugs in lipid bilayers, data indicate that the QDHisMgTx conjugate can similarly retain Kv1.3 all of which can factor into potency, toxicity, and ability to bind to different current inhibition properties that are not significantly different than those sites in cardiac ion channels. In this work, we developed parameters compatible attributed by HisMgTx alone. Towards targeted delivery of the with the CHARMM biomolecular force field for a small set of known IKr QDHisMgTx conjugate to the native channel, we have surgically im- blockers with varying risk propensities for arrhythmogenesis, in different planted C57BL6/J adult mice with osmotic mini-pumps with cannula drug ionization states, and used all-atom molecular dynamics simulations to that are bilaterally directed towards each OB. Outfitted mice have been compute kinetics and thermodynamics of their partitioning through hydrated probed for the effects of the conjugate on whole-body metabolism using lipid membranes. We aimed to elucidate drug water-membrane a continuous laboratory animal monitoring system (CLAMS) and the dis- distributions and permeability rates for their translocation into the cell, and tribution of the conjugate following delivery is currently being anatomi- thus propensities for lipophilic and aqueous access to protein targets, such as cally explored. the hERG channel. 1546-Pos Board B455 1548-Pos Board B457 Identification of Hyperekplexia Mutations that Impair the Homopenta- GABAA Receptor Subtype Selectivity of the Proconvulsant Rodenticide meric Assembly of the Glycine Receptor GLYRA1 TETS Anke Dopychai, Simone Heidenreich, C. Flore Pokam, Gunther€ Schmalzing. Brandon Pressly, Hai Minh Nguyen, Heike Wulff. Molecular Pharmacology, RWTH Aachen University, Aachen, Germany. UC Davis, Davis, CA, USA. Hyperekplexia or startle disease is a rare monogenic neuromotor disorder The rodenticide tetramethylenedisulfotetramine (TETS) is a potent convulsant characterized by a generalized stiffness in the newborn (‘‘stiff-baby syn- (lethal dose in humans 7-10 mg) that is listed as a possible threat agent by the drome’’) and excessive startle reflexes in response to unexpected acous- United States Department of Homeland Security. TETS has previously been tic or tactile stimuli. Hyperekplexia-causing mutations have been studied in vivo for toxicity and in vitro in binding assays, with the latter

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demonstrating it to be a non-competitive antagonist on GABAA receptors. In molecules commonly associated with M2 states (e.g., MRC1/CD206, order to determine whether TETS exhibits subtype selectivity for a particular Arg1, FIZZ1, YM1). Such species differences must be considered GABAA receptor combination, we used whole-cell patch-clamp to determine when characterizing microglial activation. Functionally, Kv1.3 and the potency of TETS on the major synaptic and extrasynaptic GABAA recep- Kir2.1, which are commonly studies in microglia and are potential tar- tors associated with convulsant activity. The active component of picrotoxin, gets for controlling neuroinflammation, differed in prevalence and ampli- picrotoxinin, was used as a control. While picrotoxinin did not differentiate tude with activation state and species. Intriguingly, blocking Kir2.1 well between 13 GABAA receptors, TETS exhibited the highest activity on reduced a homeostatic function—migration—while blocking Kv1.3 a2b3g2 (IC50 480 nM, 95% CI: 320-640 nM) and a6b3g2 (IC50 400 nM, increased it in both species. Our results call into question several 95% CI: 290-510 nM). Introducing b1orb2 subunits into these receptor com- molecules commonly associated with classical and alternative activation binations reduced or abolished TETS sensitivity, suggesting that TETS prefer- states. Importantly, caution is needed in generalizing molecular entially affects receptors with a2/b3ora6/b3 composition. Since a2b3g2 and functional responses of microglia to activating stimuli between receptors make up 15-20% of the GABAA receptors in the mammalian CNS, species. we suggest that a2b3g2 is probably the most important GABAA receptor for the seizure inducing activity of TETS. 1551-Pos Board B460 Role of Ryanodine Receptor Channel and Mechanisms of Vascular 1549-Pos Board B458 Diseases Validation of an Automated Patch-Clamp Screening Assay on Human Yun-Min Zheng, Yong-Xiao Wang. Kir2.1 Cardiac Ion Channels Department of Molecular & Cellular Physiology, Albany Medical College, Georg Andrees Bohme, Camille Sanson, Brigitte Schombert, Albany, NY, USA. Michel Partiseti. The functional importance of ryanodine receptors (RyRs) is well established Integrated Drug Discovery, Sanofi R&D, Vitry Sur Seine, France. in cardiac and skeletal diseases, but not in smooth muscle diseases. Here we The Kir2.1 potassium channel underlies the IK1 current which is a main explored the potentially essential role of RyRs in pulmonary hypertension. contributor to the diastolic resting potential of cardiac myocytes. These cur- Using real-time RT-PCR and Western blot analysis, we found that all three rents are also involved in the shaping of the initial depolarization and final RyR subtypes (RyR1, RyR2 and RyR3) were expressed in pulmonary artery repolarization of the action potential (AP). Drug-induced disturbances of smooth muscle cells (PASMCs). Studies with gene knockout (KO) mice re- Kir2.1 function can increase the duration of the AP and produce dangerous ar- vealed that RyR1, RyR2 and RyR3 all showed their functional activity. Con- rhythmias. Here we have developed a functional cell-based assay on an tractile and calcium responses in PASMCs following acute hypoxia for 5 automated electrophysiology platform to complete a panel of pre-clinical car- and 30 min respectively were completely blocked in RyR2 KO mice, but diosafety tests for early discovery compounds. CHO cells stably expressing only partially inhibited by RyR1 or RyR3 KO mice. RyR channel activity the human KCNJ2 gene product under the control of a tetracycline- and calcium release were largely increased in PASMCs from mice with pul- inducible promoter were recorded on a QPatch HTX platform. Series of monary hypertension induced by chronic hypoxia for 21 days. The chronic incrementing voltage steps from a 20 mV holding potential elicited large, hypoxia-induced increases in RyR channel activity and calcium release are non-inactivating negative inward currents at membrane potentials below fully abolished in PASMCs from SMC-specific RyR2 KO mice. Pulmonary the Kþ-equilibrium potential, with strong rectification above. Enhancing þ artery vasoconstriction, remodeling, and hypertension following chronic the external [K ]o concentration resulted in greater inward and outward cur- hypoxia are all nulled in RyR2 KO mice. FK506 binding protein 12.6 rents, and a cross-over of the outward currents on the I-V curves. The zero- (FKBP12.6, the RyR2 channel inhibitor) is significantly dissociated from 5 þ current potential shifted by 56 6 mV per ten-fold variation of [K ]o,in the channel in PASMCs from mice with hypoxia-induced pulmonary hyper- good agreement with the Nernst equation prediction. Addition of millimolar tension. FKBP12.6 KO promoted, while FKBP12.6 overexpression dimin- 2þ þ concentrations of Ba or Cs dose- and/or voltage-dependently blocked the ished, hypoxic pulmonary hypertension in mice. Treatment of S107, a inward currents as described for native IK1 currents. Pharmacological studies specific RyR2/FKBP12.6 stabilizer, also protects against hypoxic pulmo- using a step-ramp voltage protocol allowing stable recordings for 30 min nary hypertension. Rieske iron-sulfur protein (RISP) in mitochondrial com- demonstrated inhibitory activity in the low micro-molar range for the plex III serves as an essential, primary molecule in hypoxia-evoked reactive methoxybenzyl-methanamide Kir-inhibitor ML133 and the pentamidine- oxygen species (ROS) generation and subsequent FKBP12.6/RyR2 dissoci- derivative PA-6, although these compounds did not exhibit selectivity over ation in PASMCs. Intravenous administration of lentiviral RISP shRNAs hERG channels as claimed. eliminates hypoxia-induced pulmonary hypertension in mice. Collectively, we conclude that hypoxia can cause RISP-mediated ROS production, 1550-Pos Board B459 D FKBP12.6/RyR2 dissociation, RyR2 channel hyperfunction, sarcoplasmic Transcriptional Profiles and K Channels Differ in RAT and Mouse reticulum calcium leaking, increased calcium signaling, PASMC prolifera- Primary Microglia in Response to PRO- and Anti-Inflammatory Stimuli tion and remodeling, and ultimately pulmonary hypertension. Starlee Lively1, Doris Lam2, Lyanne C. Schlichter2. 1 Genetics & Development, University Health Network, Toronto, ON, 1552-Pos Board B461 Canada, 2Physiology AND Genetics & Development, Univ of Toronto AND Changes in IKR Amplitude (NOT Gating) is the Key Determinant for University Health Network, Toronto, ON, Canada. Ventricular Action Potential Prolongation Brain inflammation is commonly studied in rat and mouse models Don E. Burgess1,2, Jennifer L. Smith2, Ahmad S. Amin3,4, in vivo, and by examining microglial responses in vitro. There is consid- Corey L. Anderson5, Craig T. January5, Brian P. Delisle2. erable evidence that pro-inflammatory microglial activation can exacer- 1Natural Sciences, Asbury University, Wilmore, KY, USA, 2Physiology, bate tissue damage, while anti-inflammatory states help resolve University of Kentucky, Lexington, KY, USA, 3Clinical and Experimental inflammation and promote tissue repair. However, before attempting to Cardiology, Heart Center AMC, University of Amsterdam, Amsterdam, translate findings to humans, it is crucial to determine whether these ro- Netherlands, 4Rare, low prevalence and complex diseases, Ern Guard-Heart, dent species respond the same. We directly and quantitatively compared Amsterdam, Netherlands, 5Cellular and Molecular Arrhythmias Research responses of primary microglia from Sprague Dawley rats and C57BL/6 Program, Department of Medicine, University of Wisconsin, Madison, WI, mice (mRNA, selected proteins, Kir and Kv currents). Several activating USA. cytokines that increase in acute CNS injury models were used: the pro- Congenital long QT syndrome (LQTS) is primarily caused by autosomal domi- inflammatory stimulus, interferon-g þ tumor necrosis factor-a (IþT; nant mutations in cardiac ion channel genes that regulate the ventricular action ‘M1’ activation), and the anti-inflammatory stimuli, interleukin (IL)-4 potential (AP). About 35-40% of LQTS-linked mutations are in KCNH2 (‘M2a’, alternative activation), and IL-10 or TGFb1 (‘M2c’, acquired (hERG), which encodes the Kv11.1 channel that conducts the rapidly activating deactivation). Transcriptional profiling showed that, despite some species þ delayed rectifier K current (IKr) in the heart. The goal of this work was to pre- similarities, there were substantial qualitative and quantitative differ- dict the relative impact for decreases in IKr amplitude vs. changes in channel ences in response to each of the stimuli, and differences between gating on the human AP. We initially developed a 10-state Markovian model IL-10 and TGFb1 responses. There were qualitative or quantitative spe- for IKr channels based on the biophysical properties of wild type Kv11.1 chan- cies differences in resting levels and/or responses of several microglial nels expressed in HEK293 cells. The model contains 2 open states with parallel markers(Iba1,CD11b,CD68),inIþT-induced up-regulation of pro- activation/deactivation pathways. The eigenvalues of the model coefficient inflammatory molecules (e.g., iNOS, NO production, COX-2), and in matrix connected the model rate constants with experimentally determined

BPJ 8632_8635 312a Monday, February 19, 2018 time constants, and voltage-clamp simulations showed the model preserves throughput methods to quantify cardiomyocyte contraction and relaxation several important key kinetic features, including a fast and slow deactivating kinetics are less prevalent. Furthermore, there are no optical high- component. We replaced the Hodgkin-Huxley IKr model in the O’hara- throughput methods to quantify cardiomyocyte force generation and cell Virag-Varro-Rudy (OVVR) human AP model with our Markovian model. stiffness changes simultaneously during a cardiac cycle. Here, we present AP simulations at 1 Hz showed that it minimally affected the AP shape, AP a method to measure the mechanical tension produced by a monolayer cul- duration, and corresponding IKr profile. In control simulations, the time to ture of cardiomyocytes as well as the elastic modulus. By utilizing the forces 90% AP repolarization (APD90) was 264ms. Reducing the amplitude of the generated by the cells themselves to probe the material properties, our IKr component by 25%, 50%, and 75% increased the APD90 by 38ms, 93ms, method is fully non-invasive. iPSC-cardiomyocytes were cultured on and 198ms, respectively. We found that we had to adjust the activation, deformable substrates doped with fluorescent beads and were live stained deactivation, inactivation, or recovery from inactivation transitions rates by a with fluorescently-labeled wheat germ agglutinin (WGA). We acquired factor of 0.35, 39.6, 1.84, or 0.56, respectively to produce a 50ms prolongation high framerate videos of the iPSC-cardiomyocytes labeled with WGA in APD90. These simulations predict that the APD90 is much more sensitive to and of the fluorescently-labeled beads. From the WGA movies, we deter- changes in IKr amplitude vs. gating. These results likely help to explain the mined the strain field of the cell monolayer and its variations during the car- experimental observation that >95% of LQTS-associated KCNH2 mutations diac cycle. Similarly, the substrate deformations were measured by tracking decrease IKr amplitude. the motion of the beads, and were used to compute the cell-substrate forces using Traction Force Microscopy. From these datasets, we computed the Posters: Cardiac Muscle Mechanics and intracellular stresses using Monolayer Stress Microscopy, and estimated Structure I the elastic modulus of the cells by fitting the relation between the indepen- dently measured stress and strain maps to Hooke’s law. Our method is 1553-Pos Board B462 comparable to existing data from invasive methods such as AFM. Addition- Hypertrophic Cardiomyopathy: Variable Expression of Myosin-Binding ally, we present a high-throughput application of our method by testing a Protein C from Cell-To-Cell and Functional Imbalance among Individual series of benchmark compounds on monolayers of iPSC-cardiomyocytes Cardiomyocytes in multi-well plates. This work represents the first high-throughput David Aldag-Niebling1, Ante Radocaj1, Denise Hilfiker-Kleiner2, assay for simultaneous force and stiffness characterization in iPSC- Cristobal dos Remedios3, Bernhard Brenner1, Theresia Kraft1. cardiomyocytes. 1Molecular and Cell Physiology, Hannover Medical School, Hannover, Germany, 2Department of Cardiology and Angiology, Hannover Medical 1555-Pos Board B464 School, Hannover, Germany, 3Bosch Institute, University of Sydney, Sydney, Stable Microtubules Provide Viscoelastic Resistance to Cardiomyocyte Australia. Length Change Hypertrophic cardiomyopathy (HCM) has been related to mutations in Matthew A. Caporizzo1, Christina Y. Chen1, Alexander K. Salomon1, sarcomeric proteins. Mainly affected are b-myosin heavy chain (b-MyHC) Kenneth Bedi2, Kenneth B. Margulies2, Benjamin L. Prosser1. and myosin binding protein C (cMyBP-C). A common mechanism how 1Physiology, The University of Pennsylvania, Philadelphia, PA, USA, different mutations trigger HCM development is not clear. We asked whether 2Medicine, The University of Pennsylvania, Philadelphia, PA, USA. for an HCM-patient with cMyBP-C-mutation c.927-2A>G, which creates A debate surrounds the contribution of the stable microtubule network a premature stop-codon (i) functional imbalance among individual (MTs) to cardiomyocyte mechanics. MTs buckle during myocyte contrac- cardiomyocytes exists, and (ii) expression of cMyBP-C from cell-to-cell is tion, suggesting they play an elastic (spring-like) role in resisting myocyte variable, as seen in our previous work on HCM-related b-MyHC-mutations. contraction and aid in relaxation, but depolymerizing MTs with colchicine To test this we treated individual cardiomyocytes with the heterozygous MyBP- has yielded conflicting results. Some studies demonstrate increased contrac- C-mutation and donor cardiomyocytes with PP1 and PKA, and recorded force- tility in colchicine treated myocytes that may correlate with a modest reduc- pCa relations. This was followed by immunofluorescent double labelling of tion in tissue tension and cell stiffness, while others have not seen a cMyBP-C and a-actinin. functional or mechanical gain with colchicine. These results may be compli- Maximum isometric force of patient cardiomyocytes was reduced by 25% cated by an effect of colchicine on calcium handling. To determine the me- and Caþþ-sensitivity was increased compared to controls. Interestingly, a chanical consequences of stable MTs on myocyte function, we large cell-to-cell variation of force at submaximal activation levels was simultaneously measured calcium and contractility in colchicine treated mu- found, with some cells being essentially indistinguishable from controls, rine myocytes and probed viscoelasticity with atomic force microscopy while others generated substantially increased force suggesting reduced (AFM) and passive length tension. Colchicine provided a calcium- levels of cMyBP-C. Western blot revealed no truncated protein in the tissue. independent increase in contractile kinetics that is indicative of a viscous Fluorescence intensity ratio (IMyBPC/IAlpha-Actinin) was reduced by 35% for role for stable MTs in cardiomyoctye mechanics. Passive stretch and patient’s cardiomyocytes and significantly more variable from cell-to-cell AFM indicate a significant contribution of microtubules to cardiomyocyte compared to donors. The appearance of cMyBP-C was patchier in some pa- viscoelasticity. Specifically, at slow strain rates there is no difference in tient cardiomyocytes, as already seen before in histology of patients’ cardiac stiffness between colchicine treated and WT cardiomyocytes, consistent tissue. This suggests variable abundance of wildtype-cMyBP-C in patient with the previously observed minimal contribution of MTs to tissue passive cardiomyocytes. tension. At high strains rates colchicine treated myocytes appear softer than Since no truncated protein was found, presumably the mutation causes untreated cells. Additionally, targeting stable MTs by decreasing the amount mRNA-decay and only the wildtype-allele produces functional protein. of detyrosinated tubulin with parthenolide, or by overexpressing the tubulin If, as we propose, transcription of cMyBP-C occurs in a random, tyrosine ligase (TTL) has mechanical consequences that closely resemble burst-like fashion independent for both alleles, cMyBPC-protein could be colchicine. We find that the mechanical and functional gains observed highly variable from cell-to-cell, causing functional variability as seen with colchicine, parthenolide, and TTL overexpression translate into pri- here. In the long run, such functional imbalance from cell-to-cell would mary isolated human cardiomyocytes. The results suggest that the role of result in structural distortions like cellular/myofibrillar disarray, a hallmark stable MTs in resisting myocyte shortening is at least partially viscoelastic of HCM. in nature, whereby MTs provide breakable cross-links with the sarcomere that act to resist both rapid stretch and contraction. 1554-Pos Board B463 High-Throughput Functional Screening Assay of Force and Stiffness in 1556-Pos Board B465 IPSC Derived Cardiomyocytes A Novel Mouse Model for Titin-Based Dilated Cardiomyopathy Ricardo Serrano1, Wesley Lawrence McKeithan2,3, Mark Mercola3, Eyad Nusayr1, Joshua Strom1, Rebecca E. Slater2, Henk L. Granzier1. Juan Carlos del A´ lamo1. 1CMM, University of Arizona, Tucson, AZ, USA, 2University of Arizona, 1Mechanical and Aerospace Engineering, UCSD, La Jolla, CA, USA, Tucson, AZ, USA. 2Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA, Dilated cardiomyopathy (DCM) is the most prevalent form of cardiomyopathy 3School of Medicine, Stanford University, Stanford, CA, USA. and recent studies revealed that the sarcomeric protein titin is a major disease Cardiovascular disease is the leading cause of death in the U.S. and the rest gene and that mutations in titin are causative in 25% of DCM cases. Mecha- of the World. Physiological assays with iPSC-cardiomyocytes are powerful nisms by which titin can cause DCM are unknown and need to be studied. new tools for the development of new drugs, particularly assays for The heart co-expresses two titin isoforms, N2B and N2BA titin, that vary measuring action potential and calcium transient kinetics. However, high within their molecular spring region. N2BA titin is distinct from N2B titin

BPJ 8632_8635 Monday, February 19, 2018 313a because it expresses also the N2A element, a mechanosensory signaling hub. dilated cardiomyopathy. They are often caused by mutations in one or We targeted N2BA titin by generating mice in which titin exons 112-158 are more sarcomeric genes, such as TTN, which encodes the giant protein ti- deleted (TtnD112-158). These exons encode spring region sequences that are tin. Due to its size, TTN gene variants are also found in unaffected indi- specific to the N2BA isoform. The Left ventricle (LV) of TtnD112-158 shows viduals (as shown by the 1000 Genomes Project) and it is difficult to considerable downregulation of N2BA mRNA and protein. At 6-months of assess their pathogenicity. In this project, biophysical techniques such age (6mo), functional analysis reveals LV dilation and systolic dysfunction as X-ray crystallography, differential scanning fluorimetry (DSF) and with normal diastolic function, a phenotype consistent with DCM. The 1D-Nuclear magnetic resonance (NMR) have been applied to determine DCM is progressive and at 6-weeks of age (6wks) the LV does not reveal the structure of titin A-band domains, in order to assess the impact of sus- any abnormalities. pected and proven pathogenic variants on their stability and structure. Six Our hypothesis is that downregulation of N2BA also downregulates N2A- fibronectin type-3 domains from the titin A-band harboring rare missense dependent signaling mediators which disrupts cardiomyocyte homeostasis, mutations were expressed in E. coli,bothinwtandvariantforms.All thus causing the observed DCM. Of the mediators that bind to the N2A wt’s were confirmed to be folded by 1D NMR studies, whilst some var- element, CARP exhibited significant downregulation at the pre-DCM, 6wks iants did not fold or had structural changes induced by the missense mu- time point. To further examine the contribution of CARP downregulation on tation. Their DSF melting temperatures were lower by around 10C, we crossbred TtnD112-158 mice with CARP knockout mice to produce mice suggesting reduction in stability caused by the mutation as a common that are homozygous for the TtnD112-158 deletion and Heterozygous for the feature of genetically proven pathogenic TTN variants. X-ray structural CARP deletion. We found that even this 50% reduction in CARP allelic repre- data elucidated the structural basis of the destabilization, allowing visual- sentation was sufficient to increase the severity of DCM in 6mo LV. ization of impact of the missense mutation on the surrounding residues In summary, we generated a novel titin-based DCM mouse model and provided and tertiary structure of the protein. This experimental work will be im- functional evidence that downregulation of CARP is an early pathological plemented in improved structure-based predictions with the aim of modulator in this model. improving fidelity of variant interpretation and understanding the basic mechanisms of titinopathies. 1557-Pos Board B466 Location of Hypertrophic Cardiomyopathy-Causing Troponin T 1559-Pos Board B468 Mutations Determines Degree of Myofilament Dysfunction Ischemic Cardiomyopathy Perturbs GSK-3b Localization to the Maike Schuldt1, Jamie R. Johnston2, Michelle Michels3, Myofilament to Reduce Function Diederik W.D. Kuster1, Jose R. Pinto2, Jolanda van der Velden1. Marisa J. Stachowski1, Maria Papadaki1, Jody L. Martin1, 1Department of Physiology, Amsterdam Cardiovascular Sciences, VU Christine S. Moravec2, Jonathan A. Kirk1. University Medical Center, Amsterdam, Netherlands, 2Department of 1Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, Biomedical Sciences, College of Medicine, Florida State University, USA, 2Cardiovascular Medicine, Cleveland Clinic Lerner College of Tallahassee, FL, USA, 3Department of Cardiology, Thoraxcenter, Erasmus Medicine, Cleveland, OH, USA. Medical Center, Rotterdam, Netherlands. Cardiac dyssynchrony worsens function and mortality. Normally caused by Background: Hypertrophic cardiomyopathy (HCM) is the most common conduction abnormalities, dyssynchrony can also arise due to contractile inherited cardiac disease with a prevalence of 1:200. It is caused by >1400 heterogeneity in ischemic cardiomyopathy (ICM). The normal treatment mutations in sarcomeric genes and characterized by ventricular hypertrophy, for dyssynchrony is bi-ventricular pacing (CRT), but pacing an infarct is diastolic dysfunction, fibrosis and increased risk of sudden cardiac death. ineffective, so CRT does not fix ischemia-induced dyssynchrony (IID). Variability in disease onset and progression between patients is large. We Our previous work in dyssynchrony found CRT reactivates glycogen syn- hypothesize that the mutant protein dose and mutation location are main thase kinase-3b (GSK-3b) and restores myofilament function. We hypothe- determinants of myofilament dysfunction in HCM. sized GSK-3b mayalsomodulatemyofilamentfunctioninICM,andcould Methods: To assess the effect of mutation location, we measured force be leveraged to treat IID. Firstly, we examined GSK-3b levels in human re- development, calcium sensitivity (EC50) and length-dependent activation in jected donor (Control), ICM, and dilated cardiomyopathy (no IID, DCM) detergent-permeabilized cardiomyocytes of two patients carrying the R278C left ventricle (LV). We discovered a myofilament-localized pool of GSK- troponin T (TnT) mutation. In addition, recombinant TnT with mutations 3b,whichwas71% reduced in ICM. The regulatory mechanism of this I79N and R94C was exchanged in detergent-permeabilized donor cardiomyo- novel localization of GSK-3b was suggested by enriched phospho-Y216 cytes. By controlling the amount of troponin incorporation, we defined the ef- GSK-3b in the myofilament (compared to whole tissue), suggesting Y216 fect of mutant protein dose. phosphorylation modulates GSK-3b binding to the myofilament. Immuno- Results: Troponin exchange (1.5 mg/ml recombinant troponin complex) was fluorescence and immuno-precipitation both confirmed that p-Y216 GSK- lower with TnT-R94C (40%) compared to TnT-I79N (70%). In comparison 3b localizes to the myofilament, while un-phosphorylated does not. Adeno- with controls (EC50: 1.7850.18 mM) both mutants showed a viral transfection of myc-tagged wild-type, Y216F (unphosporylatable) and dose-independent increase in calcium sensitivity (i.e. decrease in EC50), with Y216E (constitutively phosphorylated) GSK-3b into rat neonatal ventricular TnT-R94C (1.1750.13 mM) showing an even higher increase than myocytes (NRVMs) and adult mouse myocytes also supported the necessity TnT-I79N (1.5350.13 mM). This calcium-sensitizing effect was already of Y216 phosphorylation for myofilament localization. We determined the present at a very low TnT-R94C dosage of 18%. The patients carrying the functional role of GSK-3b using skinned myocytes from cardiomyocyte- TnT-R278C mutation showed differences in EC50 (1.2750.12 mM and specific inducible GSK-3b knock-out mice, which displayed a significant 1.9750.18 mM) which may be explained by a difference in protein dosage, decrease in calcium sensitivity compared to control mice. Lastly, skinned but warrants further investigation. myocyte functional studies on Control and ICM human LV showed that Conclusion: Our troponin exchange experiments show that the maximal effect ICM myocytes were desensitized to calcium. Function was restored with on myofilament calcium sensitivity is achieved at the lowest dosage for two exogenous GSK-3b treatment, which had no effect on Control myocytes. different TnT mutations. R94C causes a larger calcium-sensitization than While GSK-3b is a promiscuous kinase in the cardiomyocyte, we have iden- I79N at an even lower dosage, indicating that mutation location rather than tified a specific regulatory mechanism (Y216 phosphorylation) that could dosage determines the impact on sarcomere function. allow precise therapeutic intervention to improve contractile function in the ICM heart. 1558-Pos Board B467 TITIN Variants in Genetic Myopathies and Cardiomyopathies- Structural 1560-Pos Board B469 and Biophysical Characterization of Pathogenic Mutations Engineered Thin Filament Mutations to Study the Sarcomere Length Roksana Nikoopour1, Martin Rees1, Mark Pfuhl1,2, Ana Ferreiro3, Dependence of Cardiac Muscle Contractility Perry Elliott4, Mathias Gautel1. Joseph D. Powers1, Farid Moussavi-Harami2, Jil C. Tardiff3, 1Randall Division, King’s College London, London, United Kingdom, Jennifer Davis1,4, Michael Regnier1. 2Cardiovascular Division, King’s College London, London, United 1Bioengineering, University of Washington, Seattle, WA, USA, 2School of Kingdom, 3Sorbonne Paris Cite, Universite Paris-Diderot, CNRS, Biologie Medicine - Cardiology, University of Washington, Seattle, WA, USA, Fonctionnelle et Adaptative UMR 8251, Universite Paris-Diderot, Paris, 3School of Medicine, University of Arizona, Tucson, AZ, USA, 4Pathology, France, 4Institute for Cardiovascular Science, University College London, University of Washington, Seattle, WA, USA. London, United Kingdom. Sarcomere length-dependent augmentation of force (the cellular basis of the Myopathies and cardiomyopathies are genetic conditions affecting skeletal Frank-Starling Law) is a critical regulatory mechanism in cardiac muscle per- and cardiac muscle, the most common of the latter being hypertrophic or formance, and is often blunted in heart failure. While many sarcomere protein

BPJ 8632_8635 314a Monday, February 19, 2018 mutations have been characterized in the context of cardiomyopathy pathogen- frequently experience fibrosis, altered cardiac output, and arrhythmias. esis, the precise role of individual proteins in regulating length dependence of These closely related diseases are primarily caused by mutations in sarco- force remains unclear. Here, we used previously characterized point mutations meric proteins that regulate cardiac contractility; however, how these muta- of regulatory proteins to probe the thin filament and elucidate the role of tropo- tions lead to the disease phenotype and to opposite effects on tissue myosin in modulating the length dependence of cardiac contractility. Twitch remodeling is unclear. Understanding these diseases will require knowledge amplitude was measured at short (2.0mm) and long (2.3mm) sarcomere of how these mutations affect cardiac contractility across multiple levels of lengths (SL) of intact cardiac trabeculae from hearts of a transgenic murine organization. While the disease presentation in intact hearts is well under- model containing a dilated cardiomyopathy-associated Tpm mutation stood, the complexity of the heart makes it difficult to attribute the disease (D230N; denoted TpmD230N). Trabeculae were mounted between a force trans- phenotypes to tissue-level reorganization or intrinsic changes in cellular ducer and length-controlling motor, perfused with oxygenated physiological contractility. To study the similarities and differences between these diseases solution (30C), and electrically stimulated at 1 Hz. At short SL, twitch force at the cellular level, we have used CRISPR/Cas9 to generate human stem cell between wild-type (WT) and TpmD230N trabeculae were not significantly lines bearing disease-causing mutations, differentiated these cells to cardio- different (2454 versus 1653 mN/mm2, respectively). At long SL, WT trabec- myocytes, and studied their structural and contractile properties. We focused ulae produced significantly higher twitch force compared to TpmD230N (5156 on two model point mutations in troponin-T that cause either HCM or DCM versus 2753 mN/mm2, respectively), demonstrating reduced length-dependent in patients. Traction force microscopy on patterned substrates with stiff- augmentation of contractility in TpmD230N trabeculae. We hypothesized that nesses that mimic the human heart was used to study the contractile proper- this is due to reduced azimuthal displacement of TpmD230N along the thin ties of single cells. Our results indicate features of the disease seen in patients filament, limiting effects of cross-bridge-mediated cooperative activation at are present at the single cell level. Specifically, the HCM mutant shows hy- longer SL. Thus, we incorporated an engineered calcium-sensitizing troponin percontractility and impaired relaxation, while the DCM mutant shows hypo- C mutation (L48Q, denoted TnCL48Q) to aid thin filament activation by contractility. Additionally, both mutants show altered sarcomeric structure. developing a TpmD230N/TnCL48Q double mutant murine model. Twitch forces The development of a cell-based system for these diseases will likely open in intact trabeculae from TpmD230N/TnCL48Q mice were increased compared to the door for the development of novel therapeutics for familial TpmD230N and were not significantly different than WT at short and long SL cardiomyopathies. (3054 and 4656 mN/mm2, respectively). Our results suggest that tropomyosin plays a unique role in modulating the length dependence of 1563-Pos Board B472 contractility in cardiac muscle. The Missense E258K-MyBP-C Mutation Increases the Energy Cost of Ten- sion Generation in Both Ventricular and Atrial Tissue from HCM Patients 1561-Pos Board B470 Giulia Vitale, Francesca Gentile, Nicoletta Piroddi, Beatrice Scellini, The Off State of the Thick Filament of Cardiac Muscle is Not Affected by Jose` Manuel Pioner, Iacopo Olivotto, Cecilia Ferrantini, Chiara Tesi, Inotropic Interventions Like the Increase in Diastolic Sarcomere Length Corrado Poggesi. or the Addition of a Beta-Adrenergic Effector Department of Experimental and Clinical Medicine, University of Florence, Vincenzo Lombardi1, Francesca Pinzauti1, Marco Caremani1, Florence, Italy. Joseph Powers1, Serena Governali1, Massimo Reconditi1, Mutations in MYBPC3, the gene coding for cardiac myosin-binding protein-C Theyencheri Narayanan2, Ger J.M. Stienen3, Marco Linari1, (cMyBP-C) are the most common cause of Hypertrophic CardioMyopathy Gabriella Piazzesi1. (HCM). The E258K-MyBP-C is a highly penetrant missense mutation with 1University of Florence, Firenze, Italy, 2European Synchrotron Radiation poorly understood molecular mechanisms. Mechanics and kinetics of contrac- Facility, Grenoble, France, 3VU University Medical Center, Amsterdam, tion as well the energetic cost of tension generation were investigated using left Netherlands. ventricular (LV) and atrial tissue from three E258K HCM patients and We study the regulation of cardiac contractility by using SAXS-USAXS at the compared to those from controls (donor hearts, aortic stenosis patients, and ID02-beamline of the European Synchrotron (ESRF, Grenoble, France) on HCM patients negative for sarcomeric protein mutations). Kinetics of tension intact trabeculae isolated from the rat ventricle to record both the nanometer- generation and relaxation were measured in single LV and atrial myofibrils scale X-ray signals from the contractile proteins along the thin (actin) and thick mounted in a force recording apparatus (15 C), maximally Ca2þ -activated (myosin) filaments and the changes of the sarcomere length (SL). Previously (pCa 4.5) and fully relaxed (pCa 9.0) by rapid solution switching (<10 ms). we demonstrated that in diastole (external [Ca2þ] 2.5 mM, 27 C) most of Maximal ATPase and isometric active tension were simultaneously measured the myosin motors are in the off-state (unavailable for actin binding and in Triton-permeabilized LV and atrial strips. In E258K, maximal tension of ATP hydrolysis), packed into helical tracks with 43-nm periodicity on the both ventricular and atrial myofibrils was reduced compared to controls. The 2þ surface of the thick filament, and that the fraction of myosin motors leaving rate of tension generation following maximal Ca activation (kACT) was faster the off-state during the twitch depends on the load through a rapid positive in both ventricular and atrial E258K myofibrils compared to controls. The rate feedback based on thick-filament mechano-sensing (Reconditi et al. PNAS of isometric relaxation (slow kREL) was also faster in E258K myofibrils, sug- 114:3240, 2017). This regulatory mechanism occurs downstream with respect gesting faster cross-bridge detachment and increased energy cost of tension to the Ca2þ-dependent thin-filament activation which controls cardiac contrac- generation. Direct measurements in ventricular and atrial skinned strips tility via the intracellular [Ca2þ] and the Ca2þ-sensitivity of the filaments. Here confirmed that tension cost was 2-3 fold higher in E258K preparations we tested the interdependency of the two regulatory mechanisms by recording compared to controls. We conclude that the E258K mutation primarily alters the X-ray signals that mark the state of the thick filament during two inotropic apparent cross-bridge kinetics and impairs sarcomere energetics. In vitro, the interventions that double the twitch force at SL 2.0 mm and external [Ca2þ]1 mutation induces similar kinetic and energetic effects in both atrial and LV sar- mM: either SL increase to 2.25 mm or addition to the solution of the b-adren- comeres. The smaller impact of the mutation on atrial muscle function ergic effector isoprenaline (107 M). In diastole none of the signals related to compared to LV muscle in vivo is likely due to the different loading conditions the off-state of the thick filament was affected by either intervention. The re- of the two chambers. sults indicate that Ca2þ-dependent thin-filament activation and thick-filament mechano-sensing act independently, further clarifying the role of the thick- 1564-Pos Board B473 filament stress-sensitivity as an energetically well-suited downstream mecha- Chronic Exercise Increases Compliant Titin and Kettin Isoform Content in nism. Supported by Ente Cassa di Risparmio di Firenze 2016-2018. Cardiac Muscle of Rat and Drosophila Models Mark Hiske, Deena Damschroder, Rober J. Wessells, Patrick J. Mueller, 1562-Pos Board B471 Charles S. Chung. Mechanical and Structural Analysis of Cardiomyopathies at the Single Wayne State University, Detroit, MI, USA. Cell Level Reduced cardiac passive stiffness correlates with increased exercise tolerance in Paige E. Cloonan, Lina Greenberg, Michael J. Greenberg. mammals. Titin, the giant elastic protein, decreases passive stiffness by expression Washington University in St. Louis, St. Louis, MO, USA. of larger titin isoforms (increased N2BA:N2B ratio) or altered post-translational Inherited cardiomyopathies are the leading cause of sudden death in young modifications (increased PKA, decreased PKC phosphorylation). Short-term and people, affecting more than one in five-hundred people, and there is currently acute exercise has been previously shown to change post-translational modifications no known cure. Two of the most common forms of familial cardiomyopa- but not the N2BA:N2B. Kettin is an elastic protein in invertebrates that is also differ- thies, hypertrophic (HCM) and dilated (DCM), show thickening and thinning entially spliced. Little is known about kettin isoform splicing in response to exercise. of the left ventricular wall, respectively. Patients with these diseases The purpose of our study was to evaluate changes in titin and kettin isoform content

BPJ 8632_8635 Monday, February 19, 2018 315a following chronic exercise or sedentary conditions. 4 week old male Sprague- valves of ApoE KO mice exhibited increased expression of calponin 2 and Dawley rats were randomly assigned to cages with (exercise, n=18) and without smooth muscle actin (SMA), a hallmark of myofibroblasts. Using primary cul- (sedentary, n=17) running wheels. After 12 weeks, hearts were harvested and the tures of sheep aortic valve interstitial cells (AVICs) as a model system, we left ventricle solubilized and proteins separated using SDS-VAGE. Titin induced differentiation into myofibroblast-like cells and studied the calcifica- N2BA:N2B ratio increased (p=0.047) in exercising animals with wheels compared tion phenotypes. We found that myofibroblast differentiation of AVICs to sedentary animals. Degraded titin to total titin ratio and total titin to myosin heavy increased the expression of calponin 2 that co-localized with SMA. By further chain content ratios were unchanged. Canton S flies (Drosophila) were exercised up assessing the effect of Cnn2 KO on myofibroblast differentiation and the effect to 3 hrs per day 5 times per week for 3 weeks using the PowerTower, a machine that of knocking-down Cnn2 on decreasing calcification phenotypes in AVICs, our repetitively induces negative geotaxis (climbing exercise). Abdominal segments study proposes a novel hypothesis that calponin 2 may contribute to the devel- from whole, flash frozen Drosophila were solubilized from 3 technical replicates opment of CAVD by promoting myofibroblast differentiation. containing 10 Drosophila each for both the exercised and un-exercised groups. Abdominal samples contain both cardiac and skeletal muscles, but are more en- 1567-Pos Board B476 riched for cardiac muscle than thoraces. Three isoforms of kettin were consistently The ACTC M305L Hypertrophic Cardiomyopathy Mutation Results in Drosophila identified using western blot. Preliminary data showed an increased expression of Hypercontractility and Impaired Relaxation of Muscles 1 1 1 2 larger kettin isoforms sizes in exercise versus un-exercised samples. These prelim- Meera C. Viswanathan , William Schmidt , Aditi Madan , Leah C. Sullivan , 2 3 3 inary studies indicate that both mammalian and invertebrate cardiac tissues have Christopher S. Newhard , Michael J. Rynkiewicz , William Lehman , 2 1 increased compliant isoforms of titin and kettin, respectively. Douglas M. Swank , Anthony Cammarato . 1Medicine, Johns Hopkins University, Baltimore, MD, USA, 2Biology, 1565-Pos Board B474 Rensselaer Polytechnic Institute, Troy, NY, USA, 3Physiology and Cardioskeletal Defects in R58Q-RLC Mouse Model of HCM Biophysics, Boston University, Boston, MA, USA. Katarzyna Kazmierczak1, Jingsheng Liang1, Zhiqun Zhou1, Sunil Yadav1, Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle char- Aldrin V. Gomes2, Danuta Szczesna-Cordary1. acterized by ventricular wall thickening, hyperdynamic contractile properties, and 1Molecular and Cellular Pharmacology, University of Miami School of impaired relaxation. It is commonly caused by mutations in sarcomeric proteins. Medicine, Miami, FL, USA, 2Neurobiology, Physiology, and Behavior, The alpha-cardiac actin ACTC M305L HCM mutation is located near the University of California Davis, Davis, CA, USA. nucleotide-binding site and residues predicted to help confine tropomyosin to an Hypertrophic Cardiomyopathy is a heterogeneous disorder that primarily affects inhibitory position along thin filaments. We generated several Drosophila models cardiac muscle. However, a severe cardioskeletal myopathy was reported for of the disorder to evaluate the effects of the lesion from the organ through molecular HCM-related IVS6-1 mutation in MYL2 (encoding human ventricular RLC), level. In all muscles tested, phenotypic disturbances correlated with mutant protein with onset and death in infancy and muscle type I hypotrophy and cardiac myop- load. For example, relative to wildtype actin, when highly overexpressed in the fly athy. This study continues to focus on R58Q (Arginine-to-Glutamine) mutation heart, the variant significantly reduced cardiac output, prolonged systole, and in the ventricular RLC shown to cause HCM in humans. Since MYL2 expresses restricted diastolic volumes. Preliminary force measurements from jump muscle fi- the same RLC protein in the hearts and in type I skeletal muscles fibers, we have bers corroborated a dose-dependent, M305L actin-induced pathological effect. examined myosin RLC function in cardiac papillary muscles (PM) and in soleus Moreover, we observed greater incremental decreases in flight ability with muscles (SOL) of a-MHC-driven transgenic mice, expressing either human car- increasing amounts of mutant versus wildtype actin expression in indirect flight diac R58Q-RLC or WT-RLC. Electrically stimulated intact fibers from R58Q- muscles (IFM). Flight was abolished when the mutant was highly overexpressed mice showed slower rates of force contraction and relaxation transients in both and destructive myosin-induced hypercontraction was evident, suggesting disrupted PM and SOL as well as slower calcium transient relaxation rates in PM compared contractile regulation and poorly inhibited actomyosin associations. However, regu- with WT. Consistent with our findings, proteomic results showed downregulation lated in vitro motility parameters, including Vmax, nH, and pCa50,ofthinfilaments of Ryr1/2 (Ca-release channel of sarcoplasmic reticulum) and calsequestrin-2, reconstituted from M305L or wildtype IFM actin and bovine cardiac troponin- the Ca-binding protein and Ryr1/2 regulator, in both the hearts and SOL of tropomyosin, were indistinguishable. Likewise, no overt differences in R58Q vs. WT. On the other hand, Ca2þ-ATPase, SERCA2a2 was only downre- computationally-derived electrostatic interaction energy landscapes between gulated in the R58Q mice hearts, explaining delayed Ca2þ-transients in PM but M305L and wildtype actin-tropomyosin filaments were observed, suggesting unper- not in SOL. Skinned fibers mechanics showed a significantly reduced maximal turbed tropomyosin-positioning in the absence of troponin. Nonetheless, over force in PM and SOL of R58Q mice compared to respective WT fibers. A trend sparsely populated beds of myosin, a significantly higher percentage of troponin- of lower force observed in PM/SOL of R58Q vs. WT was not present in fast- free M305L versus wildtype actin-tropomyosin filaments were motile, implying twitch EDL muscles. ATPase staining data showed a decreased proportion of tropomyosin is less effective at blocking actomyosin associations on mutant fila- type I to type II fibers in SOL of R58Q vs. WT mice, with no difference between ments. Mispositioning of tropomyosin may contribute to the supranormal contrac- R58Q and WT for EDL. These results suggest that the HCM-related functional tion and compromised relaxation observed in our in vivo models and, potentially, in changes observed in the R58Q hearts coincide with those seen in SOL of HCM human patients. mouse model. Supported by NIH-HL123255 (DSC). 1568-Pos Board B477 1566-Pos Board B475 Cardiac Thin Filament-Mediated Calcium Sensitization Modulates Cross- Deletion of Calponin 2 Attenuates the Development of Calcific Aortic Bridge Kinetics Valve Disease Maicon Landim-Vieira1, David Gonzalez-Martinez1, Jamie R. Johnston1, Olesya Plazyo, Jian-Ping Jin. Weikang Ma2, Olga Antipova2,3, Omar Awan1, P. Bryant Chase4, Physiology, Wayne State University, Detroit, MI, USA. Thomas Irving2,3, Jose R. Pinto1. Calcific aortic valve disease (CAVD), the second-leading cause of cardiovascu- 1Biomedical Sciences, Florida State University, Tallahassee, FL, USA, lar mortality, currently lacks treatment options other than valve replacement. 2Biological Sciences, Illinois Institute of Technology, Chicago, IL, USA. Although the pathology of CAVD involves perturbation of valvular cells by 3X-ray Science Division, Advanced Photon Source, Argonne National the mechanical stimuli including shear stress, pressure and leaflet stretch, the Laboratory, Chicago, IL, USA, 4Biological Science, Florida State University, intracellular mechanism requires further elucidation. We recently demonstrated Tallahassee, FL, USA. that knockout (KO) of the gene Cnn2 that encodes calponin isoform 2, a mecha- Previous studies have focused on examining the effects of Ca2þ sensitivity on noregulated cytoskeleton protein (Liu et al. Gene 585:143-153, 2016), attenu- cross-bridge kinetics in skeletal muscle. However, given the implications for ates atherosclerosis in ApoE KO mice (Liu et al. J Mol Cell Cardiol 99:87-99, heart disease, it is important to understand these mechanisms in cardiac muscle. 2016). Considering that CAVD and atherosclerosis share many pathogenic pro- Therefore, this study explored the effects of thin-filament mediated Ca2þ cesses with common risk factors, we hypothesized that calponin 2 may also sensitization on cardiac cross-bridge kinetics. Myofilament Ca2þ sensitivity contribute to the development of CAVD. We found that Cnn2 KO indeed was increased with either (i) cardiac preparations from a cardiac troponin C decreased calcification of the aortic valve in ApoE KO mice, an established knock-in mouse model of hypertrophic cardiomyopathy (cTnC-A8V), or (ii) model of CAVD. Although myeloid cell-specific Cnn2 KO highly effectively 300 mM bepridil (WTþBEP), a cTnC Ca2þ sensitizer. Papillary muscles attenuated vascular atherosclerosis in ApoE KO mice, it did not reduce aortic were isolated from the left ventricles of cTnC-A8V homozygote and WT valve calcification. This finding indicates a significant difference between the mouse hearts. Sarcomere length (SL) of skinned cardiac muscle preparations pathogeneses of atherosclerosis and CAVD. Calponin participates in myofibro- (CMPs) was adjusted using HeNe laser diffraction after the ends were blast differentiation, a cellular process in the development of CAVD. The aortic chemically fixed with glutaraldehyde to minimize end-compliance. All

BPJ 8632_8635 316a Monday, February 19, 2018 measurements were carried out in the presence of 3% dextran T500 and at to form weak crossbridges. We altered the Gibbs free energy to form a 21oC. At 2.1 mm SL, cTnC-A8V and WTþBEP CMPs displayed increased super-relaxed crossbridge by less than 1 kT as titin’s force decreased maximal rate of tension redevelopment (kTR) compared to WT. The maximal over sarcomere lengths from 2.25 to 1.9 mm. This caused the maximal sinusoidal stiffness (SS) was reduced only in cTnC-A8V compared to WT. steady-state force for isometric contraction at saturating Caþþ to reduce Ca2þ sensitivity of isometric tension and SS were increased in both by 25%, as data suggests (Dobesh et al, 2002). The model thus unites cTnC-A8V and WTþBEP CMPs compared to WT. Mathematical modeling existing data and provides a more quantitative understanding of how of force and kTR revealed increased rates of cross-bridge detachment (g)in titin interacts with the thick filament at different sarcomere lengths. both cTnC-A8V and WTþBEP. Myofilament lattice spacing (LS) and We anticipate that this model insight and more extensive data will steady-state isometric tension in CMPs were recorded simultaneously. LS of lead to deeper understanding of cardiac length-dependent activation. skinned WT CMP increased with force. Starting at similar LS at pCa 8, WT (Supported by the Center for Engineering MechanoBiology through a and cTnC-A8V CMPs exhibited comparable peak increases in LS with Ca2þ grant from NSF’s STC program (CMMI):15-48571). activation. WTþBEP CMP started at larger LS at pCa 8 and thus exhibited a smaller peak increase. These data suggest an important role of the thin filament in modulating not just the number of cross-bridges during Ca2þ-activation but 1571-Pos Board B480 also their kinetics. NIH-HL128683. Understanding Calcium Sensitization and Desensitization using a Cardiac Troponin Chimera Fangze Cai1, Peter Hwang2, Brian Sykes1. 1569-Pos Board B478 1Biochemistry, University of Alberta, Edmonton, AB, Canada, 2Medicine, Characterization of a Fluorescent Levosimendan Analog Binding to University of Alberta, Edmonton, AB, Canada. Cardiac Troponin Heart failure is the end stage of almost all heart disease, characterized by an Brittney Klein, Brian D. Sykes. inability to pump enough blood to satisfy the needs of the body. Systolic heart Biochemistry, University of Alberta, Edmonton, AB, Canada. failure is caused by impaired cardiac muscle contraction, and insufficient A compound that modulates the calcium sensitivity of and specifically diastolic relaxation leads to diastolic heart failure. The ability to sensitize or targets the cardiac sarcomere has been a sought after therapeutic tool for sys- desensitize cardiac muscle to calcium has therapeutic potential in treating tolic heart failure. Cardiac troponin controls the contraction of the heart by cardiac dysfunction. However, most treatments for heart failure regulate the initiating a cascade of conformational changes in response to shifting intra- calcium based signaling pathways, which can increase stress in the failing cellular levels of calcium. Recent structural studies have examined an array heart. The ideal drug would directly alter the calcium response of the cardiac of compounds that target the interface between the calcium binding and inhib- sarcomere. We propose to understand the calcium sensitization and/or itory subunits of cardiac troponin, cTnC and cTnI, respectively. Issues desensitization of compounds directly targeting cardiac troponin, the thin encountered include insolubility, lack of specificity, inconsistency of binding filament based calcium-dependent protein switch that turns heart muscle constants amongst methods and the necessity to individually screen contraction on and off. compounds. We are currently investigating DNF, a compound incorporating A number of small molecules are known to bind to the interface of structural features of Bepridil, diphenylamine and Levosimendan. We have between the regulatory N-terminal domain of cTnC (cNTnC) and the characterized DNF binding to cardiac troponin complexes and chimeras switch region of cTnI. Therefore, we constructed a cNTnC-cTnI switch using mass spectrometry, nuclear magnetic resonance and fluorescence region chimera (cChimera) for structure, dynamics and drug binding spectroscopy. studies by NMR spectroscopy. The solution structure shows that DNF’s configuration includes a reactive cyano group that provides specificity, a cChimera faithfully reproduces the native interface between cNTnC carboxyl group for increased solubility and is highly conjugated causing fluo- and cTnI switch region, with the cNTnC in an open conformation rescence in the visible range. Binding of DNF to the regulatory N-terminal even in the absence of a bound drug suggesting its potential as a domain of cardiac troponin C exhibits a high affinity towards both the C35S, screening tool. We determined the structure of 3-methyldiphenylamine C84S and C35S mutants and enhances fluorescence. At low DNF concentra- (3-mDPA), one the highest binding affinity compounds, bound to tions, the binding affinity was determined to be 1 mM and was verified using 1 1 15 cChimera. The cChimera-3-mDPA complex shows that the drug binding H NMR and fluorescence spectroscopy. Using 2D H, N NMR chemical shift preserves the native interaction between cNTnC-cTnI. We have mapping, we showed DNF to be in slow exchange with cNTnC but with a investigated the dynamics and structure of a well-known calcium weaker KD given low compound solubility. DNF is also capable of forming desensitizer W7 bound to cChimera, providing the details of interactions a reversible covalent thioimidate bond with C84 of cTnC, but is less stable between W7 and the cTnI switch peptide. The structures help elucidate than the Levosimendan complex. These studies led to the development of a the details and mechanism of calcium sensitization and desensitization competitive drug binding assay using DNF as a fluorescent probe for cNTnC. targeting cardiac troponin. Using this assay, we examined the relative binding affinities of several diphenylamine compounds. Posters: Myosins 1570-Pos Board B479 Regulated Model of Steady-State Cardiac Length-Dependent Activation 1572-Pos Board B481 Timothy Alcid1, William C. Hunter2. Is the Myosin Head Conformation Coupled to the Thick Filament Back- 1New Jersey Institute of Technology, Newark, NJ, USA, 2Biomedical bone Structure? Engineering, New Jersey Institute of Technology, Newark, NJ, USA. Kenneth A. Taylor1, Zhongjun Hu2, Dianne W. Taylor1, Robert J. Edwards3. Recent data suggests length-dependent activation derives from titin 1Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, interacting with super-relaxed myosin crossbridges on the thick filament. USA, 2Molecular Biophysics Program, Florida State University, Tallahassee, This feature was incorporated into an existing model of crossbridge FL, USA, 3Department of Cell Biology, Duke University Medical Center, dynamics (Caremani et al, 2015) by replacing the detached post-ATP- Durham, NC, USA. hydrolysis state with an equilibrium mixture of rapidly interacting Some striated muscles thick filaments show evidence of structural changes in super-relaxed, detached, and weakly bound crossbridges. The model response to activation or tension. Relaxed thick filaments from Lethocerus also included: (1) cooperative activation of the thin filament by both flight muscle change their helical angle by 0.15/crown (145A˚ repeat) in Caþþ and strongly bound crossbridges, (2) loosely coupled interaction response to externally applied tension. The change in helical angle, which is that allows Caþþ to dissociate from troponin C (TnC) before strong a structural parameter defined by the myosin rods within the filament backbone, crossbridges detach, (3) Caþþ bound more strongly to TnC when a is accompanied by intensity changes in the 145A˚ layer line, which is sensitive strong crossbridge is also bound nearby. As suggested by data (Sun & to the arrangement and order of the myosin heads. The intensity change could Irving, 2015), the equilibrium between super-relaxed and detached indicate that putting the relaxed muscle under tension alters the order or crossbridges equaled 3:1 for a long sarcomere length. We supposed arrangement of at least some of the myosin heads. Vertebrate striated muscle that the reduced forces exerted by titin on the thick filament at low thick filaments, which are not helical, change their axial repeat from 143A˚ to sarcomere length made it more likely to form a super-relaxed 145A˚ , the latter being the axial spacing in Lethocerus thick filaments. We crossbridge. As the force exerted by titin increases, the super-relaxed reasoned that if externally applied tension could change the helical angle and myosin transitions to an unfettered detached state and becomes available the myosin head arrangement, that relaxed Lethocerus thick filaments with

BPJ 8632_8635 Monday, February 19, 2018 317a partially disordered heads might display an altered helical angle coupled to opment by single point mutations in myosin II motors, particularly nonmuscle changes in myosin head arrangement. The 3-D images obtained by cryoEM, myosin II, and underpin the allosteric nature of myosin motors. The results provide the first structural evidence that the arrangement of myosin heads correlate well with experimental data and support the growing body of evidence within the interacting heads motif is coupled to the structure of the thick that many disease-associated mutations manifest their effect on protein struc- filament backbone. We find that a 0.16 change in helical angle disorders tural dynamics. the blocked head preferentially, which suggests a mechanism for how tension affects the dynamics of the myosin heads and their contribution to stretch 1575-Pos Board B484 activation and shortening deactivation. We suggest the blocked head Impact of Hypertrophic Cardiomyopathy Mutations and the Role of preferentially binds the thin filament followed by the free head when the Myosin Binding Protein-C on the Sequestered State of Myosin blocked head produces force. The shift in blocked head dynamics from ordered Darshan V. Trivedi*, Saswata S. Sarkar*, Makenna M. Morck, to disordered is achieved by tension applied by the antagonist muscle. Arjun A. Adhikari, Kathleen M. Ruppel, James A. Spudich. Supported by NIH and AHA. Biochemistry, Stanford University, Stanford, CA, USA *Equal Contribution 1573-Pos Board B482 The heart contracts by the virtue of multiple ATP-dependent interactions be- Myosin Phosphorylation-Mediated Rescue of Cardiac Function in Familial tween the actin-containing thin filaments and myosin-containing thick fila- Hypertrophic Cardiomyopathy ments. This sliding of the thin and thick filaments generates force necessary Sunil Yadav, Katarzyna Kazmierczak, Jingsheng Liang, Chen-Ching Yuan, to pump blood throughout the body. It is imperative for the heart to modulate Zhiqun Zhou, Lauro Takeuchi, Rosemeire Kanashiro-Takeuchi, this force-generating mechanism thus fine-tuning its power output. An abroga- Danuta Szczesna-Cordary. tion of this fine-tuning may lead to hypercontractility of the cardiac tissue Molecular and Cellular Pharmacology, University of Miami, Miami, FL, which in-turn leads to diseases like Hypertrophic Cardiomyopathy (HCM). USA. For the first time, we report biochemical evidence outlining the molecular basis Hypertrophic Cardiomyopathy (HCM) is one of the most prevalent heart dis- of this fine-tuning which involves an intramolecular interaction between the eases characterized by ventricular and septal hypertrophy, myocardial fibrosis myosin head and tail region. This interaction holds myosin heads in reserve, and myofibrillar disarray. Genetic forms of HCM are mainly caused by muta- only to be released under dynamic needs of the heart. Another protein of the tions in major sarcomeric proteins such as b-myosin heavy chain (b-MHC), thick filament called Myosin Binding Protein-C also interacts with myosin myosin protein binding C (MyBP-C), regulatory and essential light chains thus additionally reigning more myosin heads on the thick filament. We demon- (RLC/ELC), troponin, tropomyosin and actin. Our study focusses on RLC mu- strate that many HCM-associated mutations abrogate these interactions thus tations that cause HCM and strives to provide insight into the importance of bringing more myosin heads into play leading to the observed clinical myosin RLC phosphorylation for normal heart function and its potential rescue hypercontractility. role in HCM. Studies from HF patients and RLC animal models of HCM show severely reduced levels of cardiac myosin phosphorylation that coincide with 1576-Pos Board B485 depressed heart function. We used a highly effective Adeno-Associated Virus, A Mixed-Kinetic Model Describes Unloaded Velocities of Smooth, Skeletal, serotype-9 (AAV9), to deliver phosphomimic S15D-RLC protein into the and Cardiac Muscle Myosin Filaments in Vitro hearts of HCM-D166V(Asp/Val) mice and examined recovery of heart Richard Brizendine, Gabriel Sheehy, Diego Alcala, Sabrina Novenschi, function by echocardiography, invasive hemodynamics and muscle fiber Josh Baker, Christine Cremo. mechanics. A significant improvement of cardiac function, including a decrease Department of Pharmacology, University of Nevada, Reno School of in left ventricle isovolumetric relaxation time (shown to be prolonged in HCM), Medicine, Reno, NV, USA. relaxation constant, Tau and increase in cardiac output (CO) and end-systolic In vitro motility assays, where purified myosin and actin move relative to one pressure-volume relationship (improved contractility) were observed in another, are used to better understand the mechanochemistry of the actomyosin AAV-injected vs. control HCM-hearts. An increase in maximal contractile ATPase cycle. We examined the relationship between the relative velocity (V) force in skinned papillary muscles from AAV9-S15D-RLC injected of actin and myosin and the number of available myosin heads (N) or [ATP] for HCM-D166V mice versus control D166V mice indicated recovery of smooth (SMM), skeletal (SKM) and cardiac (CMM) muscle myosin filaments contractile function in AAV-treated mice. These results are consistent with moving over actin as well as V from actin filaments moving over a bed of our proof-of-principle study in transgenic mice expressing the phosphomimic monomeric SKM. These data do not fit well to a widely-accepted model that S15D-RLC mutation in the background of D166V-RLC. D166V-induced predicts V is limited by myosin detachment from actin (d/ton,), where d equals abnormal changes in heart function, morphology and structure were step size and ton equals time a myosin head remains attached to actin. To abrogated in S15D-D166V mice. Our gene therapy study is novel and account for these data we have developed a mixed-kinetic model where V is significant and will provide insight into the phosphorylation-mediated rescue influenced by both attachment and detachment kinetics. The relative contribu- from heart diseases that among other symptoms are manifested by reduced tions at a given V vary with the probability that a head will remain attached to RLC phosphorylation. Supported by NIH-HL123255 (DSC) and AHA- actin long enough to reach the end of its flexible S2 tether. Detachment kinetics 17PRE33650085 (SY). are affected by L/ton, where L is related to the tether length. We show that L is relatively long for SMM, SKM, and CMM filaments (59 5 3, 22 5 9, and 22 1574-Pos Board B483 5 2 nm, respectively). In contrast, L is shorter (8 5 3 nm) when myosin mono- The Impact of Disease-Related Mutations on the Structural Dynamics and mers are attached to a surface. This suggests that movement of the S2 domain Allostery in Myosin Motors may be an important mechanical feature of myosin filaments that influences un- Julia Weder, Matthias Preller. loaded shortening velocities of muscle. Biophysical Chemistry, Hannover Medical School, Hannover, Germany. Structural dynamics and intrinsic allosteric regulation play crucial roles for the 1577-Pos Board B486 biological function of proteins. Single point mutations, associated with the Single Molecule, Optical Trapping Studies of Omecamtiv Mercarbil on development of severe diseases, are suggested to affect in many cases the Human Cardiac Myosin’s Force Production inherent protein dynamics. However, the underlying mechanisms remain Michael S. Woody1, Michael J. Greenberg2, Bipasha Barua3, mostly unclear. In this study, we performed comparative in silico analyses of Donald A. Winkelmann3, Yale E. Goldman4, E. Michael Ostap4. the effect of disease-related point mutations on the structural dynamics of 1Graduate Group in Biochemistry and Molecular Biophysics, Perelman myosin motors. Myosins are ATP-dependent molecular motors that are shown School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, to be highly regulated by intrinsic allosteric mechanisms. Distant sites in the 2Department of Biochemistry and Molecular Biophysics, Washington motors are remotely interconnected and communicate with each other in order University in Saint Louis, St. Louis, MO, USA, 3Department of Pathology to trigger largescale conformational changes of the protein, leading finally to and Laboratory Medicine, Robert Wood Johnson Medical School, Rutgers force production and directed movement. Comprehensive analysis of the University, Piscataway, NJ, USA, 4Pennsylvania Muscle Institute, Perelman conformational landscapes of wildtype and in silico mutated motors using mo- School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. lecular dynamics simulations showed significant disturbances of crucial allo- The cardiac myosin small-molecule activator, omecamtiv mercarbil (OM), is in steric communication pathways in the motors and marked changes in the stage three clinical trials for the treatment of chronic heart failure, however the conformational dynamics caused by the disease-associated point mutations. exact mechanism of its action is not settled. Initial reports suggested that OM Our data provide novel insights in the molecular mechanisms of disease devel- increases the rate of phosphate release and the myosin duty ratio, resulting in

BPJ 8632_8635 318a Monday, February 19, 2018 increased force production due to an increased number of myosins strongly- 1580-Pos Board B489 bound to actin during contraction. Studies on muscle fibers and animal models Biochemical and Functional Characterization of the Interaction of Myo1c corroborated these results as measured by force production and stroke volume, with 14-3-3 without changes in kinetics. However, in vitro motility assays published subse- Huan-Hong Ji, E. Michael Ostap. quently indicated that the sliding velocity is greatly reduced in the presence of The Pennsylvania Muscle Institute and Department of Physiology, Perelman OM in this experimental geometry. We utilized single molecule optical trap- School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. ping techniques to study the step size and kinetics of actively cycling, recom- Myosin-IC (Myo1c) and 14-3-3, an adaptor protein proposed to associate with binant human cardiac myosin under load in the presence of OM. We measured Myo1c, have been implicated in regulating the delivery of GLUT4-containing an increase in actin-attachment lifetime of myosin and a decrease in the length compartments to the plasma membrane in response to insulin. However, molec- of the powerstroke from 6 nm to <1 nm in the presence of 10 mM OM. The ular and functional details of the Myo1c-14-3-3 interaction have yet to be decreased actomyosin dissociation rate and unitary displacement provide a described. It has been proposed that 14-3-3 binding to Myo1c is mechanism for the inhibitory action of OM in the in vitro motility assay. Addi- phosphorylation-dependent with a binding site at S701, near the first tionally, these results suggest that OM inhibits a kinetic step that occurs in be- calmodulin-associated, IQ-motif of the motor. Here we show that 14-3-3 bind- tween phosphate release and ADP release, as solution kinetic experiments ing to Myo1c increases with increasing calcium concentration. However, 14-3- indicate that OM does not slow the rates of ADP release or ATP-induced acto- 3 binding does not appear to displace calmodulin from Myo1c at concentrations myosin dissociation. Single molecule optical trapping is providing a way to where calcium-bound calmodulin normally remains associated. 14-3-3 induces observe the entire cycle of myosin in the presence of OM and is thus helping dissociation of actin from coverslip-bound Myo1c in the in vitro motility assay, to elucidate its therapeutic mechanism. resulting in the inhibition of directional actin gliding. Surprisingly, phosphor- ylation of S701 with CAM kinase-II, which has been suggested to regulate 1578-Pos Board B487 14-3-3 binding, has little effect on the Myo1c-14-3-3 interaction or the ability Hydration of Magnesium is Required for Myosin VI Phosphate Release of 14-3-3 to inhibit motility. Our data suggest that 14-3-3 is able to interact with Mauro L. Mugnai, Devarajan Thirumalai. Myo1c and inhibit its activity in a calcium dependent, but phosphorylation in- Chemistry, The University of Texas at Austin, Austin, TX, USA. dependent manner. Myosin motors utilize the free energy released by the hydrolysis of ATP to move along the filamentous actin. In order to do so, myosins undergo a che- 1581-Pos Board B490 momechanical cycle during which binding and release from actin and nucle- Tools to Study Nonmuscle Myosin-2 Motor Function Revisited 1 1 2 2 otides (ADP and ATP) are coupled to conformational changes favoring the Sarah Heissler , Neil Billington , Xuefei Ma , Robert Adelstein , James Sellers1. movement towards one end of the track. ATP binds with the phosphate 1 2 groups towards the inside of the motor. After ATP hydrolysis, the motor NHLBI/LMP, NIH, Bethesda, MD, USA, NHLBI/LMC, NIH, Bethesda, binds actin and the phosphate is released first. Because ADP obstructs the MD, USA. entrance pathway, the phosphate needs to find an alternative route. Recently, Nonmuscle myosin-2 is the major enzyme that powers the contractility of the a new crystal structure of myosin VI was resolved, which is believed to actin cytoskeleton in fundamental biological processes including cytokinesis represent the conformation capable of releasing the phosphate from the bind- and cell motility. The protein consists of two myosin heavy chains that each ing site. In order to determine the structural basis of phosphate release, we associate with one essential and one regulatory (RLC) light chain. performed numerous multi-microsecond-long simulations starting from the Phosphorylation of the latter on a conserved serine and threonine is regulatory new structure. Within the time scale of our simulations, the release occurred for nonmuscle myosin-2 and associated with the activation of the enzymatic only if we rotated the phosphate at the beginning of the simulations. The ma- activity of its motor domain. Tools to generate constitutively active or inactive jor finding is that phosphate release of always occurs after hydration of the nonmuscle myosins-2 for cell biological experiments therefore include (i) the magnesium. The simulations also predict a few different escape routes, sug- generation of phosphomimetic RLC mutants but also (ii) the introduction of gesting that there is plasticity in the pathways in this critical step in the cat- loss-of function mutations in the myosin motor domain. Here we show that alytic cycle of Myosin VI, and presumably in other members of this super ‘‘phosphomimetic’’ RLC mutations in which we replace the phosphorylatable family. serine and threonine with glutamate or alanine to mimic phosphorylation or non-phosphorylation fail to recapitulate the respective RLC state. 1579-Pos Board B488 Moreover, we show that in contrast to previous studies, the frequently used Optical Control of Fast and Processive Engineered Myosins: Optimization disease-associated N93K mutation in nonmuscle myosin 2-A is not a complete and Characterization in Vitro and in Living Cells loss-of-function mutation. Instead, the mutation modestly decreases the Paul V. Ruijgrok, Rajarshi P. Ghosh, Muneaki Nakamura, Robert Chen, enzymatic activity of nonmuscle myosin-2A. The inability of RLC Vipul Vachharajani, Jan Liphardt, Zev Bryant. "phosphomimetics" and the N93K mutant to recapitulate constitutively active Bioengineering, Stanford University, Stanford, CA, USA. or inactive nonmuscle myosin-2 underline the need for new tools to control Spatiotemporal control of cytoskeletal transport can provide new possibilities myosin motor function in cells. Based on a molecular approach to the for dissecting cellular processes or for constructing complex artificial devices. structure-function relationship of nonmuscle myosins-2, we suggest genetic Optogenetic approaches have been used for both controlled recruitment of disruption of a conserved salt bridge in the myosin motor domain that is pivotal motors to cellular cargos [1] and direct modulation of motor speed and for its enzymatic activity. We find that the disruption of the salt bridge elimi- direction [2]. Here we have worked to create optimized and diversified nates the actin-activation of myosin’s motor activity. This approach is believed engineered myosin motors with velocities that can be optically controlled using to be universal for all nonmuscle myosins-2 and is superior to the use of RLC dynamic changes in lever arm geometry. Previous designs for light-activated ‘‘phosphomimetics’’ and the disease-associated N93K mutation to inhibit gearshifting [2] were non-processive, and suffered from either low velocities myosin motor activity. (< 10 nm/s) or modest degrees of velocity modulation (15%) in response to light. These limitations preclude many potential applications in cell biology, 1582-Pos Board B491 devices, and reconstituted systems. We have now engineered (i) non-processive ATPase Activity of Diaphragm Muscle Fibres Isolated From the Rabbit myosin motors that combine large optical modulation depths with high Containing the R403Q Mutation in the Heart velocities and (ii) processive myosin motors with optically controllable Md Rezuanul Haque Saikat, Yu-Shu Cheng, Dilson Rassier. directionality. We have characterized a series of optimized constructs using Kinesiology & Physical Education, McGill University, Montreal, QC, in vitro motility assays of propelled actin filaments, single-molecule tracking Canada. of processive complexes, and live cell imaging of motors tagged with Introduction: Respiratory muscle weakness is present in condition of hyper- fluorescent protein arrays [3]. For non-processive plus-end directed myosins, trophic cardiomyopathy (HCM). The Mg2þ-ATPase activity of myosin and we measure up to 4X speed increases in the presence of blue light, reaching heavy meromyosin (HMM) ultimately regulates the speed of muscle velocities of 4-5 microns/s. For processive myosins, we demonstrate contraction and performance. In this study, we analyzed ATPase activity of controllable bidirectional processive transport, and report on cellular myosin isolated from the diaphragm of rabbits with the R403Q mutation in localization under optical stimulation. [1] P. van Bergeijk, et al. (2015) Nature beta-myosin heavy chain, which causes HCM. Methods: Myosin filaments 518 (7537) ‘‘Optogenetic control of organelle transport and positioning’’ [2] were isolated from the diaphragm of rabbit with R403Q mutation and from M. Nakamura et al. (2014) Nat. Nanotechnol 9, 693 ‘‘Remote control of myosin wild-type (WT) rabbit. HMM fragments were prepared from myosin and and kinesin motors using light-activated gearshifting’’ [3] R.P. Ghosh et al. myofibrils isolated by homogenization. The protein concentration was (2017) ‘‘A Fluorogenic Array Tag for Temporally Unlimited Single Molecule measured by the Bradford protein assay. The Mg2þ-ATPase activity of Tracking’’, BioRxiv doi: 10.1101/159004. actomyosin was reconstituted from rabbit actin, myosin or HMM in a medium

BPJ 8632_8635 Monday, February 19, 2018 319a containing (in mM): 30 KCl, 0.1 CaCl2, 0.5 MgCl2, 2 DTT, 20 Imidazole-HCl cosity of a cell or by forming co-polymeric filaments with NM2B. The (pH 7.2) with a slightly modified Fiske and Subbarow method. The co-filaments exhibit mechanical properties that are intermediate between ATPase reaction was started after 10 min of incubation at 25C by adding 1 those of the individual myosins. This demonstrates that cells can fine mM of ATP and was terminated after 10 min by adding 2.5% tune the mechanical output of a myosin filament by controlling the iso- trichloroacetic acid. The amount of Pi liberated was evaluated colorimetrically forms present in the filament. at 595nm. Results & discussion: The Mg2þ-ATPase activity of R403Q myosin (0.186 mmol/mg/min) was 2.8 times lower than the activity of WT 1585-Pos Board B494 myosin (0.53 mmol/mg/min) (p<0.05). R403Q HMM fragments also reported Variation in Stride Length of Myosin-5A Revealed by Interferometric a drop in Mg2þ-ATPase (0.086 mmol/mg/min) compared to the WT HMM Scattering Microscopy (iSCAT) (0.186 mmol/mg/min) activity (p<0.05). However, there were no significant Joanna Andrecka1, Adam Fineberg1, Daniel Cole1, Alistair Curd2, differences between the Mg2þ-ATPase activity of R403Q and WT myofibrils. Kavitha Thirumurugan2, Yasuharu Takagi3, James R. Sellers3, The result of this study suggests that the R403Q mutation in the heart leads to a Peter J. Knight2, Philipp Kukura1. reduced contraction of the diaphragm. 1Physical & Theoretical Chemistry, University of Oxford, Oxford, United Kingdom, 2Astbury Centre for Structural Molecular Biology, University of 3 1583-Pos Board B492 Leeds, Leeds, United Kingdom, National Heart, Lung, and Blood Institute, Mapping Intrinsic Communication Pathways in the Myosin Motor National Institutes of Health, Bethesda, MD, USA. Domain Associated with Force Generation Myosin-5a is a molecular motor that can take 36 nm steps along actin filaments, Wiebke Ewert1, Peter Franz1, Georgios Tsiavaliaris1, Matthias Preller1,2. matching their 13-subunit helical repeat. EM studies have found that in addition 1Institute for Biophysical Chemistry, Hannover Medical School, Hannover, to this 13 subunit separation, myosin is also found with 11 and 15 subunit sep- Germany, 2Centre for Structural Systems Biology (CSSB), German Electron arations between its heads. Attempts to characterise the step length distribution Synchrotron (DESY), Hamburg, Germany. for a mobile motor with optical trapping or tracking of fluorescent labels, how- Myosin uses the chemical energy of ATP hydrolysis and the cyclic ever, have only exhibited broad distributions of movements with a peak at 37 interaction with actin filaments to produce mechanical force for directed nm corresponding to a step length of 13 subunits, lacking the spatiotemporal movement. A key step in this mechanochemical cycle is the power stroke, precision to reveal shorter or longer steps. To address this inconsistency, we a rotational movement of the lever arm that occurs following hydrolysis took advantage of the unique spatiotemporal precision of iSCAT and tracked with myosin bound to actin. The details of the structural changes within myosin-5a with a 20 nm gold particle bound to the motor domain of just one the myosin motor domain induced upon actin binding and cleft closure of the two heads. Achieving 1 nm localisation precision at 100 Hz frame rate are not yet well understood. Here we show mapping of the pathways of allowed us to reveal the strides taken by individual motors. Detailed analysis signal propagation and allosteric communication in myosin II at the of the iSCAT traces revealed a family of peaks separated by intervals of 5.5 atomic resolution level using molecular dynamic simulations of nm, corresponding to strides spanning 22, 24, 26, 28 and 30 subunits as pre- equilibrium states and conformational transitions. Structural perturbations dicted from the EM data. Additionally, we found strides corresponding to 32 in combination with free energy calculations allowed us to characterize and 34 actin subunits, extending possible separations between the two heads pathways of correlated rearrangements of critical structural elements in to 17 actin subunits. Importantly, we could reveal the sequence of strides the myosin motor domain, mediating the communication between that individual molecules take on a trace-by-trace basis. These show that the actin-binding region and the transducer as well as the active site. Our multimodal distribution does not arise from heterogeneous molecules, with data suggest a key role for the W-helix in propagating the information some that take predominantly short and others taking long strides, but instead of actin binding through the twisting of the transducer eventually to the all molecules taking a mixture of stride lengths. Dwell time analysis of the converter domain in preparation of the lever arm swing. The present study distinct stride lengths supports a model in which strain between the heads reg- provides novel insights into allosteric communication and the molecular ulates ADP release, promoting the high processivity and directionality of mechanism of the coupling between cleft closure and initiation of the po- myosin-5a. wer stroke. 1586-Pos Board B495 1584-Pos Board B493 High Resolution Cryo-EM Structures of Actin-Bound Myosin States Tuning the Mechanical Output of Nonmuscle Myosin-2 Filaments Reveal the Mechanism of Myosin Force Sensing Luca Melli1, Neil Billington1, Attila Nagy2, Hajer Ennomani3, Ahmet Mentes1, Andrew Huehn2, Xueqi Liu2, Adam Zwolak1, Yasuhara Takagi1, Laurent Blanchoin4, James R. Sellers1. Roberto Dominguez1, Henry Shuman1, E. Michael Ostap1, 1Cell Biology and Physiology Center, National Heart, Lung and Blood Charles V. Sindelar2. Institute, Bethesda, MD, USA, 2Vaccine Production Program Laboratory, 1Pennsylvania Muscle Institute and Department of Physiology, Perelman National Institute of Arthritis and Infectious Disease, Bethesda, MD, USA, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, 3Univ. Grenoble-Alpes, CEA, CNRS, INRA, Grenoble, France, 2Department Molecular Biophysics and Biochemistry, Yale University, New 4CytoMorphoLab, Biosciences and BiotechnologyInstitute of Grenoble, Haven, CT, USA. Univ. Grenoble-Alpes, CEA, CNRS, INRA, Grenoble, France. Myosin motors are able to dynamically adjust their power outputs in Three nonmuscle myosin-2 (NM2) paralogs participate in many mamma- response to mechanical loads, with different isoforms differing substantially lian cellular phenomena. Each NM2 paralog forms 310nm bipolar fila- in their force sensitivities. While it is known that force sensing is related to ments containing either 30 (NM2A/NM2B) or 16 myosins (NM2C). rotation of the myosin lever arm, it is not known how this motion is coupled The three paralogs are all slow enzymatically and mechanically to the active site, or how this coupling differs among myosins. We used compared to other myosins, but have distinct kinetic signatures with cryo-electron microscopy to solve near-atomic resolution structures of a NM2B having the highest duty ratio. NM2B bipolar filaments show highly force-sensitive motor, myosin-Ib (Myo1b), before (AM.ADP state) robust processive movements on single actin filament in vitro with a and after (AM state) the lever arm swing accompanying ADP release. run length of about 2 mm and a velocity of 43 nm/s. Rings of branched Comparison of these structures reveals that Mg.ADP stabilizes a partially actin filaments formed in the presence Arp2/3 are contracted by addition closed conformation of the nucleotide cleft in which elements of switch I of NM2B filaments. In addition, NM2B filaments bundle long parallel tightly coordinate the Mg cation in a distinctly different fashion than for actin filaments that emanate from the rings. To determine the number other myosin nucleotide states. This partial closure of the nucleotide cleft of NM2B motor domains that are necessary for processive movement traps the N-terminal subdomain in a twisted orientation that presents a novel we co-assembled NM2B molecules with headless Halo-tagged-myosin interface to the lever arm, helping to stabilize an intermediate state of the rods allowing us to vary the number of motor domains in a bipolar fila- power stroke. Moreover, structural classification of our cryo-EM data ment. More than five NM2B motors/half filament are required for proc- indicates that the specialized N-terminal extension of myo1b, which we essive movements. This number agrees well with calculations based on identified previously as a structural component important for ADP release, the duty ratio of single headed NM2B S1. As the number of myosin mo- selectively stabilizes the ‘rigor’ orientation of the lever arm in both AM.ADP tor domains are reduced in the co-filaments the run-length, but not the and AM (rigor) states of the motor by dynamically exchanging in and out of a velocity decreases. In contrast, under the same buffer conditions, ‘sandwiched’ position between the motor and lever arm helix. These NM2A filaments do not move processively. This is not surprising given observations give insights into force sensing by myo1b and also provide a the lower duty ratio of NM2A. Processive movements with NM2A can framework in which to understand the diversity of force sensing among be achieved by including methylcellulose in the assay to mimic the vis- members of the myosin superfamily.

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1587-Pos Board B496 surface-bound myosin molecules is generally used to compare different Force Produced by Smooth and Skeletal Muscle Myosin Filaments myosin isoforms and constructs or to study disease-associated mutations Measured with Micro-Fabricated Cantilevers of e.g. human cardiac myosin. However, we previously found that sliding Yu-Shu Cheng, Md Rezuanul, Haque Saikat, Dilson Rassier. velocity of actin filaments in standard in vitro motility assays is much Department of Kinesiology and Physical Education, McGill University, slower than unloaded shortening velocity in muscle fibers even when Montreal, QC, Canada. experimental conditions and myosin isoforms are identical. Besides the Introduction: The muscle myosin uses energy derived from ATP hydrolysis unphysiological random orientations of myosin molecules moving actin to perform mechanical work and move actin filaments, thereby generating filaments in such assays the absence of regulatory proteins like force. The goal of this study was to investigate the force production by troponins and tropomyosin on actin filaments can lead to deceleration of isolated myosin filaments at different ATP concentration, using a new actin sliding. system that we developed in our laboratory that allows mechanical To overcome such limitations reconstituted thick and thin filaments have measurements and visualization of the filaments simultaneously. We also been utilized to mimic the native environment in muscles. Instead of us- measured the actomyosin ATPase activity of the filaments to correlate ing regulated thin actin filaments reconstituted from mixed or expressed with force. Methods: Two types of myosin filaments were used in this study; components of various sources we used native thin filaments prepared smooth myosin filaments were isolated from the ABRM of M. edulis,and from defined muscle samples. Sliding of native thin filaments from skeletal myosin filaments were isolated from rabbit psoas muscles. An slow skeletal and cardiac ventricular muscle was characterized on tissue experimental chamber was mounted on an inverted microscope with purified myosin of the same sources. We observed that although both fluorescence imaging. The force produced by the myosin filaments upon slow muscle types are supposed to contain the same myosin isoform interaction with fluorescence actin at four different concentration of ATP, sliding was faster on cardiac myosin. We also found that the source 0.5, 0.75, 1.0 and 1.25mM was calculated using the displacement of the of native thin filaments exert a wide influence on the sliding velocity. cantilevers of known stiffness. (Kalganov et al., 2013). Mg-ATPase activity While cardiac thin filaments accelerate sliding, slow skeletal muscle was measured using methods previously described (Shelud’ko, et al., 2007). thin filaments decrease the sliding velocity both on cardiac and slow Results and Discussion: Forces in both types of myosin filaments were skeletal myosin molecules. increased consistently with increasing ATP concentration from 0.5mM to From our results we conclude that an influence of myosin and native filaments 1.25mM during actomyosin interactions. The force produced by smooth sources should be considered, e.g. when disease-associated mutations in car- and skeletal muscle myosin filaments were 67.05pN/mm and 54.62pN/mm diac myosin are studied. at 0.5mM ATP, and 103.27pN/mm and 112.51pN/mmat1.25mMATP, respectively. The force generated by smooth and skeletal muscle myosin 1590-Pos Board B499 filaments were increased approximately 54.0% and 100% when ATP Measuring the Force of Single and/or Multiple Myosin 5 by Using a Single concentration increased from 0.5mM to 1.25mM. ATPase activity of smooth Beam Optical Trap and skeletal myosin was significant different (p<0.05), suggesting that Justin J. Raupp, Yuwen Mei, Takeshi Sakamoto. higher ATP concentration will be hydrolyzed to enhance the mechanical Physics, Wayne State University, Detroit, MI, USA. work during actomyosin interactions. The transportation of vesicles is important for the development, organiza- tion, and functioning of all cellular organisms. The failures of vesicle 1588-Pos Board B497 transportation cause many diseases and chronic illnesses associated with Can We Reproduce the Latch-State in Vitro at the Molecular Level? the malfunctioning of these systems. The molecular motors responsible Zsombor Balassy1, Linda Kachmar2, Gijs Ijpma2, Anne-Marie Lauzon3. for these vesicle transportations supply the energy for locomotion by 1Biomedical Engineering, McGill University, Montreal, QC, Canada, exchanging chemical energy into mechanical force. To measure the forces 2McGill University, Montreal, QC, Canada, 3Experimental Medicine, McGill generated by these molecular motors we use an optical trap to capture University, Montreal, QC, Canada. polystyrene beads that have myosin molecules attached to their surface. Smooth muscle (SM) has a unique property, called the latch-state, during To control the number of myosin on the surface, the concentration of which force is maintained for long periods of time at low energy consump- myosin can be changed. This will be a stepping stone to studying a tion and low myosin activation (phosphorylation) levels. This property has particular transport vesicle, zymogen granules, which are large digestive been observed in whole muscle studies. Theories to explain the latch-state enzymes secreted by the pancreas. Some fundamental questions exist as were extrapolated to the molecular level but were never verified. One to how these vesicles move throughout the cell. Some motor proteins such theory states that, during the cross-bridge cycle, if SM myosin gets de- are processive (myosin 5A), meaning that they take several steps before phosphorylated while attached to actin, it will remain attached, in a load- falling off. Others are non-processive (myosin 5C). Our lab has shown bearing mode. In an attempt to reproduce the latch-state at the molecular that two myosin 5c molecules attached to a DNA scaffold can move level, we used the in vitro motility assay, which consists of measuring the processively, suggesting that perhaps multiple myosin 5c may work velocity of fluorescently labeled actin filaments when propelled by myosin together to transport extremely large vesicles. Investigating the molecules on a coverslip, to which we added a microfluidic chamber to mechanism of transportation and the force generation involved can be inject myosin light chain phosphatase (MLCP) to efficiently dephosphory- done by combining the use of fluorescence imaging and optical tweezer late myosin, without creating bulk flow. We used a mixture of SM and skel- techniques. In order to model the forces involved in vesicle transport, etal (SK) muscle myosin, the latter not being regulated by phosphorylation. modified polystyrene beads (diameter: 200 nm 3 mm) with motor The rationale behind this protocol was that if the latch-state occurs, we proteins such as myosin 5 can be held in an optical trap to calculate should observe a transient decrease in actin filament velocity, due to the the forces generated. These force measurements can be used to estimate load induced by the attached, dephosphorylated SM myosin. This decrease the number of motors involved for processive movement along actin in velocity would eventually resume after the detachment of these latch filaments. bridges. Note that at a [SM] =25 mg/ml, the injection of MLCP stopped the actin movement in 150sec, indicating myosin dephosphorylation. 1591-Pos Board B500 Foramixtureof[SM]=25mg/ml/[SK] =80 mg/ml, we measured a velocity The Force-Dependent Activity of Multiple Myosin VI Monomers of 2.150.2 mm/s before injection, that was significantly increased at 150sec Ellen Rumley, David Altman. (2.450.2 mm/s; P=0.01) and at 250sec (2.750.5 mm/s; P=0.001). Thus, our Physics, Willamette University, Salem, OR, USA. data do not show evidence of a load bearing phase after myosin dephosphor- Myosin VI motors are proteins associated with sensory function, ylation. Future studies will address the involvement of the actin regulatory endocytic vesicle transport, as well as a broad range of other cellular proteins. Funded by NSERC. functions. A mutation in myosin VI has also been linked to a familial hypertrophic cardiomyopathy. It has been hypothesized that myosin VI 1589-Pos Board B498 may exist as either a monomer or dimer in vivo, and that regulating Influence of Sample Source: Sliding Velocity of Different Native Thin Fil- the dimeric state of the motor may be a means of modifying its function. aments on Tissue Purified Slow Skeletal and Cardiac Myosin This has motivated our studies probing the kinetic similarities between Maral Mohebbi, Petra Uta, Theresia Kraft, Tim Scholz. assemblies of multiple myosin VI monomers and their dimeric Molecular and Cell Physiology, Hannover Medical School, Hannover, counterparts. Past results indicate force-dependent behavior of myosin Germany. VI dimers in response to an external force applied to its cargo via an In vitro motility assays are a powerful method for studying the function of optical trap system. Our research involves observing whether the effects motor proteins such as myosins. The actin sliding in vitro motility assay on of external forces applied to the cargo of multiple myosin VI monomers

BPJ 8632_8635 Monday, February 19, 2018 321a generates similar force-dependent (‘‘anchoring’’) behaviors. Unique 1594-Pos Board B503 kinetic properties could indicate that these different myosin Ensemble Behavior of Actomyosin Crossbridges configurations achieve different functions. Presented here are the initial Khoi D. Nguyen, Madhusudhan Venkadesan. results from our studies investigating the force-dependent activity of Mechanical Engineering and Material Sciences, Yale University, New multiple monomers working in concert. Haven, CT, USA. The ability of muscles to switch their mechanical behavior between an 1592-Pos Board B501 elastic solid and a viscous fluid plays an important role in motor control. Mutations in the Converter Domain of Myosin V Demonstrate Coupling The elastic solid-like response of muscle helps stabilize posture, whereas Between Lever Arm Swing and Phosphate Release the viscous fluid-like response enables antagonist muscles to offer mini- Laura K. Gunther, Shane D. Walton, Wanjian Tang, William C. Unrath, mal resistance during fast movements. Therefore, functional consider- Darshan Trivedi, Christopher M. Yengo. ations imply that muscle is capable of switching between elastic versus Department of Cellular and Molecular Physiology, Penn State University viscous behaviors depending on its activation level, i.e. intracellular cal- College of Medicine, Hershey, PA, USA. cium concentration that affects the fraction of available binding sites for Myosin V is an actin based molecular motor that utilizes ATP to convert myosin. Both these mechanical regimes arise from the ensemble dy- chemical energy into mechanical work. The converter domain plays a namics of crossbridges that comprise the sarcomere. A bound crossbridge crucial role in the allosteric communication between the lever arm and responds like a spring, and over short timescales, the sarcomere responds the actin- and nucleotide-binding regions. Interestingly, mutations in like an elastic solid against external perturbations. Over long timescales, the converter domain of human beta cardiac myosin are associated the crossbridge unbinds and dissipates the stored elastic stress, causing with hypertrophic and dilated cardiac myopathies. Thus, we have made the sarcomere to yield like a viscous fluid against external stresses. the corresponding mutations, R712G and F750L, in the converter Here, we model the sarcomere as an ensemble of crossbridges, and domain of myosin V to investigate the effect of these mutations on show mathematically that the emergent behavior resembles a linear visco- the myosin kinetics, structural dynamics, and motor properties. The elastic material (generalized Maxwell model). We find that the timescale R712G and F750L mutations did not change the actin concentration at of transition between elastic- versus viscous-dominated regimes is inde- which the ATPase activity is one-half maximal (KATPase)andthe pendent of activation. The strain rate (i.e. the timescale) at which the maximum ATPase rate was increased 15% in the F750L mutant but un- ensemble can be stretched with minimal elastic resistance at low activa- changed in the R712G mutant. In vitro motility assays revealed sliding tions, is also the same timescale over which internal elastic stresses are velocities similar to WT for both R712G and F750L. Actin-activated dissipated at high activations. Therefore, in a functional context, the abil- phosphate release was increased (43%) in the R712G mutant, while ity to yield against fast stretches comes at the expense of the ability to the actin-dependence of phosphate release was disrupted in the F750L maintain postural stiffness. We hypothesize that this trade-off between mutant. We followed the motion of the lever-arm during the ATP the elastic and viscous regimes can be mitigated and the timescale of binding (recovery stroke) and actin-activated product release (power switching modulated by activation upon including the load-dependence stroke) steps using stopped-flow FRET. The R712G mutation reduced of myosin unbinding kinetics. the rate of the recovery stroke by 30% while the power stroke was increased by 60%, whereas the F750L mutation had the opposite effect 1595-Pos Board B504 on the recovery and power strokes. Mechanosensitivity studies Force and Calcium Regulation of a Single Myosin-5B Motor demonstrated that both mutants are more sensitive to the addition of Lucia Gardini1,2, Sarah M. Heissler3, Claudia Arbore1,4, Yi Yang5, load in the in vitro motility assay. Our results provide direct evidence James R. Sellers3, Francesco S. Pavone1,4, Marco Capitanio1,4. that the converter domain is crucial for lever arm rotation and we pro- 1LENS, Sesto Fiorentino, Italy, 2National Institute of Optics–National vide further evidence that the lever arm gates phosphate release. Research Council, Firenze, Italy, 3National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA, 4Department of Physics 1593-Pos Board B502 and Astronomy, University of Florence, Sesto Fiorentino, Italy, 5Hunan Observing the Behavior of a Single Myosin Head Within a Myosin Agricultural University, Changsha, China. Filament Moving on Actin In mammals, there are three class-5 myosin molecular motors, 5A, 5B, Richard Brizendine, Josh Baker, Christine Cremo. and 5C. Myosin-5A, a cargo transporter that is mainly expressed in brain Department of Pharmacology, University of Nevada, Reno School of and melanocytes, has been thoroughly investigated during the last 15 Medicine, Reno, NV, USA. years. Molecular and cellular studies elucidated most of its molecular We developed a novel unloaded in vitro motility assay in which myosin fil- mechanisms to define the basis of myosin-5A processivity and its regula- aments move over surface-attached actin (Brizendine et al, PNAS 2015). tion by force and calcium. Conversely, although myosin-5B is ubiqui- For three types of muscle myosin II (smooth, skeletal, and cardiac), the rela- tously expressed and involved in fundamental cellular processes such as tionship between myosin filament velocity (V) and the number of myosin the trafficking of recycling endosomes and collective actin network dy- heads available to interact with actin (N) can be accounted for by a novel namics, its basic biophysical properties have not been extensively investi- mixed-kinetic model where V isinfluencedbybothattachmentanddetach- gated and no single molecule studies have been reported in the literature ment kinetics (see Brizendine et al, Abstract this meeting). A key model var- so far. iable is the flexibility and effective length (L) of the subfragment 2 (S2) Here, we implemented single molecule manipulation and imaging methods region of the myosin tail, which can presumably bend or buckle to allow to dissect the molecular mechanisms of myosin-5B function. Single actin-attached myosin heads to place very little drag load on the moving fila- recombinant myosin-5B motors were labelled with quantum dots and observed ment. This model predicts that a given myosin head within a moving fila- in an in vitro motility assay with fluorescence microscopy. We found that ment will appear stationary for the duration of time it remains attached to myosin-5B is a processive motor that walks in 36 nm steps at 691525 nm$s1 actin (ton), followed by a jump forward a distance L relative to the maximum velocity, covering an average distance of 78052 nm before filament backbone. To test these predictions, we prepared three dissociating from actin. By using ultra-fast force-clamp laser tweezers, we different smooth muscle myosin (SMM)-derived QD-labeled constructs found that this motor is stalled by 2 pN opposing forces, and reverses its (regulatory light chain (RLC), light meromyosin (LMM), and S2) and directionality at forces >2 pN. Interestingly, myosin-5B mechanosensitivity assembled them individually and simultaneously into co-filaments with un- differs from that of myosin-5A, but has many features in common with that labeled native SMM. We will track the QD motion with nm accuracy during of kinesin-1. In particular, myosin-5B’s run length is markedly and filament motility to compare the behavior of myosin heads (RLC-QD) and asymmetrically sensitive to force, a property that might be central for motor the headless S2-QD relative to the global filament motion (LMM-QD). As ensemble coordination. Lastly, we prove that Ca2þ does not affect the 1 [ATP] is varied, ton is predicted to change according to ton =(k-AD) þ enzymatic activity of the motor unit, but abolishes myosin-5B processivity 1 (kt[ATP]) ,wherek-AD is the ADP release rate and kT is the rate of attach- through calmodulin dissociation. Our results provide new fundamental ment of ATP to acto-myosin, whereas L will be invariant at 60 nm, a value information on the molecular mechanisms regulating cargo transport in cells based upon our previous work (Brizendine et al, PNAS 2015). by myosin-5B.

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Posters: Cell Mechanics, Mechanosensing, and ture called the glycocalyx. While the glycocalyx is known to impede cell adhesion by acting as a steric barrier, the mechanical response of the glycoca- Motility I lyx and its molecular constituents to force is largely unknown. Therefore, we have developed a physical model of the glycocalyx to understand and predict 1596-Pos Board B505 its deformation and reorganization under compressive loads, such as those Biophysics of Mechanosensitive Cadherin Adhesion and its Regulation that would be experienced by cells in confined three-dimensional environ- Andrew V. Priest, Ramesh Koirala, Chi-Fu Yen, Sanjeevi Sivasankar. ments. Simulations of our model predict that the glycocalyx can sustain sub- Physics and Astronomy, Iowa State University, Ames, IA, USA. stantial pressures without undergoing significant deformations, but beyond a Cadherin cell-cell adhesion proteins play critical roles in embryogenesis and critical pressure it experiences a pressure-sensitive response, followed by in maintaining tissue integrity. Defects in cadherin adhesion occur in meta- increased resistance at high strains. This suggests that cells could switch be- static cancers. Cadherins mediate adhesion by binding in two conformations, tween weakly adherent and highly adherent states depending on the degree X-dimers and strand-swap dimers. These binding conformations have of confinement and the compression of the glycocalyx. Additionally, the sim- distinctly different adhesive properties: while X-dimers form catch bonds ulations predict the formation of load-bearing clusters of glycoproteins in that become longer-lived in the presence of a pulling force, strand-swap di- favorable regions away from the cytoskeletal membrane attachments. We mers form slip bonds that weaken upon pulling. It has been proposed that elucidate the effects of the biophysical properties of the glycocalyx and the the cell, switches between X-dimer and strand-swap dimer conformations cell membrane on the mechanical behavior. Increasing the stiffness and the in order to regulate adhesion. However, little is known about the molecular density of glycoproteins and the membrane bending modulus and the cytoskel- mechanisms for these conformation changes and for cadherin adhesion regu- etal density or decreasing the glycoprotein length increases the collective stiff- lation. Here, we use an integrated approach that couples live-cell, single ness of the glycocalyx and the pressure required to generate significant molecule Atomic Force Microscope force measurements with precise, deformations. Together, our results demonstrate how bulk material properties cell-biological manipulations of cadherin-cytoskeleton interactions, to char- of the glycocalyx emerge from the physical properties of its molecular constit- acterize the biophysical mechanisms by which cadherins switch between uents and how these constituents rearrange under load. The understanding alternate conformations on the cell surface and subsequently regulate adhe- developed here has broad relevance in cell migration, adhesion, communica- sion. We demonstrate that cadherin interactions with the cytoskeleton tion, and other receptor-mediated processes in the glycocalyx. tunes ectodomain conformation using an ‘inside-out’ mechanism. Our data also resolves the mechanistic details for mechanosensitive adhesion 1599-Pos Board B508 regulation. Frequency and Curvature of the Flagellar Waveform of Chlamydomonas Reinhardtii are Stable during Regrowth 1597-Pos Board B506 Mathieu Bottier1,2, Susan K. Dutcher2, Philip V. Bayly1. Mechanochemical Modeling as an Explorative Tool to Study Tissue 1Department of Mechanical Engineering and Materials Science, Washington Morphogenesis University in Saint Louis, Saint Louis, MO, USA, 2Department of Genetics, Francesco Atzeni1,2, Richard S. Smith3, Christof Aegerter1, Washington University in Saint Louis, Saint Louis, MO, USA. Damian Brunner2. Cilia and flagella are highly conserved organelles that generate propulsive, 1Physics Institute, University of Zurich, Zurich, Switzerland, 2Institute of oscillatory waveforms that propel cells or move fluids. The mechanism of oscil- Molecular Life Sciences, University of Zurich, Zurich, Switzerland, 3Max lation is a mystery; to distinguish competing models we studied the effects of Planck Institute for Plant Breeding Research, Cologne, Germany. regrowth on frequency and waveform in the alga Chlamydomonas reinhardtii Dorsal closure is a morphogenetic process in Drosophila embryogenesis, using high-speed video-microscopy with bright field optics. Wild-type unifla- whereby an epidermis opening closes. The epidermal opening is filled gellate mutant Chlamydomonas cells were deflagellated by pH shock. Using with the Amnioserosa (AS) tissue, which drives dorsal closure by apical a method described previously [Bayly et al. Cytoskeleton, 2010] we obtained constriction of its individual cells. Apical constriction in turn is driven by high-resolution, quantitative descriptions of the waveform at different lengths. apical surface area oscillations of AS cells, which are the result of period- We recorded 97 videos of beating flagella that varied from 0.7 to 10.9 mm. ically forming, transient actomyosin foci. It is currently unclear whether Videos were recorded at 2000 frames/s with 169 nm spatial resolution. We neighboring cells coordinate their oscillations and how the cell scale discovered that the beat frequency is reduced for flagella < 4 mm but oscillates behavior leads to tissue scale dynamics. To better understand these aspects, around 60 Hz (60.2 5 20.7 Hz) when flagella are longer than 4 mm. The we developed a coupled finite element simulation of actomyosin dynamics, average curvature is small and variable in flagella < 4 mm, and reaches a force generation, and material behavior. Thereby, we model the dynamics of plateau at 0.20 5 0.08 rad/mm for flagella > 4 mm. Other quantities such the actomyosin network in AS cells with reaction-diffusion equations at the as beat amplitude, mean total angle, torque, power, and average force applied subcellular level in 2D. The actomyosin concentration determines the by the flagellum on the fluid in both the x and y directions, all increase with strength of local contractions within the AS cell surfaces. Based on our in flagellar length. These results indicate that frequency and curvature are impor- vivo observations, we model the dynamics of actin binding proteins (eg. tant conserved features of the flagellar waveform during regrowth. Because the Arp2/3) as an activator-depleted substrate system, and couple it to actin average curvature is consistent, the total bend angle increases with flagellar network formation. Analytical methods describe the system behavior in length (waveforms of small flagella are not scaled versions of the waveform the absence of mechanical deformation, and predict how the system will of longer flagella). These results suggest that the local mechanics of the evolve in coupled simulations. We model the surrounding epidermis as a axoneme are stable in flagella > 4 mm and that changes in flagella length alone linear viscoelastic material, so that it adapts in response to the pulling force can explain qualitative changes in the waveform shape. of the AS tissue, as shown experimentally. Our simulations reproduce dorsal closure and make clear predictions that ask for experimental testing. In 1600-Pos Board B509 particular, a sequential arrest of cellular oscillations simply emerges, similar Dual Biomembrane Force Probe Enables Single-Cell Mechanical Analysis to what was observed in vivo. So far, a dpp-signaling gradient was thought of Signal Crosstalk between Multiple Molecular Species to control this phenomenon. Addressing this experimentally provides sup- Lining Ju1, Yunfeng Chen2, Kaitao Li2, Cheng Zhu2. port for the model prediction. Interestingly, 3D finite element simulations 1Heart Research Institute, University of Sydney, Camperdown, Australia, predict that the emerging sequential pulsation arrest generates a contractility 2Georgia Institute of Technology, Atlanta, GA, USA. gradient, which controls AS tissue morphogenesis. Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The per- 1598-Pos Board B507 turbing preparation procedures often alter the sample from their native state Equilibrium Structure and Mechanics of the Cellular Glycocalyx on the cell. 2) Long processing time before the final readout makes it difficult Jay G. Gandhi, Donald L. Koch, Matthew J. Paszek. to capture transient signaling events (<1 min). 3) The experimental environ- Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, ments are force-free, therefore unable to visualize mechanical signals in real USA. time. In contrast to these biochemistry and cell biology methods that are usually Cells can employ distinct modes of cell migration, ranging from mesenchymal- population-averaged and non-real-time, here we introduce a novel single-cell like or amoeboid-like single cell motility to collective migration. Each mode is based nanotool termed dual biomembrane force probe (dBFP). The dBFP pro- characterized by a unique balance of cell-matrix and cell-cell adhesion. Carbo- vides precise controls and quantitative readouts in both mechanical and chem- hydrates called glycans are concentrated on the cell surface in a complex struc- ical terms, which is particularly suited for juxtacrine signaling and

BPJ 8636_8639 Monday, February 19, 2018 323a mechanosensing studies. Specifically, the dBFP allows us to analyze dual re- The dominant thinking of the mechanism for the apical constriction is the ceptor crosstalk by quantifying the spatiotemporal requirements and functional purse-string model which explains the shrinkage of apical area is due to the consequences of the up- and down-stream signaling events. In this work, the contractile force from circumferential actomyosin bundle around the apical sur- utility and power of the dBFP has been demonstrated with four important face. However, recent studies of morphogenesis gastrulation in Drosophila and dual receptor systems that play key roles in immunological synapse formation, Xenopus hasve shown that the apical constriction of ventral furrow cells can be shear-dependent thrombus formation, and agonist driven blood clotting is driven by pulsed actomyosin contraction within medio-apical surface. Here respectively. we used MDCK cysts, which consist of cellular monolayers enclosing a fluid- filled lumen, to study apical constriction. We found that the tight junctions, 1601-Pos Board B510 which form continuous belt structure at subapical regions of a cell, become Mechanical Property Change of Red Blood Cell Membrane under tortuous as cysts mature. We quantified the tortuous tight junctions and found Photosensitizer Mediated Oxidative Stress of CIS Porphyrin that the tight junctions become straighter when we disrupt the actomyosin 1 2 2 3 Koji Kinoshita , Gustavo Campos , Tayana Tsubone , Vita Solovyeva , contractility with pharmaceutical perturbation. Together with theoretical simu- Jonathan Brewer3, David Needham1, Rosangela Itri2. Ò 1 lations based on Surface Evolver , we can explain the morphological change SPSE-Center, Department of Molecular Medicine, University of Southern by the change of apical surface tension. In addition, we performed laser abla- Denmark, Odense, Denmark, 2Instituto de Fisica, Universidade de Sa˜o Paulo, 3 tion at the apical face and found that the apical surface area increase after abla- Sa˜o Paulo, Brazil, MEMPHYS-DaMBIC-Center, Department of tion. The expansion rate increase with increasing tortuosity and decreasing Biochemistry and Molecular Biology, University of Southern Denmark, apical area of the cells. This observation fits our model of actomyosin contrac- Odense, Denmark. tility on the apical surface. Our proposed cytoarcheture of medioapical actomy- Various cytoskeletal networks have been investigated to understand the cell osin network may shed light to how cytoskeleton regulates the paracellular mechanics because it can directly apply for therapeutic researches of immune transportation of tight junctions. or cancer disease. However, due to a lack of sufficient signal-transduction mea- surement techniques, even for the well-studied red blood cell (RBC), the rela- 1604-Pos Board B513 tionships between the cytoskeleton physiology and mechanical properties of A Breakdown of Cellular Mechanisms Required for Cell and Focal the cell membrane – i.e., ‘‘signaling’’ – are still not well understood. Here, Adhesion Area Sensitivity to Substrate Stiffness we applied a newly designed micropipette manipulation technique to make dy- Magdalena Stolarska1, Aravind Rammohan2. namic membrane elasticity measurements in the presence of a therapeutic 1Mathematics, University of St. Thomas, St. Paul, MN, USA, 2Corning, Inc., photosensitizer-mediated oxidant, cis-porphyrin. We found that the shear Corning, NY, USA. 5 6 modulus of the RBC was dynamically changed from 6.7 0.5 x 10 N/m Cell spread area and focal adhesion (FA) sizes are known to increase with sub- (absence of the porphyrin) to a non-measureable or plastic-like behavior value strate stiffness (Yeung et al., Cell Motil. Cytoskeleton., 2005). Different models m (presence of 0.05 M porphyrin and irradiation) with the degree of oxidative have been developed, some of which can predict increases in cell spread area stress controlled by the irradiation. We also found that the porphyrin oxidative with substrate stiffness while others can predict increases in only the FA stress could induce unique morphological change before hemolysis of the RBC. area. Here, we systematically add one mechanism at a time to start recapitu- We hypothesize that dynamical cytoskeletal network reformation with oxida- lating both these behaviors together. We start with an adapted model (Walcott tive stress could cause these behaviors. Therefore, to visualize the network pro- et. al. Biophysical J., 2011) of FA growth implemented in a 2D cell that spreads teins, we also applied the techniques of Stimulated Emission Depletion (STED) at a constant rate. This model demonstrates that while the total FA area in- microscopy. We consider that this further characterization of the mechanical creases with substrate stiffness the cell spread area is not sensitive to the stiff- properties of the RBC membrane will help to understand more complicated ness. In order for the cell spread area to increase with substrate stiffness the cytoskeletal network of immune, cancer or even neuron cells for therapeutic spreading model must include a coupling between the spreading rate, the FA development in the future. More details will be shown at the meeting. complex evolution, and intracellular stresses (Vernerey & Farsad, J. Math. 1602-Pos Board B511 Bio., 2013). When the spreading rate is coupled with cellular stresses and FA Cell Growth Rate Dictates the Onset of Glass to Fluid-Like Transition and evolution, we demonstrate the ability of our model to qualitatively reproduce Long Time Super-Diffusion in an Evolving Cell Colony the increase in cell spread area and FA area with substrate stiffness. Further Abdul Malmi Kakkada, Xin Li, Himadri S. Samanta, Sumit Sinha, we try to generate biological insight on the role of the model parameters in their Dave Thirumalai. ability to reproduce cellular behavior for various experimentally studied Dept. of Chemistry, University of Texas, Austin, Austin, TX, USA. cellular systems such as 3T3 fibroblasts and human mesenchymal stem cells. Collective migration dominates many phenomena, from cell movement in Based on this we also articulate the gaps in the current model and make a living systems to abiotic self-propelling particles. Studying early stages of tu- case for potentially bringing in additional mechanisms that can account for mor evolution, we clarify the principles involved in cell dynamics and highlight the role of stress fiber orientation (Deshpande et al, PNAS, 2006) which can their implications in understanding similar behavior in seemingly unrelated soft enhance our ability to understand the interplay between the different mecha- glassy materials and possibly chemokine-induced migration of CD8þ T cells. nisms in predicting cellular responses across a broader range of cell types. We performed simulations of tumor invasion using a minimal three- dimensional model, taking into account cell elasticity and adhesive cell-cell in- 1605-Pos Board B514 teractions to establish that cell growth rate-dependent tumor expansion leads to Magneto-Active Substrates for Local Mechanical Stimulation of Living the emergence of distinct topological niches. Cells at the periphery move with a Cells 1 1 2 1 higher velocity perpendicular to the tumor boundary, while the movement of Alexis E. Coullomb , Cecile M. Bidan , Mario Fratzl , Philippe Moreau , 1 1 1 1 interior cells is slower and isotropic. The mean square displacement, D(t), of Alain H. Lombard , Irene Wang , Martial Balland , Thomas Boudou , Nora M. Dempsey2, Thibaut Devillers2, Aurelie Dupont1. cells exhibits glassy behavior at times comparable to the cell cycle time, but, 1 2 exhibit super-diffusive behavior, D(t) ta, at longer times. We establish the LIPhy, Saint Martin d’He`res, France, I. Neel, CNRS, Grenoble, France. universality of super-diffusion in a class of seemingly unrelated non- Cells can feel and react to the mechanical properties of their environment such equilibrium systems. Our findings for the collective migration, which also sug- as stiffness or geometry by translating mechanical cues into biochemical ones, gests that tumor evolution occurs in a polarized manner, are in quantitative inducing biochemical and mechanical responses. This process, called mecha- agreement with in vitro experiments. Although set in the context of tumor in- notransduction, drives critical functions such as cell differentiation, prolifera- vasion, our findings should be relevant in describing collective motion in tion and migration. In order to assess to which extend cellular response growing cell colonies especially in active systems where creation and annihila- depends on the temporal and spatial characteristics of the stimulation, it is tion of particles play a role. essential to control temporally and spatially the mechanical cues. Methods have been proposed to apply global and continuous deformations, or local de- 1603-Pos Board B512 formations though discrete substrates. Here we propose a novel method to The Apical Constriction Force of Madin-Darby Canine Kidney (MDCK) apply mechanical stimuli in a local and dynamic way to cells plated on the Cells continuous surface of deformable substrates. These substrates are made of a Ching-chung Hsueh1, Ivan Alex Priela Lazarte2, Mathieu Prouveur3, soft elastomer (PDMS) in which iron micro-pillars are embedded and actuated Wen-Hsiu Wu4, Ying-Ting How1, Keng-Hui Lin5. by two electromagnets. The amplitude of the surface deformation is controlled 1Physics, National Taiwan University, Taipei, Taiwan, 2Physics, National by the input current in the coils, and monitored by tracking fluorescent particles Central University, Chungli, Taiwan, 3Mines ParisTech, Paris, France, underneath the surface. Traction Force Microscopy (TFM) allows us to esti- 4Physics, National Tsing-Hua University, Hsin-Chu, Taiwan, 5Physics, mate the stress generated by the pillars, and the cellular mechanical response. Academia Sinica, Taipei, Taiwan. Cells adhering to the magneto-active substrates can be stimulated both in

BPJ 8636_8639 324a Monday, February 19, 2018 traction and compression in the range of contractile cell stresses. Thanks to the ical cues and leading to wide spread secondary tissue damage. This study in- compatibility with standard fluorescence techniques, we can furthermore vestigates the biophysical response of primary neutrophils from healthy and observe the biochemical response of cells with fluorescence techniques such septic patients to defined chemotactic gradients confined in three- as quantitative Fluorescence Resonance Energy Transfer (FRET). dimensional collagen matrices. Materials and Methods: Primary neutrophils isolated from the whole blood of 1606-Pos Board B515 patient donors are embedded in 3D collagen matrices with microspheres. A sus- Cell Membrane Transmits High-Level Integrin Tension to Mediate Cell tained gradient of chemoattract, such as N-Formylmethionine-leucyl-phenylal- Rear De-Adhesion during Keratocyte Migration anine (fMLP) mimicking bacterial infection, is introduced across the collagen Yuanchang Zhao1, Yongliang Wang1, Anwesha Sarkar1, Xuefeng Wang1,2. 1 2 gel by diffusion. Time-lapsed acquisition of confocal z-stacks are recorded for Physics and Astronomy, Iowa State University, Ames, IA, USA, Molecular, 2 hours and the displacement fields of neutrophils in collagen gels are resolved Cellular, and Developmental Biology Interdepartmental program, Molecular by 3D confocal imaging utilizing the fast iterative digital volume correlation Biology Building, Iowa State University, Ames, IA, USA. (FIDVC) algorithm. Integrin-transmitted forces play crucial parts in the shape, survival, Results and Discussion: When compared with naı¨ve cells, LPS-activated neu- signaling and migration of the eukaryotic cells. In fish epidermal kerato- trophils mimicking sepsis, demonstrated significant out of plane stresses as cytes, a classic model for cell migration, integrin participates in the pro- shown by increases in the overall cell rotation and rotation angle during migra- cesses of adhesion, translocation and deadhesion during cell migration. tion through collagen matrices. These measurements were limited to short time Although adhesion and translocation processes are well studied, the cellular and length scales; subjecting the data analysis to sampling errors. Utilizing pro- force for rear deadhesion doesn’t have a precise picture yet because its range longed chemotactic gradients to drive cell motion, this current study improves and distribution are poorly understood. Thus, we made integrative tension upon their observations by tracking matrix deformations induced by persis- sensor (ITS), a single molecular tension sensor which is based on double tently migrating neutrophils over multiple cell lengths, while simultaneously strand DNA and is able to convert cellular force to fluorescence, with quantifying biophysical parameters critical to cell motility such as velocity, 0.4uM spatial accuracy and pN force sensitivity. We found that the large volume, directionality and mean squared displacement. The use of primary force, from 54pN to 100pN, is exclusive distributed at rear edge, which cells from trauma and septic patients further elicits the role of biomechanical leaves two tracks as the ‘footprint’ of the migrating cell. We further demon- regulation in the loss of chemotactic cues experienced by neutrophils in the strated that the force is transmitted by the cell membrane instead of actomy- diseased state. osin, as commonly thought as the source of cell’s retraction force for less motile cells, which means the cell membrane can supersede actomyosin to transmit large force for cell migration and deadhesion. A simple model 1609-Pos Board B518 has come out that the keratocytes can detach integrins from the substrate Pressure-Ineduced Activation of the Swimming Motility of Magnetotactic without the participation of actomyosin. Bacterium Masayoshi Nishiyama1, Ruan Juanfang2, Takayuki Kato2, Toru Minamino2, 1607-Pos Board B516 Keiichi Namba2, Akitoshi Seiyama1, Long-Fei Wu3, Yoshie Harada2. Integrin Molecular Tensions in Live Cells are Altered by Substrate 1Kyoto University, Kyoto, Japan, 2Osaka University, Osaka, Japan, Rigidity 3Aix-Marseille University, Marseille, France. Anwesha Sarkar, Xuefeng Wang. The marine bacterium MO-1 cell has a flagellar apparatus with two lophotri- Physics and Astronomy, Iowa State Univ, Ames, IA, USA. chous bundles. Each apparatus is a tight bundle of seven flagellar filaments Mounting evidence suggests that mammalian cells are capable of sensing in a sheath [1]. The unique motility machinery is sensitive to environmental the rigidity of their local environment and responding to it. However, it re- factors including physical and chemical conditions. Here, we performed a mains unclear whether cells sense cellular force alteration caused by matrix motility assay of marine bacterium MO-1 at high-pressure. In general, appli- compliance (stress sensing) or sense cytoskeleton remodeling caused by ma- cation of pressure could change the structure and function of motility ma- trix deformation (strain sensing). Because integrins are the major mechano- chineries [2]. We enclosed MO-1 cells in a high-pressure chamber for sensitive receptors that transmit cellular force at the cell-matrix interface, optical microscopy [3]. At ambient pressure condition (0.1 MPa), most cells the tension transmitted by integrin molecules (integrin tension) may serve did not show any directional motions. When pressure was increased to 80 as fundamental mechanical signal in cell mechanotransduction and rigidity MPa, many cells started to swim in solution. The fraction of swimming cells sensing. Therefore, measuring and mapping integrin molecular tension in drastically increased with time, and reached to a peak of 70 %, and then live cells on elastic substrates will shed light to the mechanism of cell rigid- gradually decreased. The translational speed of the swimming cells at the ity sensing. In this work, we applied integrative tension sensor (ITS), a peak was about 50 mms1, which was almost half of the speed of swimming sensor converting molecular tension above a threshold to fluorescent signal, cells at 0.1 MPa. The speed also gradually decreased with time. The to calibrate and map integrin tension directly by fluorescence imaging. In pressure-induced activation of cell motility occurred remarkably in the experiments,ITSwith54pNtensionthresholdwasgraftedonPDMS(Poly- limited pressure region of 60-100 MPa. High-speed video microscopy re- dimethylsiloxane) substrates with Young’s modulus ranging from 1kPa to vealed that MO-1 cells swim constantly in a helical trajectory at high- 1.8 MPa. Integrin tension above 54 pN in live cells was mapped with pressure conditions. But, the pitch in a helical motion at 80 MPa was about high resolution (0.4 mm) and sensitivity on these substrates. The results 2 times larger than that at 0.1 MPa. This result indicated that the flagellar show that cell spreading area and integrin tension activity both increased motion induced by high pressure is slightly different from that under normal with increasing PDMS gel stiffness for CHO-K1, NIH-3T3 and skin fibro- pressure. The detailed mechanism would be revealed by monitoring the mo- blast cell lines. Also, keratocytes migrated faster on softer PDMS substrates tion of individual sheaths. and integrin tension activity was the highest on the stiffest PDMS sample. [1] Ruan J., T. Kato, ., K. Namba. 2012. Proc. Natl. Acad. These experiments for the first time monitored integrin tension in live cells Sci.USA.109:20643-8. on elastic substrates, and clearly demonstrated that the substrate rigidity af- [2] Nishiyama M. 2017. Biophys Chem., DOI: 10.1016/j.bpc.2017.03.010. fects integrin tension activity, providing evidence in favor of stress sensing [3] Nishiyama M., and Y. Arai. 2017. Methods Mol Biol. 1593:175-184. doi: hypothesis. 10.1007/978-1-4939-6927-2_13. 1608-Pos Board B517 Four Dimensional Traction Measurements of Chemotactic Neutrophils in 1610-Pos Board B519 Hydrogels Hepatitis C Virus Alters Nuclear Mechanics by Down-Regulating Michael W. Harman1, Christian Franck1, Jonathan Reichner2. Lamin A/C 1Engineering, Brown University, Providence, RI, USA, 2Division of Surgical Sreenath Balakrishnan1, M.S. Suma2, Geetika Sharma3, Saumitra Das3, Research, Rhode Island Hospital, Providence, RI, USA. G.K. Ananthasuresh2. Introduction: Neutrophils are the primary innate defense against infection 1BioSystems Science and Engineering, Indian Institute of Science, and tissue trauma, initially becoming activated through the localized inflam- Bengaluru, India, 2Mechanical Engineering, Indian Institute of Science, matory response. Typically, rapid in onset, and brief, the inflammatory Bengaluru, India, 3Microbiology and Cell Biology, Indian Institute of response efficiently clears microbial infection and initiates tissue repair. How- Science, Bengaluru, India. ever, in cases of severe traumatic injury or septic infection, neutrophils Nuclear mechanics is a critical regulator of cell behavior. Nuclear lamina, become excessively activated, dysregulating their environmental biomechan- along with the cytoskeleton, serve as a transducer of mechanical and

BPJ 8636_8639 Monday, February 19, 2018 325a chemical signals from the exterior of the cell and the cytoplasm to the tions. Our results show that netrin-1 reduces chemokinetic motility of chromatin and hence govern gene expression. Here, we show that Hepatitis human neutrophils, which is accompanied with reduced cell polarization C Virus (HCV) alters the morphology and the stiffness of the nuclei by and spreading. In addition, netrin-1 reduces neutrophil chemotaxis to down-regulating lamin A/C. We observed changes in nuclear morphology N-Formyl-Met-Leu-Phe (fMLP) on fibronectin substrate but interestingly and used computational modeling to predict the causes for the observed not on collagen substrate. By contrast, netrin-1 promotes the migration of changes in the shape of the nuclei. We confirmed these predictions biome- human breast cancer cells. Furthermore, we found that netrin-1 reduces chanically using AFM and biochemically using protein expression studies. neutrophil chemotaxis to the supernatant of human breast cancer cell cul- By subjecting cells to shear stress due to flow, we show that the nuclei of ture. Collectively, this microfluidic cell migration study provided quantita- HCV infected cells assume increasingly elliptical shape even at low shear tive characterizations of the effects of netrin-1 on the motility and stress corresponding to interstitial flow. Our results show a possible dereg- chemotaxis of neutrophils and breast cancer cells, and further suggested ulation of the mechanobiology of the cells by HCV through disruption of the potential role of netrin-1 in regulating neutrophil recruitment to breast the lamin and rendering the nuclear morphology susceptible to shear stress. cancer microenvironments. By engineering cells lacking lamin, recent studies have shown a host of functions of the nuclear lamina in the context of chromatin regulation. 1613-Pos Board B522 We believe that HCV is a natural pathogen which is exploiting the dereg- How Filaments Density Impacts Force Generation and Protrusion Rate of ulation of all these important functions of the nuclear lamina for its Lamellipodium in Motile Cells pathogenesis. Setareh Dolati. Max Delbruk Center of Molecular Medicine, Berlin, Germany. 1611-Pos Board B520 In Migrating cells, the Arp2/3-complex is thought to be responsible for forma- Blood Shear Stress Selects Metastasis-Initiating Cells with Metastatic tion and maintenance of the lamellipodium. However, studies show in addi- Advantages tion to Arp2/3 activity formins also contribute to actin filament nucleation Xin Tang, Jing Jin, Shiying Huang, Ying Xin, Youhua Tan. and elongation in the lamellipodium of B16-F1 melanoma cells and their ac- Department of Biomedical Engineering, Hong Kong Polytechnic University, tivity strongly impacts force generation. Loss of formins reduces actin density, Hong Kong, China, Hong Kong. lamellipodium width and protrusion velocity of B16-F1 melanoma cells, Metastasis is the most lethal and malignant state of cancer, which accounts while Arp2/3 activity and the actin network assembly rate are not affected for over 90% of cancer-related deaths. Tumor cells from the primary by the absence of formins. Knocking out FMNL2 and FMNL3 individually lesion metastasize to distant organs mainly through hematogenous dissemi- and both together shows a correlation between actin filament area density nation. The presence and frequency of circulating tumor cells in blood are and protrusion rates. Also, by manipulating Arp2/3 activity in B16-F1 mela- correlated with poor prognosis and overall survival. It has been proposed noma cells, the same correlation between the protrusion rate and the filament that circulating tumor cells contain a rare subset of metastasis-initiating area density in the lamellipodium has been observed. Here, we mathemati- cells (MICs) with the ability to generate clinically significant metastatic tu- cally model the lamellipodium as a viscoelastic gel representing an actively mors in distant organs. These cells must hold survival advantages in the met- polymerizing and cross linked network of actin filaments. Taking the density astatic cascade and cancer stem cell properties that are essential in of filaments as a control parameter, we suggest a mechanism that explains metastasis formation. Although the influence of blood circulation has been how the formin’s contribution to the actin area density leads to their corre- extensively studied, the roles of blood shear flow in metastatic potential of sponding contribution to the protrusion rate in the lamellipodium and how tumor cells are unclear. In particular, it remains largely elusive whether the structure of the actin network and properties like actin filament volume and how hematogenous shear stress selects MICs with metastatic advantages density and assembly rate of the network affect the dynamics of the from bulk tumor cells. This study developed a circulatory microfluidic lamellipodium. system, in which single breast cancer cell suspensions with differential met- astatic potential were treated under various conditions to eradicate the ma- jority of cells and optimize the levels of shear stress and circulating time. 1614-Pos Board B523 Tumor cells with high metastatic potential survived better in fluid shear High-Throughput Mechanotransduction in Drosophila Embryos with a flow in a force-magnitude and circulating-time dependent manner. Impor- Microfluidic Device Ardon Z. Shorr1, Utku So¨nmez2, Jonathan S. Minden1, Philip R. LeDuc3. tantly, the surviving cells exhibited metastasis-initiating abilities, including 1 high migratory and invasive potential, self-renewal and tumorigenicity, sur- Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA, 2Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA, vival advantages, and chemoresistance. Mechanistically, fluid shear stress 3 significantly affected epithelial-mesenchymal transition (EMT) plasticity Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA. and YAP/TAZ activity that influenced tumor cell survival in blood circula- Living systems experience and respond to a range of mechanical forces. Fail- tion. Silencing YAP/TAZ or critical transcription factors in EMT attenuated ures in mechanotransduction have been linked to several pathologies, including MIC properties of the surviving tumor cells selected by blood shear stress. developmental disorders, muscle and bone loss, neurological disorders, Taken together, these results suggest that blood shear stress selects microgravity-related disorders, and age-related disorders. A major barrier in MICs with stemness and metastatic advantages that depend on EMT and understanding mechanotransduction is that the proteins involved in transducing YAP/TAZ. mechanical perturbation remain unknown. One way to learn these pathways is through the Drosophila embryo. 1612-Pos Board B521 During Drosophila gastrulation, patterned gene expression coordinates the The Effect of Netrin-1 on Neutrophil and Breast Cancer Cell Migration movement of developing tissue; for example, the transcriptional regulator and their Migratory Interaction Twist controls a sequence of events that leads ventral cells to apically Jolly Hipoilito1, Hagit Peretz-Soroka1, Aniel Moya Torres2, Evan Booy2, constrict, initiating invagination of the mesoderm. Embryos unable to ex- Ke Yang1, Monika Gupta2, Markus Meier2, Sean McKenna2, Manuel Koch3, press Twist fail to properly gastrulate. Surprisingly, gastrulation can be Susy Santos4,Jo¨rg Stetefeld2, Francis Lin1. rescued with a local mechanical stimulation which triggers the ectopic 1Physics and Astronomy, University of Manitoba, Winnipeg, MB, Canada, expression of Twist. This can serve as an experimental paradigm to under- 2Chemistry, University of Manitoba, Winnipeg, MB, Canada, 3Institute for stand mechanotransduction. Dental Research and Oral Musculoskeletal Biology, University of Cologne, However, the mechanical induction of Twist has only been observed by manip- Cologne, Germany, 4Victoria General Hospital, Winnipeg, MB, Canada. ulating one embryo at a time, which precludes proteomic techniques where Netrin-1 is well-known for its chemoattractive and chemorepulsive proper- hundreds of embryos are required. To make these techniques more tractable, ties for axon guidance. Early studies report that netrin-1 inhibits granulocyte we developed a microfluidic device to precisely compress hundreds of migration. On the other hand, netrin-1 can promote cancer cell migration Drosophila embryos concurrently. Here we describe the design considerations and invasion. The underlying mechanisms are not well understood, which and validation of this device. The device permits sufficient oxygenation of the requires more in-depth characterizations of netrin-1 mediated immune and embryos, and embryos show normal survival rates after compression by 5-20%. cancer cell migration. The present study, for the first time, employed micro- We found that mechanical compression of embryos can induce Twist expres- fluidic devices that we recently developed to quantitatively investigate the sion as measured by a Twist:GFP mutant. This experimental system allows effects of netrin-1 on the motility and chemotaxis of human blood neutro- for better molecular characterization of how embryos respond to mechanical phils and human breast cancer cells under well-controlled gradient condi- stress.

BPJ 8636_8639 326a Monday, February 19, 2018

1615-Pos Board B524 that MLCK and ROCK respectively regulate SFs located in the cellular center Mechanical Feedback Coordinates Cell Wall Expansion and Assembly in and periphery, it is unclear how these kinases differ in their effects on RLC Yeast Mating Morphogenesis phosphorylation state and how each phosphospecies influences the mechanics Samhita Banavar1, Carlos Gomez2, Michael Trogdon3, Linda Petzold3, of central and peripheral SFs. Here we address these questions by combining Tau Mu Yi2, Otger Campas3. graded induction of ROCK or MLCK expression with phospho-RLC immuno- 1Physics, University of California, Santa Barbara, Santa Barbara, CA, USA, analysis and mechanical measurement of single SFs in living cells. Immuno- 2Molecular, Cellular, and Developmental Biology, University of California, blots and quantitative immunofluorescence imaging reveal that graded MLCK Santa Barbara, Santa Barbara, CA, USA, 3Mechanical Engineering, overexpression preferentially produces graded increases in p-RLC, which lo- University of California, Santa Barbara, Santa Barbara, CA, USA. calizes to peripheral SFs, while graded ROCK overexpression increases pp- The shaping of individual cells requires a tight coordination of cell mechanics RLC, which localizes to central SFs. Single-SF laser ablation studies revealed and growth. However, it is unclear how information about the mechanical that MLCK increases the effective viscosity and stored elastic energy in pe- state of the wall is relayed to the molecular processes building it, thereby ripheral SFs but not central SFs, while ROCK preferentially increases these enabling the coordination of cell wall expansion and assembly during parameters in central SFs. Graded variations in kinase activities revealed sur- morphogenesis. Combining theoretical and experimental approaches, we prising and previously unappreciated nonlinearities between phosphorylation show that a mechanical feedback coordinating cell wall assembly and expan- levels and SF viscoelasticity. To establish causal relationships between sion is essential to sustain mating projection growth in budding yeast (Saccha- RLC phosphospecies and SF mechanics, we overexpressed RLC mono- and romyces cerevisiae). Our theoretical results indicate that the mechanical di-phosphomimetic mutants. We found that phosphomimetic p-RLC localized feedback provided by the Cell Wall Integrity pathway, with cell wall stress to and selectively influenced the mechanics of peripheral SFs while di- sensors Wsc1 and Mid2 increasingly activating membrane-localized cell phosphomimetic pp-RLC localized to and selectively influenced the me- wall synthases Fks1/2 upon faster cell wall expansion, stabilizes mating pro- chanics of central SFs. This work supports a model in which MLCK regulates jection growth without affecting cell shape. Experimental perturbation of the peripheral SF viscoelasticity via p-RLC while ROCK regulates central SF osmotic pressure and cell wall mechanics, as well as compromising the me- viscoelasticity via pp-MLC. chanical feedback through genetic deletion of the stress sensors, leads to cellular phenotypes in agreement with the theoretical predictions. Our results 1618-Pos Board B527 show that while the existence of mechanical feedback is essential to stabilize High-Throughput Cell Deformability Screening to Identify Novel mating projection growth, the shape and size of the cell are insensitive to the Anti-Cancer Compounds feedback. Navjot Kaur Gill, Kendra Dee Nyberg, Dongping Qi, Bobby Tofiq, Robert Damoiseaux, Amy C. Rowat. 1616-Pos Board B525 Molecular, Cellular and Integrative Physiology MCIP, University of A Computational Framework to Accurately Predict Multivalent Enthalpy California, Los Angeles UCLA, Los Angeles, CA, USA. and Configurational Entropy Landscapes of Multivalent Interactions of Cell mechanical phenotype, or mechanotype, is emerging as a valuable label- Cell Mimetics free biomarker for cancer diagnosis, prognosis, and treatment response. To Aravind R. Rammohan1, Sungmin Ha2, Mathew Mckenzie1, advance mechanotype as a marker for screening requires a method to simulta- Natesan Ramakrishnan2, Ravi Radhakrishnan2. neously measure the deformability of 102 cell samples that interfaces with ex- 1Corning Inc., Corning, NY, USA, 2UPENN, Philadelphia, PA, USA. isting high throughput screening facilities. Here we present the high throughput We present a multiscale modeling framework to predict the adhesion of model parallel microfiltration (HT-PMF) method, which enables parallel measure- particles from nano-to-micron scales of different shapes and compliances that ments of cell deformability. HT-PMF relies on the pressure-driven filtration mimic functionalized nanoparticles to biological cells by virtue of being deco- of cells through a porous membrane with micron-scale pores. More deformable rated with receptors/antibodies in their surface. The model derives cell surface cells passage through the pores or interpillar gaps more quickly than less receptor interactions with functionalized peptides using atomistic simulations. deformable cells, resulting in a smaller retention volume and larger filtrate vol- These interaction potentials are then fit to predefined functions which are used ume, which are quantified using a plate reader. To demonstrate the ability of to characterize the interaction between the particle/cells with substrates and to HT-PMF to screen cells based on cell deformability, we show the differential measure the free energy of adhesion/binding constants. We also show that filtration of human ovarian cancer cells that have acquired drug resistance, these spring constants can alternately be obtained via a Principal Component that overexpress transcription factors (Snail, Slug) that are implicated in Analysis of the ligand bound intergrin complex. Once these atomistic details epithelial-to-mesenchymal transition; as well as cells treated with the common are incorporated into our mesoscopic model we are able to show that differ- anti-cancer agent, paclitaxel, and cytoskeleton perturbing drugs. We also use ences in amino acid sequences do contribute to more macroscopic changes in HT-PMF for deformability-based screening of cisplatin-resistant ovarian can- the average multivalency and the corresponding enthalpy of binding. Specif- cer cells treated with the library of pharmacologically active compounds (LO- ically we find that the average increase in multivalent interactions directly PAC1280) to identify lead compounds that reverse their more deformable correlate with the increase in enthalpic interactions. Expanding on this we phenotype. We validate the lead compounds using secondary, orthogonal find that anisotropy in particle shape with flatter and more pancake like par- assays including invasion and dose response assays. To further advance HT- ticles show a much stronger binding and a greater tendency for multivalent PMF for automated screening, we also demonstrate the use of an easy-to- interactions. This provides insights on why biological cells would prefer to fabricate polydimethylsiloxane (PDMS) membrane, which consists of an array adopt this more spread configuration. We also share some results on extending of 96 individual filtration devices, each with an array of micropillars with the results to a more typical cellular scale. In this work we show a computa- micron-scale gaps that are fabricated using soft lithography. Taken together, tional tool where we couple chemistry of peptide, membrane fluctuations and our results demonstrate a simple and scalable mechanotyping method that in- cellular sizes to estimate the overall binding affinities of these model cells to terfaces with commercial equipment such as multichannel pipettes, multiwell functionalized surfaces. The model as it stands can be a tool for development plates, and plate readers for HT screening of cells to identify novel anti- of functionalized surfaces aimed at cell based assays and or cell based cancer agents. therapies. 1619-Pos Board B528 1617-Pos Board B526 A Constitutive Flow Relation for Lymphatic Endothelium Rock and MlCK Tune Regional Stress Fiber Mechanics via Preferential Emily A. Margolis, Cassandra M. Chua, Joe Tien. Myosin Light Chain Phosphorylation Biomedical Engineering, Boston University, Boston, MA, USA. Elena Kassianidou, Jasmine H. Hughes, Sanjay Kumar. The lymphatic vasculature consists of a blind-ended network that drains excess Bioengineering, University of California, Berkeley, Berkeley, CA, USA. fluid, solutes, and cells from tissues. Each lymphatic vessel is lined with endo- Non-muscle myosin II (NMMII) is the key force-generating component of thelial cells (ECs), which form a permeable boundary between the tissue inter- actomyosin stress fibers (SFs), which transmit tension throughout the cyto- stitial fluid and the lymph within the vessel. While much is known about how skeleton and to the extracellular matrix. NMMII motor activity is strongly blood vessel-derived ECs react to flow-induced mechanical signals, it is less promoted by phosphorylation of its regulatory light chains (RLCs) at Ser19 clear to what extent lymphatic ECs do. In this work, we study how the mechan- (p-RLC) and additionally enhanced by further phosphorylation at Thr18 ical environment affects the hydraulic properties of lymphatic endothelium. (pp-RLC). The main RLC regulatory kinases are myosin RLC kinase Measurement of pressure-driven flow across lymphatic EC monolayers on a (MLCK) and Rho-associated kinase (ROCK). While prior work suggests collagen gel yielded the endothelial hydraulic conductivity as a function of

BPJ 8636_8639 Monday, February 19, 2018 327a the transendothelial pressure difference and/or flow speed. For basal-to-apical Eukaryotic cells undergo shape changes during their division and growth. pressure differences up to 1 cm H2O, lymphatic ECs displayed a log-linear rela- This involves flow of material both in the cell membrane as well as in the tionship between endothelial hydraulic conductivity and flow speed, which in- cytoskeletal layer beneath the membrane. Such flows result in redistribution dicates that small increases in fluid uptake can lead to large increases in of phospholipid and acto-myosin cortex at the cell surface. This brings our conductivity. Furthermore, this relation appears to be intrinsic to the ECs and focus to the growth of the inter-cellular surface during cell division in C. independent of collagen stiffness. This work thus reveals the constitutive elegans embryo. The growth of this surface leads to the formation of a flow-conductivity relation for lymphatic endothelium, which may help clarify double-layer of separating membranes between the two daughter cells. how the lymphatic system functions and may improve the engineering of arti- The division plane typically has a circular periphery and the growth starts ficial lymphatic-containing tissues. from the periphery as a membrane invagination, which grows radially in- ward like the shutter of a camera. The growth is typically not concentric, 1620-Pos Board B529 in the sense that the closing internal ring is located off-center. Cytoskeletal Reduced Motility of Swimming Algal Cells at Increased Medium Viscosity proteins anillin and septin have been found to be responsible for initiating Kara M. Clark, Victoria Hodge, Gang Xu. and maintaining the asymmetry of ring closure but the role of possible Engineering and Physics, University of Central Oklahoma, Edmond, OK, asymmetry in the material flow into the growing membrane has not yet USA. been studied in detail and need to be investigated further. Motivated by Motile cilia are whip-like subcellular organelles in the human body that beat experimental evidence of such flow asymmetry, here we explore the patterns actively to move fluids or materials in airways, brain ventricles, and the of internal ring closure in the growing membrane in response to asymmetric oviduct. As a result, these cilia grow and bend under a wide range of viscous boundary fluxes. Highlighting the importance of the flow asymmetry we resistance. The goal of this project is to quantify the changes in ciliary show that many of the asymmetric growth patterns observed experimentally motility in response to altered mechanical environment. Using the single- can be reproduced by our model, which incorporates viscous nature of the celled biflagellated green alga Chlamydomonas reinhardtii, we quantified membrane and contractility of the associated cortex. their cellular motility, as the indicator of ciliary motility, at various increased viscosities controlled by different concentrations of methylcellu- 1623-Pos Board B532 lose in the medium. We found that the ciliated cells, grown at either normal Cell Migration through a Confined Micro-Environment: An Attempt to or increased viscosities, exhibit similarly reduced cellular velocities when Understand the Motion of Metastatic Cells swimming at increased medium viscosities. The results suggest conserved Carlotta Ficorella1, Rebeca Martı´nez Va´zquez2, Paul Heine1, ciliary motility despite changing mechanical environment during growth Eugenia Lepera2, Jing Cao3, Roberto Osellame2, Joseph A. K€as1. or motion. 1University of Leipzig, Leipzig, Germany, 2Istituto di Fotonica e Nanotecnologie (IFN) - CNR, Milan, Italy, 3ICMMO, CNRS-UPSud, 1621-Pos Board B530 Universite Paris Sud in Universite Paris Saclay, Paris, France. Influence of Bending of Microvilli on Leukocyte Rolling Adhesion in Shear Traditionally, metastasis were thought to arise during the late stage of Flow - a Simulation Study tumour progression. Studies on human cancer, such as breast carcinoma, Tai-Hsien Wu, Dewei Qi. have shown that cancer cells in a primary tumour exhibit a metastatic Paper and Chemical Engineering, Western Michigan University, Kalamazoo, phenotype, suggesting that metastatic spread may occur in earlier stages MI, USA. of tumorigenesis. Micro-fabricated devices have started to be employed in Receptor-mediated adhesion plays an important role in many physiological recent years to simulate the migration path of cancer cells in vitro and processes and biotechnology-related applications, such as leukocyte recruit- outline the mechanisms of cell motility through confined micro- ment to sites of inflammation and selective capture of target cells environments. Previous work suggests that the nuclear envelop can rupture from blood. Experimental and numerical studies have confirmed that a va- in confinement causing DNA damage and genomic instability, one of the riety of factors, such as cell deformability, association and dissociation hallmarks of cancer. In this study we observe healthy and cancerous cell rates of adhesion bonds, receptor and ligand densities, and flow rate, affect lines (MCF-10A, MDA-MB-231, MDA-MB-231 with stable cytosol ex- the dynamics of cell adhesion. Since most of receptors are located at the pressing GFP) with varying motility, adhesion and mechanical properties, microvillus tips, deformation of microvilli could potentially affect the adhe- migrating in chips equipped with funnel-shaped micro-constrictions. In sion status of cells. Therefore, some simulation work was performed to addition to phase contrast imaging, the cells are also stained with Sir- investigate the influence of microvillus deformation on cell adhesion. How- DNA to track the exact position of the nucleus, and Sir-Actin to enable ever, these studies addressed only the tensile effects of microvilli. This live cell imaging of the cytoskeletal structure. Absence of nuclear jamming study presents an advanced leukocyte model, which consists of a combina- and dissolution of the cytosol are the main requirements for migration tion of five numerical models: Lattice Boltzmann Method (LBM) for through the micro-channels to occur. The cells exhibit different kinds of blood flow; Coarse-Grained Cell Model (CGCM) for leukocytes; Lattice motility when moving on the chips, such as protrusion-based and bleb- Spring Model (LSM) for microvilli; Immersed Boundary Method (IBM) based amoeboid motion. However, they show a repeating migration pattern for the blood-cell interactions; and Adhesive Dynamics (AD) for stochastic when making their way through the micro-constrictions. Gaining further binding between P-selectin and PSGL-1. This innovative model introduces insight into the dynamics behind cancer cell migration through confining the flexural stiffness of microvilli and investigates the influence of bending spaces may help to delineate strategies to restrict the spread of metastatic of microvilli on leukocyte adhesion. Results indicate that the flexural cells from the primary tumour. stiffness of microvilli alter the rolling velocity and, under certain conditions, the adhesion status of leukocytes in shear flow. This means that as the flexural stiffness of microvilli increases, the leukocytes roll faster or Posters: Bacterial Mechanics, Cytoskeleton, and detach from the ligand-coated substrate easier. This finding represents a Motility crucial factor involved in the separation of target cells from whole bloods. This information plays a critical role in the scicentific understanding of 1624-Pos Board B533 inflammation as a disease-causing mechanism within the biophysical Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor landscape of the human body and will allow for increased diagnostic Minyoung Heo1, Ashley L. Nord1, Delphine Chamousset1, Erwin van Rijn2, efficacy. Hubertus J.E. Beaumont2, Francesco Pedaci1. 1Single Molecule Biophysics Dept., Centre de Biochimie Structurale, 1622-Pos Board B531 Montpellier, France, 2Kavli Institute of Nanoscience, Delft University of Asymmetric Flows in the Intercellular Membrane During Cell Division Technology, Delft, Netherlands. Vidya V. Menon1, Sundar R. Naganathan2, Mandar M. Inamdar3, Fluorescent fusion proteins open a direct and unique window onto protein Anirban Sain4. function. However, they also introduce the risk of perturbation of the function 1Center for Research in Nanotechnology and Science, Indian Institute of of the native protein. Successful applications of fluorescent fusions therefore Technology Bombay, Mumbai, India, 2The Francis Crick Institute, London, rely on a careful assessment and minimization of the side effects, but such United Kingdom, 3Civil Engineering Department, Indian Institute of insight is still lacking for many applications. This is particularly relevant in Technology Bombay, Mumbai, India, 4Department of Physics, Indian the study of the internal dynamics of motor proteins, where both the chemical Institute of Technology Bombay, Mumbai, India. and mechanical reaction coordinates can be affected. Fluorescent proteins

BPJ 8636_8639 328a Monday, February 19, 2018 fused to the stator of the Bacterial Flagellar Motor (BFM) have previously with other cellular processes. These interactions serve for flexibility in a been used to unveil the motor subunit dynamics. Here we report the effects changing environment and may possibly be the source of cell-to-cell pheno- on single motors of three fluorescent proteins fused to the stators, all of which typic variability. We exploited the advantage of the chromosomally encoded altered BFM behavior. The torque generated by individual stators was reduced HU-eGFP to monitor nucleoid segregation in live E. coli cells using time- while their stoichiometry remained unaffected. MotB fusions decreased the lapse microscopy. The cells were grown in linear grooves formed in the switching frequency and induced a novel bias-dependent asymmetry in the agarose gel pad, convenient for the image processing of fluorescence intensity speed in the two directions. These effects could be mitigated by inserting a profiles along individual cell lineages. Analysis of these profiles produced linker at the fusion point. These findings provide a quantitative account of nucleoid localization trajectories for each cell lineage and was used to obtain the effects of fluorescent fusions to the stator on BFM dynamics and their alle- the main characteristics of the segregation process, namely, the start of parti- viation- new insights that advance the use of fluorescent fusions to probe the tioning, rates of separation and final positions, as well as variability of these dynamics of protein complexes. characteristics throughout the population. Moreover, the same characteristics were determined for non-dividing, filamentous cells growing under the same 1625-Pos Board B534 conditions. Comparison between dividing and filamentous cells is aimed to Modeling Colony Pattern Formation under Differential Adhesion and Cell reveal the involvement of physical boundaries and diffusion barriers in the Proliferation control of nucleoid segregation dynamics. Our findings shed light on the Jiajia Dong1, Stefan Klumpp2. 1 role of these factors both in the mechanism of segregation and its variability Physics and Astronomy, Bucknell University, Lewisburg, PA, USA, in bacterial cells. 2Institute for Nonlinear Dynamics, University of Goettingen, Goettingen, Germany. 1628-Pos Board B537 Proliferation of individual cells is one of the hallmarks of living systems, Calcium Channel Blockers Effect on Motility : a Novel Target in and collectively the cells within a colony or tissue form highly structured Biomedical Research patterns, influencing the properties at the population level. We develop a Negar Motayagheni. cellular automaton model that characterizes bacterial colony patterns WFIRM, Winston Salem, NC, USA. emerging from the joint effect of cell proliferation and cell-cell differential Introduction adhesion. Through simulations and theoretical analysis akin to interface Each year in the United States many people become infected with bacteria that growth, we show that this model gives rise to novel properties consistent are resistant to antibiotics and many people die as a result of infection. They with recent experimental findings. We observe slower than exponential grow on medical devices such as heart valves, and catheters also take root growth in the case of a single cell type as well as new colony patterns in within wounds and implants and cost the health care system millions per year. the case of two cell types. In particular, engulfment of one cell type by Calcium homeostasis is a key factor for multiple functions including motili- the other is strongly enhanced compared to the prediction from the equilib- ty,aggregation and biofilm formation in bacteria. We report here that swimming rium differential adhesion hypothesis in the absence of proliferation. These motility and biofilm formation in pathogenic Escherichia coli and Proteus mir- observations provide new insights in predicting and characterizing colony abilis is inhibited by verapamil, a calcium channel blocker. The core concep- morphology using experimentally accessible information such as single tion for this study is that Calcium Channel Blockers will prevent bacterial cell growth rate and cell adhesion strength. motility and biofilm formation. 1626-Pos Board B535 Methods Brauns Lipoprotein Facilitates OmpA Interaction with the Escherichia coli We tested the effect of verapamil (3 and 6 mM), on swimming motility and Cell Wall swarming in an in vitro model. Firdaus Samsudin1, Alister Boags1, Thomas J. Piggot1,2, Syma Khalid1. Next In a laboratory model we tested Verapamil effect on p .mirabilis ability to 1School of Chemistry, University of Southampton, Southampton, United biofilm formation on a plastic catheter. Lastly we evaluated the ability of bac- Kingdom, 2CBR Division, Defence Science and Technology Laboratory, terial migration within the catheter using bridge test. Salisbury, United Kingdom. Results Gram-negative bacteria such as Escherichia coli are protected by an Our results revealed that verapamil inhibits swimming motility in E. coli also in extremely complex cellular envelope containing two layers of membranes P. mirabilis. Immersing catheters in Verapamil caused a significant decrease in separated by an aqueous periplasmic space housing a network of cell wall. bacterial migration(44mm vs 0-12mm). More importantly it was able to These membranes are embedded with various proteins that are linked to the decrease biofilm formation on plastic catheter in a dose dependent manner. cell wall either covalently or non-covalently. Little is currently known on Conclusion how these interactions are maintained together in such a crowded environ- Bacterial colonization complicates the care of many patients .We identified for ment. Here we used atomistic molecular dynamic simulations to reveal the de- the first time that verapamil decreases bacterial aggregation and motility and tails of simultaneous binding between the outer membrane porin OmpA and biofilm formation of tested bacterial pathogens in a plastic catheter as well the Braun’s lipoprotein with the cell wall. Braun’s lipoprotein tilts and bends, as bacterial migration in a dose dependent manner. resulting in a shift of the cell wall towards the outer membrane. This enables both OmpA monomer and dimer to interact with the cell wall. In the absence 1629-Pos Board B538 of Braun’s lipoprotein, however, OmpA monomer shows propensity to form Measuring the Effective Temperature of Single Magnetotactic Bacteria as contacts with the outer membrane instead. Dimerisation is required to a Tool to Study Non-Thermal Biological Noise 1 2 3 3 strengthen the electrostatic attraction towards the underlying layer of the Lucas Le Nagard , Solomon Barkley , Xiaohui Zhu , Adam P. Hitchcock , Cecile Fradin1. cell wall to initiate binding. Overall, our study uncovers molecular details 1 of the structure-dynamics-function relationships of the various components Physics & Astronomy, McMaster University, Hamilton, ON, Canada, 2Physics, Harvard John A. Paulson School of Engineering and Applied of the cell envelope, which are necessary for future development of novel 3 therapeutics to fight bacterial infections. Sciences, Cambridge, MA, USA, Chemistry & Chemical Biology, McMaster University, Hamilton, ON, Canada. 1627-Pos Board B536 Magnetotactic bacteria are motile prokaryotes that synthesize magnetosomes, Nucleoid Segregation Dynamics and its Variability in Dividing and which are magnetic single domain crystals surrounded by a lipid membrane. Filamentous E. Coli These organelles confer to the cells a permanent magnetic moment that makes Anteneh H. Abebe1, Itay Gelber2, Alex Aranovich1, Mario Feingold2, them align passively in external magnetic fields and therefore behave as active Itzhak Fishov1. micro-compasses that swim along magnetic field lines. 1Life Sciences, Ben Gurion University of the Negev, Beer-Sheva, Israel, We use single-cell tracking experiments based on optical microscopy 2Physics, Ben Gurion University of the Negev, Beer-Sheva, Israel. to study the magnetic properties of these bacteria. For each cell, we first Segregation of the replicating chromosome from a single to two nucleoid measure the magnetic moment by applying an alternating magnetic field bodies is one of the major processes in growing bacterial cells. It is tightly and studying the angular motion of the bacterium during field reversal. correlated with cell growth and division and this correlation is crucial for We then analyze the statistics of cell alignment with a constant field. Our cell viability. The mechanism of segregation is still enigmatic: although it data show that considering the susceptibility of the magnetosomes is needed is believed that the underlying driving force is purely thermodynamic, it is to obtain reliable results, therefore we first measure it on dead cells. The obvious that the segregation dynamics is tuned by intricate interactions orientation histogram of each live bacterium is then fitted to a Boltzmann

BPJ 8636_8639 Monday, February 19, 2018 329a distribution with temperature as the only fit parameter. A value close rate trajectory taken is dilution dependent. Moreover, the maximum growth to room temperature is obtained for dead cells while a much larger effective rate is also initial OD or dilution dependent. Inoculating at a lower initial den- temperature is needed for live cells, showing that these bacteria are a sity allows the bacterial culture to have a higher maximum growth rate. In our great tool for measuring non-thermal noise originating from biological model, simple nutrient depletion is able to recapitulate these experimental processes. findings qualitatively. In addition to the initial inoculum size affecting the maximum growth rate, other factors such as the variable presence of glycerol 1630-Pos Board B539 can affect the robust measurement of lag. We observed that the presence of Mechanical Perturbations to the Gut Microbiota glycerol in day 1 of growth affects the lag times in day 2 for B. subtilis Carolina Tropini, Justin Sonnenburg, KC Huang, Katharine Ng. 168 dramatically. Traditionally this might not have been a problem in B. sub- Stanford University, Stanford, CA, USA. tilis because most labs streak out cultures and start liquid culture from a col- The consortium of microbes living in and on our bodies is intimately ony, or inoculate into large volumes. However, for an experimental method connected with human biology and deeply influenced by physical forces. that is tractable to working with libraries and high throughput measurements Despite incredible gains in describing this community, and emerging knowl- these solutions aren’t possible. edge of the mechanisms linking it to human health, understanding the basic physical properties and responses of this ecosystem has been compar- 1633-Pos Board B542 atively neglected. Most diseases have significant physical effects on the gut; Role of Bacterial Electrophysiology in Biofilm Development diarrhea alters osmolality, fever and cancer increase temperature, and bowel Bradley Prythero1, R. Andrew Weekley2, Giancarlo N. Bruni1, diseases affect pH. Furthermore, the gut itself is comprised of localized Joel M. Kralj1. niches that differ significantly in their physical environment, and are in- 1Molecular Cellular Developmental Biology, Univ of Colorado Boulder, habited by different commensal microbes. Understanding the impact of Boulder, CO, USA, 2BioFrontiers Institute, Univ of Colorado Boulder, common physical factors is necessary for engineering robust microbiota Boulder, CO, USA. members and communities; however, our knowledge of how they affect Contrary to the view of bacteria as free swimming, solitary individuals, most the gut ecosystem is poor. As a model of a biophysical perturbation, we species of bacteria spend the majority of their time embedded within multi- are investigating how osmotic diarrhea affects the host and the microbial cellular structures called biofilms. Biofilms are formed by communities of community by causing mechanical changes to the cellular environment. Os- bacteria that secrete polysaccharides, proteins, and DNA to build gel-like motic diarrhea is extremely prevalent, caused by the use of laxatives, lactose matrices that form protective microenvironments for the cells. Previous intolerance, or celiac disease. In our studies we monitored osmotic shock to studies of biofilms have focused mostly on broader biofilm morphology or the microbiota using a comprehensive and novel approach, which combined on the molecular composition of the matrix. In addition, due to limitations in vivo experiments to imaging, physical measurements, computational in technology, most traditional studies of bacteria physiology focused on analysis and highly controlled microfluidic experiments. By bridging several global, population-wide dynamics and ignored the heterogeneity of individ- disciplines from biology and physics, we developed a mechanistic under- ual cells. Single-cell imaging of biofilms using optogenetic sensors can fill standing of the processes involved in osmotic diarrhea, linking single-cell this conceptual gap in our understanding of how the physiology of individ- biophysical changes to large-scale community dynamics. Our results ual cells change as they transition into the biofilm phenotype. Presented here indicate that physical perturbations can profoundly and permanently is a new technique to image developing biofilms as they grow from a single change the competitive and ecological landscape of the gut, and affect cell, and a new set of computational tools to track individual cells and their the cell wall of bacteria differentially, depending on their mechanical lineages. Using these tools, we have discovered long-term Ca2þ oscillations characteristics. in the cytosol of Escherichia coli following cell division. These occur with a 1631-Pos Board B540 period of about 40 minutes which correlates closely with the reported repli- Dynamics of Growth, Cell Division, and Phenotypic Switching cation rate of the genomic DNA. Ofescherichia Coliat Elevated Concentration of Magnesium Sulfate Sudip Nepal1, Azarin Yazdani2, Vincent Chevrier2, Pradeep Kumar1. 1634-Pos Board B543 1Physics, University of Arkansas, Fayetteville, AR, USA, 2Space and Investigating the Effect of Antimicrobial Peptides on Biofilm Survival Planetary Science, University of Arkansas, Fayetteville, AR, USA. Thelma Mashaka, Catherine B. Volle. Cells undergo various environmental fluctuations that result in specific Biology, Cottey College, Nevada, MO, USA. cellular responses. We study the growth, morphology, viability, and revers- In the environment, bacteria may be found in two forms. Present in liquid or ibility of Escherichia coli (E. coli) in an elevated concentration of magne- semi-solid materials will be free swimming planktonic bacteria. However, sium sulfate. The population doubling time increases with increasing salt other bacteria adhere to surfaces, forming a biofilm. Because of their complex, concentration. At intermediate salt concentration, 15%, three phenotypic three-dimensional structure and protective extracellular polymeric substance, subpopulations of normal, elongated, and decreased cell length are biofilms represent a significant problem in industries such as food processing, observed. However, at high salt concentrations (20 - 25%), entire population health care, and machine manufacturing. Indeed, because of their structure, bio- switches to smaller cells. The average length of the cells and heterogeneity films are also an ideal environment for development of antibiotic resistance. as measured by the variance of cell length are maximum at 15% salt concen- However, with the need for new antibiotic compound becoming more and tration and decrease on either side monotonically. The viability of the cell more critical, only some studies investigate the effects of antibiotics on bio- decreases with the salt concentration and a complete population collapse films. Preliminary research indicates that planktonic cells of the Gram- is observed at the salt concentration beyond 26%. We further study the negative Escherichia coli (E. coli) respond differently to antibiotics than their reversibility of E. coli in terms of growth rate and morphology when the biofilm counterparts. Thus, we are investigating differences in susceptibility to high salinity stress is removed. The cells are reversible for all the salt con- antibacterial peptides for the Gram-positive Micrococcal luteus planktonic and centrations and the time scale of reversibility increases with increasing salt biofilm cells using minimum inhibitory concentration assays as well as fluores- concentration. cent and atomic force microscopy. 1632-Pos Board B541 Posters: Membrane Pumps, Transporters, and Factors Affecting Bacterial Growth Constants Esha Atolia. Exchangers II Stanford University, Stanford, CA, USA. Bacterial population growth can largely, if not completely, be characterized 1635-Pos Board B544 by three fundamental growth constants: (1) Maximum Growth Rate, (2) Regulation of Mammalian Large Neutral Amino Acid Transporter LAT1 Lag Time, and (3) Final Optical Density (OD). Due to their ability to capture by its Partner CD98 growth dynamics, these fundamental growth constants have traditionally also Qingnan Liang1, Pattama Wiriyasermkul2, Matthias Quick2, Ming Zhou1. been used as measurements of fitness. However, these metrics can be incred- 1Biochemistry, Baylor College of Medicine, Houston, TX, USA, 2Columbia ibly sensitive to the experimental choices in seemingly unintuitive ways. We University, New York City, NY, USA. quantified these fundamental growth constants in different growth conditions The large neutral amino acid transporter 1 (LAT1) mediates uptake of many for B. subtilis and E. coli and extract the maximum growth rate, lag time, and of the essential amino acids. LAT1 is a heterodimer composed of two pro- final OD. Growing E. coli from different dilutions, we observe that the growth teins, SLC7A5 and CD98. SLC7A5 is a polytopic membrane protein with

BPJ 8636_8639 330a Monday, February 19, 2018

12 predicted transmembrane helices, and can transport amino acids even in the 1638-Pos Board B547 absence of CD98. CD98 is a bitopic glycoprotein with one helix spanning the Exploring the Transport Mechanism of the Human AE4 (SLC4A9) - - membrane and a large, glycosylated ectodomain. Uptake of amino acids against CL /HCO3 Exchanger their concentration gradient by LAT1 is coupled to the concurrent export of Marcelo A. Catala´n1, Juan Jose Viveros2, Fernanda Fernandez2, glutamine (antiport), and is driven by the higher intracellular glutamine con- Lisandra Flores2, Sebastian Brauchi2,3, Gaspar Pen˜a-Munzenmayer€ 2,3. centration. LAT1 is overexpressed in cancer cells and inhibition of LAT1 ac- 1Universidad Arturo Prat, Iquique, Chile, 2Universidad Austral de Chile, tivity reduces cancer cell proliferation. In addition, LAT1 is highly expressed Valdivia, Chile, 3Millennium Nucleus of Ion Channels-associated Diseases at the blood brain barrier to facilitate amino acid uptake into the brain, and pro- (MiNICAD), Santiago, Chile. þ - - þ vides a route for delivering drugs into the brain. However, the mechanism of The SLC4 family includes Na -independent Cl /HCO3 exchangers and Na - - antiport and the role of CD98 in LAT1-mediated transport remains unknown. coupled HCO3 transporters. SLC4 proteins are crucial for important physio- We co-expressed SLC7A5 and CD98 and purified the complex. We confirmed logical processes, including CO2 transport, intracellular and extracellular pH - that the two proteins are covalently linked by a disulfide bridge. However, we regulation and HCO3 transport in epithelia. The AE4 (SLC4A9) protein plays also found that the two remain assembled even when the disulfide bridge is dis- a role in NaCl reabsorption and Cl--dependent fluid secretion in distal nephron rupted, indicating that there are other non-covalent interactions between the and salivary glands respectively; however, its transport mechanism remains - - two proteins. We were also able to purify SLC7A5 and CD98 individually. controversial. It has been proposed that AE4 mediates Cl /HCO3 exchange, þ - - - Future work will focus on how CD98 assembles with SLC7A5 and its influence Na -HCO3 cotransport or Cl /cation-HCO3 exchange suggesting that shares - - on LAT1-mediated transport. functional features with both the Cl /HCO3 exchanger AE1(SLC4A1) and the þ - Na -HCO3 co-transporter NBCe1 (SLC4A4). Our sequence alignments show 1636-Pos Board B545 that AE1, NBCe1 and AE4 share high amino-acidic identity in the transmem- Structure-Based Ligand Discovery for the Human Oligopeptide brane domain. Functionally relevant residues in AE1 and NBCe1 are Transporter 1, PEPT1 conserved in AE4, suggesting that the ion transport mechanism should be en- Claire Colas1, Masayuki Masuda2, Kazuaki Sugio2, Seiji Miyauchi2, coded by the same set of residues. We explored the transport mechanism of Yongjun Hu3, David E. Smith3, Avner Schlessinger1. 1 the human version of AE4 using molecular modeling, site-directed mutagen- Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New esis and functional assays. Our molecular simulations show that the proposed York, NY, USA, 2Faculty of Pharmaceutical Sciences, Toho University, 3 anion-binding site in AE1, between the helical ends of TM 3 and TM10, is Funabashi, Japan, Department of Pharmaceutical Sciences, University of also present in AE4. Three residues at this region have been postulated to Michigan, Ann Arbor, MI, USA. be crucial for anion transport in AE1 and NBCe1. We mutated homologous In humans, peptides derived from dietary proteins and peptide-like drugs are D709, T448 and I758 in AE4 and found that only T448 is functionally impor- transported via the proton-dependent oligopeptide transporter hPepT1 tant. Additional mutations of S754 and T756 in this region dont show func- (SLC15A1). hPepT1 is located across the apical membranes of the small intes- tional changes compared to WT AE4. These results suggest that the tine and kidney, where it serves as a high-capacity low-affinity transporter of a predicted ion-binding site in AE4 requires T448 to support the transport cycle broad range of di- and tri-peptides. hPepT1 is also overexpressed in the colon of but, in contrast to AE1 and NBCe1, residues at equivalent positions 709 and Inflammatory Bowel Disease (IBD) patients, where it mediates the transport of 758 are not important for function. harmful peptides of bacterial origin. Therefore, hPepT1 is a drug target for pro- drug substrates interacting with intracellular protein or inhibitors blocking the 1639-Pos Board B548 transport of toxic bacterial products. In this study, we construct multiple struc- How Structural Elements Added in Evolution From Bacterial tural models of hPepT1 representing different conformational states that occur Transporters Serve Human SLC6 Homologs during transport and inhibition. We then identify and characterize five ligands Asghar Razavi, George Khelashvili, Harel Weinstein. of hPepT1 using computational methods such as virtual screening and QM- Weill Cornell Medical College of Cornell University, New York, NY, USA. polarized ligand docking (QPLD), and experimental testing with uptake kinetic Much of the structure-based mechanistic understandings of the function of measurements and electrophysiological assays. Our results improve our under- neurotransmitter transporter family (SLC6A) proteins emerged from the standing of the substrate and inhibitor specificity of hPepT1. Furthermore, the study of their bacterial LeuT-fold homologs. It has become evident, howev- newly discovered ligands exhibit unique chemotypes, providing a framework er, that structural differences such as the long N- and C-termini of the eu- for developing tool compounds with optimal intestinal absorption as well as karyotic neurotransmitter transporters are likely to impart phenotypic future IBD therapeutics against this emerging drug target. properties not shared by the bacterial homologs. These structural "additions" to the common molecular architecture have been shown to be involved in 1637-Pos Board B546 functions, including reverse transport (efflux), regulated by phosphorylation Molecular Dynamics Simulations Reveal Specific Interactions of the Band and by interactions with lipids found only in the eukaryotic cell membranes. 3 Anion Exchanger with Lipids and Glycophorin A To learn how the phenotypes of the eukaryotic transporters are enabled by Antreas C. Kalli1, Reinhart A.F. Reithmeier2. molecular mechanisms involving these structural additions we have used 1Leeds Institute of Cancer & Pathology, University of Leeds, Leeds, United 2 large-scale molecular dynamics simulations of the wild type and mutant Kingdom, Biochemistry, University of Toronto, Toronto, ON, Canada. constructs of the human dopamine transporter (hDAT), and comparative Band 3 (AE1, SLC4A1) is a major glycoprotein found in red blood cells and is Markov State Model analysis. The results reveal a rich spectrum of interac- also found in the kidney. It catalyzes the electro-neutral exchange of bicar- tions of the hDAT N-terminus and distinguish different roles of the distal bonate and chloride across the membrane. Band 3 is a dimer and each subunit and proximal segments of the N-terminus, and interactions with the C-termi- is comprised of a cytosolic domain and a membrane domain that contains 14 nus, in modulating functional phenotypes not shared with the bacterial transmembrane helices. Despite much available structural and functional LeuT-like homologs. data, the dynamic changes that occur within Band 3 during the transport pro- cess and its interactions its lipid environment and other proteins remain 1640-Pos Board B549 elusive. In this study, we have used molecular dynamic simulations to Identification of the slc26a6 and NaDC-1 Transporters Binding Site assemble Band 3 in increasing complex lipid bilayers that resemble the red Ehud Ohana. blood cell plasma membrane with a high concentration of cholesterol in Clinical Biochemistry and Pharmacology, Ben Gurion University of the both leaflets; phosphatidylcholine and sphingomyelin in the outer leaflet; Negev, Beer Sheva, Israel. and phosphatidylethanolamine and phosphatidylserine in the inner leaflet. Succinate is a tricarboxylic acid cycle intermediate that is gaining attention as a Our results suggest that negatively charged phospholipids interact strongly universal metabolic marker of ischemia and a pivotal signaling molecule. with Band 3 forming an anionic annulus around the protein in the inner bilayer Importantly, since succinate is a charged molecule, succinate transport across leaflet. Cholesterol was also found to associate preferentially with Band 3. membranes is mediated by transport proteins. The major succinate transporters The dynamic interaction of Band 3 with anionic lipids and cholesterol may are NaDC-1 and the organic anion transporters (OAT1 and OAT3) expressed regulate its stability and anion exchange activity. Molecular dynamics simu- on the apical and basolateral membranes of epithelia, respectively. We have lations were also used to examine the interaction of Band 3 with glycophorin previously reported that NaDC-1 forms a complex with slc26a6, a Cl-/oxalate - - A (GPA). This interaction creates the Wright blood group antigen and also fa- and Cl /HCO3 exchanger, to regulate succinate and citrate transport through cilitates trafficking of Band 3 to the cell surface. Understanding the molecular interaction. However, the interaction site and mechanism of regulation remain details of the interaction of Band 3 with GPA in a lipid bilayer may provide unknown. Using protein modeling methods we have predicted the protein- new insights into the nature of the Wright blood group antigen and how GPA protein interaction site between NaDC-1 and slc26a6. We generated site muta- can rescue the trafficking of certain Band 3 mutants. tions in the putative binding site residues on both slc26a6 and NaDC-1 and

BPJ 8636_8639 Monday, February 19, 2018 331a monitored decreased interaction between the two transporters. Moreover, We potential cardiotoxicity of compound, in the early phase of drug discovery monitored NaDC-1- mediated succinate transport and found that mutations in campaigns. This approach can be extended to both patient-specific and het- the putative binding site relieve slc26a6 inhibition of NaDC-1. Together, our erologous backgrounds. results suggest that slc26a6/NaDC-1 interaction is mediated via charged resi- dues found on intracellular domains of both NaDC-1 and slc26a6. Mutations 1643-Pos Board B552 in the binding site impair slc26a6 regulation of NaDC-1 mediated succinate Dissecting the Thermodynamics of Transport of a Sodium-Calcium transport. Our findings describe the mechanism which leads to increased uri- Exchanger nary oxalate and reduced urinary citrate which cause Ca2þ-oxalate kidney stone Irina Shlosman1, Fabrizio Marinelli1, Joseph A. Mindell2, formation. Jose D. Faraldo-Go´mez1. 1Theoretical Molecular Biophysics Section, NHLBI/NIH, Bethesda, MD, 1641-Pos Board B550 USA, 2Membrane Transport Biophysics Section, NINDS/NIH, Bethesda, D D Effect of Adenylyl-Imidodiphosphate on K /Ca2 -Exchanger Activity in MD, USA. Human Red Blood Cells Sodium-calcium exchangers (NCX) play a critical role in a variety of physio- Daniel R. Landi Conde1,2, Naileth D. Gonza´lez Sanabria2, logical processes involved in calcium signaling. NCXs are ubiquitous Jesu´s G. Romero2. secondary-active transporters that couple the downhill movement of Naþ to 1Biological Science, Florida State University, Tallahassee, FL, USA, the translocation of Ca2þ in the opposite direction. Mammalian NCXs have 2Instituto de Biologı´a Experimental, Universidad Central de Venezuela, been widely studied functionally; however, structural insight into their trans- Caracas, Bolivarian Republic of Venezuela. port mechanism comes from the crystal structure of the prokaryotic homolog Human red blood cells (RBCs) do not present organelles. These cells are NCX_Mj. This prokaryotic exchanger has been proposed to be a good model 8mm in diameter and present an outstanding deformation capacity, allowing for the NCX family; yet, functional understanding of this protein remains them to enter blood capillaries -with a diameter between 2 and 4mm-, which incomplete. Here, we study purified NCX_Mj reconstituted into liposomes implies being the subject of a great mechanical stress. Since these cells do and demonstrate, under well-controlled experimental conditions, that this ho- not possess a nucleus, they cannot undergo a classical apoptosis process. molog in fact shares key functional features of the NCX family. Transport as- However, RBC lifespan is 12051 days, with a well-regulated disincorpora- says and reversal-potential measurements enable us to delineate the essential tion process. A cyclic process, such as RBC passage through the capillaries, characteristics of this antiporter, and to establish that its ion-exchange stoichi- has been proposed as the mechanism that controls RBC senescence. Our ometry is 3Naþ:1Ca2þ. Together with previous studies, this work reaffirms the þ group has presented evidence for the existence of a mechano-activated K notion that NCX_Mj is a functional representative of the NCX family and thus þ þ channel and a K /Ca2 exchanger, suggesting these proteins as the center a valid model system to investigate the mechanism of ion recognition and mem- þ of RBC senescence process (‘‘hypothesis of K ’’). Here we present the ef- brane transport in sodium-calcium exchangers. fect of adenylyl-imidodiphosphate, a non-hydrolyzable ATP analog, on the Kþ/Ca2þ exchanger. We found that the analog produces an increase in 1644-Pos Board B553 exchanger activity (with a maximum of 174% in the direct mode and How the Substrate Occupancy of a Membrane Transporter Determines 138% in the reverse mode). This increase corresponds to an increase in the Viability of its Alternating-Access Mechanism and thus its Functional permeability (139% in the direct mode and 81% in the reverse mode), and Specificity in the activation process (35% in the direct mode and 58% in the reverse Fabrizio Marinelli, Emel Ficici, Jose Faraldo-Go´mez. mode), making the mechanism more sensitive to membrane potential Theoretical Molecular Biophysics, National Heart, Lung, and Blood Institute, changes. Finally, the analog slows down the deactivation process in the Bethesda, MD, USA. þ þ direct mode, increasing t up to 131% (reverse mode unaffected). These re- Na /Ca2 exchangers (NCX) are important membrane antiporters involved in D þ sults are evidence for the existence of two ATP-binding sites on the Kþ/ Ca2 homeostasis. In typical physiological conditions, their role is to extrude 2þ 2þ þ Ca exchanger: one with high affinity (submicromolar Km) and one with cytosolic Ca in exchange for extracellular Na . Here, we assess the mecha- low affinity (tens of millimolar Km). Furthermore, results indicate that these nisms of ion recognition and exchange of a prokaryotic NCX homolog using binding sites are not catalytic but regulatory, because the effect is observed conformational free-energy landscapes of outward- and inward-facing confor- þ þ independently from ATP hydrolysis. mations of the transporter bound to Na or Ca2 in various occupancies as well as in an apo state. These calculations, based on advanced molecular dynamics 1642-Pos Board B551 simulations, elucidate the factors that control the alternating-access mechanism Transported by Light: Optogenetic Control of NCX1 of this antiporter. The inward-facing conformation, not known experimentally, Riccardo Rizzetto, Viviana Agus, Silvia Cainarca, Lucia Rutigliano, was independently deduced from enhanced sampling molecular simulations Loredana Redaelli, Lia Scarabottolo, Jean-Francois Rolland. and repeat-swap molecular modeling. Our results reveal that only specific AXXAM S.p.A., Bresso, Italy. ion occupancies, namely either 3 Naþ or 1 Ca2þ, permit the transporter to ac- þ 2þ The cardiac Na -Ca exchanger (NCX1) is one of the key modulators of the cess an occluded state that is intermediate between the outward- and inward- 2þ cardiomyocytes’ Ca homeostasis and its reverse-mode has been related to facing state. By contrast, the apo state or other ion occupancies lock the protein several disorders (e.g. heart failure). It therefore represents an important target into either the outward- or inward-facing conformations, thereby stalling the for both cardiac safety and drug discovery. Robust high-throughput screening transport mechanism. Our analysis indicates that the feasibility of the occluded (HTS) assays are required to allow early stage of drug discovery, but the so state, and therefore the alternating-access transition, is determined by the pre- far available assays are poorly specific and involve complex solution exchange cise balance of ion-protein and water-protein interactions, which is strongly protocols. Our aim was to develop a new HTS-compatible strategy to trigger dependent on the occupancy state of the protein. In summary, this study pro- the NCX1 reverse-mode activity by the use of optogenetics in HEK-293 cells vides a molecular-level explanation for the observation that NCX operate and to validate this approach in human induced pluripotent stem (iPS) cells- through a strictly-coupled ‘‘ping-pong’’ mechanism, and for their ion- derived cardiomyocytes assays, in order to have a more physiological human exchange stoichiometry, namely 3Naþ:1Ca2þ. More broadly, our analysis sug- background. gests that the functional specificity of membrane transporters featuring an As a proof of concept, we generated a pure HEK-293 clone stably express- alternating-access mechanism, i.e. whether they function as symporters, anti- ing both Channelrhodopsin (ChR2) and NCX1, and we confirmed their func- porters or uniporters, and their precise stoichiometry, is in large part explained tionality with manual patch clamp in voltage-clamp mode and standard by the feasibility of the conformational transitions that result in occlusion of the TETRA 2þ fluorescence protocols at FLIPR , measuring intracellular Ca substrate-binding sites to both sides of the membrane. changes or membrane depolarization. Our rationale is that following the ChR2 light stimulation we promote conditions that favour the reverse- 1645-Pos Board B554 mode function of NCX1, which can be evaluated by monitoring intracellular EAAT3 Investigated using SSM-Based HTS Electrophysiology on the Ca2þ. The same ‘‘Opto-NCX1’’ protocol was used in iPS-derived cardio- SURFE2R 96SE myocytes co-cultured with ChR2-expressing HEK-293 cells. The contribu- Maria Barthmes, Andre Bazzone, Stephan Holzhauser, Michale George, tion of NCX1 to the global Ca2þ cycling of the resulting syncytia was Niels Fertig, Andrea Bruggemann.€ evaluated by pharmacological tools at FLIPRTETRA. Our results indicate Nanion Technologies, Munich, Germany. that ChR2 activation by blue light is successful in triggering NCX1 The excitatory amino acid transporter 3 (EAAT3) is involved in the reverse-mode function. This is confirmed by the effect of specific blockers, neuronal re-uptake of glutamate and plays a central role in the regulation such as KBR-7943. Thus, we have developed a new optical tool suitable of excitatory neurotransmission and synaptic plasticity. EAAT3 also trans- to run HTS, looking for novel molecules acting on NCX1, and to evaluate ports cysteine, necessary for the synthesis of glutathione and GABA.

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EAAT3 is expressed not only throughout the brain, but in many organs such occluded transition. A recently-reported structure of the outward-open as intestines, liver and heart. Here it seems to provide the main pathway of state of human serotonin transporter (hSERT), provides a starting point aspartate. Several connections of EAAT3 to severe neuronal disorders like for molecular dynamics (MD) simulations of this transition. Initial MD epilepsy and schizophrenia have been described, as well as to metabolic dis- simulations revealed stability and convergence of this conformation over turbances concerning in the maintenance of aspartate and cysteine levels. time. However, the endpoint of this pathway closure event is not yet This makes EAAT 3 not only an important target for functional research, known. Therefore, we present a carefully-refined homology model of but also a potential drug target. hSERT based on a combination of two templates (hSERT, outward-open Here, we present data on EAAC1, a mouse homologue of EAAT3, using a and the bacterial leucine transporter LeuT, outward-occluded) in an novel high throughput instrument for SSM (solid supported membrane)- outward-occluded state. This homology model will be used as the endpoint based electrophysiology, the SURFE2R 96SE. SSM-based electrophysiology of enhanced sampling simulations of the transition between the outward- is a label-free electrical measuring method with very high sensitivity which open and the outward-occluded state of hSERT. Combined with knowledge enables the resolution of low turnover transport and even binding-events. about substrates and inhibitors sharing the same scaffold interacting with Using the purified membrane of EAAC1 expressing CHO cells, we were this transporter, our approaches will lead to a better understanding of the able to determine substrate affinities and their interaction and to compare transporter mechanism. the effect of six known inhibitors directly with each other. We evaluated the assay stability and success rate. Furthermore, we were able to resolve 1648-Pos Board B557 substrate binding and to confirm the described anion conductance of the Translocation of Potassium in Initiating Reset of Monoamine Transporter transporter. Proteins from Microsecond Molecular Dynamics Simulations Using the SURFE2R 96SE with CHO cells we were able to generate an effi- Emily M. Benner, Jeffrey D. Evanseck. cient, robust and very flexible assay, which is an ideal tool for the biophysical Duquesne University, Pittsburgh, PA, USA. and pharmacological characterization of EAAC1 and even suitable for drug Our intent is to characterize the complete transport mechanism of the serotonin screening approaches. transporter (SERT) computationally, employing both a single bilayer and dual bilayer system to investigate this phenomenon. The work presented here represents the data collected from our most recent dual bilayer system from 1646-Pos Board B555 m The Split Personality of Glutamate Transporters: a Channel and a 178,044 atom simulations after 1 s of simulation time. Herein, we utilize the Transporter human SERT protein crystal structure (PDB 5I6X), embedded in POPE lipid membrane. Ions were added to either side of the membrane at physiological Renae M. Ryan. University of Sydney, Sydney, Australia. concentrations, maintaining the membrane potential at 67 mV, representative Glutamate is the predominant excitatory neurotransmitter in the mamma- of resting potential. Potential was determined using the Goldman-Hodgkin- Katz equation. Systems were run on both XSEDE and Pittsburgh Supercomput- lian central nervous system and activates a wide range of receptors to m mediate a complex array of functions. To maintain efficient synaptic ing Center resources. After 1 s molecular dynamics simulations using the signaling and avoid neurotoxicity, extracellular glutamate concentrations CHARMM force field, we observe translocation of potassium to the central are tightly regulated by a family of glutamate transporters termed Excit- substrate binding site in both SERT and the dopamine transporter (DAT). In atory Amino Acid Transporter (EAATs). Altered glutamate transmission, both cases, potassium is interacting with a conserved aspartic acid residue in the pocket (D98 in SERT and D79 in DAT). The translocation of potassium and specifically disrupted EAAT function, has been implicated in a range m of disease states including; Alzheimer’s disease, episodic ataxia, epilepsy to the central binding pocket after 1 s supports experimental data that suggests and stroke. potassium is necessary for reset of SERT. Simulations are currently running to In addition to clearing glutamate from the extracellular space, EAATs can also characterize the complete transport mechanism. A more complete understand- function as chloride (Cl-) channels, which contributes to ionic/osmotic balance ing of the transport process can aid in drug discovery and design for treatments and can affect cell excitability. The dual transporter/channel functions are of disorders associated with these proteins, such as depression and related mediated by distinct conformational states of the transporter and we have map- disorders. - ped the Cl permeation pathway to the interface of the transport and scaffold 1649-Pos Board B558 domain of the glutamate transporters. The EAATs use a unique mode of trans- - Molecular Characterization of the CTR Copper Transporter port termed the ‘twisting elevator’ mechanism and we hypothesize that the Cl Kehan Chen1, Gang Wu2, Ah-Lim Tsai2, Ming Zhou1. channel is activated during the elevator movement. Our aim is to develop a 1Biochemistry, Baylor College of Medicine, Houston, TX, USA, model for the dual functions of the glutamate transporters through structural 2Hematology, The University of Texas Health Science Center at Houston, and functional analysis of human (EAAT1) and prokaryotic (GltPh) trans- Houston, TX, USA. porters. We have created a range of double cysteine mutants in cysteine-less Copper (Cu) is an essential trace element for cells but it is also highly toxic. EAAT1 and GltPh to explore the movement of the transport domain during sub- Therefore, Cu uptake, cellular distribution, and efflux are tightly regulated. strate translocation and to elucidate the conformational state/s that support an - CTR (copper transporter) protein is one of the major players in cellular Cu open Cl channel. uptake. It is a membrane protein selectively transporting Cu ions into the cytosol. CTR has no homology to other known transition metal ion trans- 1647-Pos Board B556 porters, and our understanding of its transport mechanism is still limited. Selectivity Profiling of the Human Monoamine Transporters: A current CTR model at 7 A˚ resolution shows that it forms a homotrimer Investigation of the Serotonin Transporter Mechanism with a central pore. However, the resolution is not sufficient to reveal details Eva Hellsberg1, Lucy R. Forrest2, Anna Stary-Weinzinger3, on ion binding sites and the mechanism of ion translocation. Previous Gerhard F. Ecker1. studies have also suggested that CTR favors Cuþ over Cu2þ, however, 1 Pharmaceutical Chemistry, University of Vienna, Vienna, Austria, Cuþ is sensitive to oxygen and its transport has not been studied in any 2 Computational Structural Biology Unit, National Institute of Neurological detail. We developed assays to measure binding and transport of Cuþ and Disorders and Stroke (NINDS), National Institutes of Health (NIH), other transitional metal ions in CTR, and will report the results from these 3 Bethesda, MD, USA, Pharmacology and Toxicology, University of Vienna, studies. Vienna, Austria. The human monoamine transporters play an important role in the central 1650-Pos Board B559 and peripheral nervous system by regulating signals among neurons. Coupling Spectroscopic Data for a Secondary Transporter with Numerous compound classes have been identified to interact with these Simulations to Assess the Role of a Key Acidic Residue proteins, either used in a therapeutic setting or abused as illicit drugs. Vanessa Leone1, Izabela Waclawska2, Burkhard Endeward3, However, based on the physicochemical properties of these ligands alone, Thomas Prisner3, Christine Ziegler2,4, Lucy R. Forrest1. the mechanistic distinction between transporter substrates and inhibitors is 1National Institute of Neurological Disorders and Stroke, Bethesda, MD, not always clear. For example, it remains to be established whether the USA, 2The Max Planck Institute of Biophysics, Frankfurt am Main, closure of the extracellular pathway facilitated by substrate can also occur Germany, 3Goethe University, Frankfurt am Main, Germany, 4Regensburg when such inhibitors are bound. It therefore seems necessary to pinpoint University, Regensburg, Germany. the molecular features of the substrate required for the transport-related The glycine betaine symporter BetP regulates the osmotic pressure conformational change, and in particular, the outward-open to outward- in Corynebacterium glutamicum, a soil bacterium used extensively in

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biotechnology. Our collaborators have resolved X-ray structures of BetP in ing a double-Trp mutant (LacYww) (2), we describe an almost occluded, several conformations, including outward-open, inward-open, and closed outward-open conformation with bound sugars [b-D-galactopyranosyl-1- states, both apo and holo, creating a unique data set for a secondary trans- thio-b-D-galactopyranoside, TDG (3) or p-nitrophenyl-a-D-galactopyrano- porter. Although remarkable, these structural insights do not establish key side, NPG (4)] and a complex of LacYww with a camelid nanobody microscopic properties of the transporter during the transport cycle, for (LacYww/Nb9047/NPG) (Unpublished data), which confirms many example the protonation state of ionizable residues. In BetP, the residue biochemical and biophysical findings. Asp470 is of particular interest, due to proximity to one of the driving ion Structural data from the LacYww mutants clearly show how the side chains binding sites, and a location along the cytoplasmic pathway that implies Glu269 (helix VIII) primarily, and Glu126 (helix IV), Trp151 (helix V), changes in protonation state may affect ion binding or cavity hydration, Arg144 (helix V), His322 (helix X), and Asn272 (helix VIII) provide speci- and thereby pathway opening. Molecular simulations could be instrumental ficity, while Phe20, Met23, and Phe27 (helix I) increase binding affinity for in addressing the true protonation state(s). However, a classical simulation NPG nonspecifically. strategy involving validation against the experimental structure is rendered Analysis of the three structures clearly suggests that Glu325 is linked to the immaterial by the low pH (5.5) of the crystallization conditions, under galactoside-binding site via a hydrogen bond with His322Nd. The His322NεH which the aspartate is unquestionably protonated. Therefore, to identify is hydrogen donor to 3-OH of the galactopyranoside, because if Glu269 is nega- the contribution of Asp470 at physiological pH (7.5), we have utilized tively charged, the 3-OH in turn acts as hydrogen donor to Glu269. If the site Double Electron-Electron Resonance (DEER) data for BetP proteolipo- returns to the occluded state without bound substrate, it must lose an Hþ, somes carried out by our collaborators at different ion and substrate concen- implying a drop in the effective pKa. It does so hypothetically through interac- trations. Since it is not trivial to integrate the spectroscopic data with tion of protonated Glu325 with Arg302 and water molecules and loses the Hþ the structural data, we coupled molecular simulations of BetP structures to the side that is closing. Although Glu325 and Arg302 are essential for pro- with DEER measurements. The approach employed here, named EBMetaD tonation/deprotonation and His322 is essential for binding, all three may be (Marinelli & Faraldo-Go´mez, BJ 2015) enforces sampling of the molecular required for the pKa change in Glu325 upon closing to the occluded, system only within the DEER distance distribution, applying the minimum substrate-free conformation. bias required to reproduce the distribution. Our computational studies allow us to identify the most probable ionization state of Asp470 1653-Pos Board B562 for different BetP conformations with different ion and betaine occupancies, Crystal Structure of an EIIC Trapped in an Inward-Facing Conformation and provide important insights into the sodium-coupled transport Zhenning Ren1, Jumin Lee2, Zhichun Xu1, Yin Nian3, Liya Hu1, mechanism. Jason McCoy4, Allan Ferreon1, Wonpil Im2, Ming Zhou1. 1Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, 1651-Pos Board B560 TX, USA, 2Biological Sciences and Bioengineering, Lehigh University, A Highly Conserved NAD Binding Site in Prokaryotic Multi-Drug Mate Bethlehem, PA, USA, 3Institute of Zoology. CAS, Kunming, China, 4Broad Transporters Institute, Cambridge, MA, USA. Emel Ficici, Wenchang Zhou, Jose D. Faraldo-Go´mez. Enzyme IIC (EIIC) is a membrane embedded sugar transporter and is part NIH/NHLBI, Bethesda, MD, USA. of the phosphoenolpyruvate-dependent phosphotransferase system. Crystal Multi-drug and Toxic-compound Extrusion (MATE) proteins are a recently structures of two EIICs, bcChbC and bcMalT, have been reported. recognized and largely uncharacterized family of secondary-active trans- Both transporters have a substrate binding domain, and the substrate binding porters. Present in both eukaryotes and prokaryotes, MATE transporters pro- domain assumes an inward facing conformation in bcChbC and an outward- tect the cell by catalyzing the efflux of a broad range of cytotoxic facing conformation in bcMalT. This has led to the hypothesis that substrate compounds, including man-made antibiotics and anti-cancer drugs. These translocation can be achieved by rigid-body motions of the substrate binding transporters are therefore potential pharmacological targets against drug- domain. However, to understand the transport mechanism, it is necessary to resistant pathogenic bacteria and tumor cells. The efflux activity of obtain different conformations of the same transporter. Here we present a MATE transporters is powered by a transmembrane electrochemical ion crystal structure of bcMalT whose substrate binding domain is trapped in gradient, but their molecular mechanism and ion-specificity are not well un- an inward-facing conformation by a mercury ion that bridges two strategi- derstood, in part due to the scarcity of high-quality structural information. cally placed cysteine residues. To find out if the crosslinked structure is dis- Here, we use computational methods to study PfMATE, from the archaeon torted significantly from its native state, we did the follow two experiments. Pyrococcus furiosus, whose structure is the best resolved among all avail- First, all-atom molecular dynamics simulations were performed for the in- able. Seemingly at odds with the notion that this transporter is solely ward facing conformation with or without the cysteine crosslink, and both coupledtoHþ, our analysis of the original crystallographic data and addi- structures remain stable after 1 microsecond. Second, single-molecule Fos- tional molecular dynamics simulations unequivocally point to the existence ter resonance energy transfer (smFRET) showed that large distance changes of Naþ-binding site in the N-lobe of this transporter. Moreover, we find this predicted by the crosslinked structure indeed occur on bcMalT without the site to be broadly conserved among prokaryotic MATEs, including members crosslinking. The structure of the inward facing bcMalT therefore supports ofthefamilyknowntobecoupledtoNaþ gradients, such as NorM and the hypothesis that the substrate binding domain moves as a rigid body to ClbM, for which a Naþ-binding site had not been conclusively identified. shuttle the substrate across the membrane, and provides details of the We further posit this site in the N-lobe may mediate Hþ coupling instead conformational changes. at sufficiently low Naþ concentrations, as has been noted for other Naþ- dependent transport systems. In summary, our study provides new insights 1654-Pos Board B563 into the structural basis for the complex ion dependency of MATE Molecular Mechanism of Sugar Transport by a SWEET Transporter transporters. Liang Feng. Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA. 1652-Pos Board B561 SWEET sugar transporters play critical roles in many physiological Lactose Permease: Mechanism through Structures processes in plants and animals. They also serve as excellent models for Hemant Kumar1, H. Ronald Kaback2, Robert M. Stroud1. understanding the alternating access transport mechanism. We captured 1Biochemistry and Biophysics, University of California San Francisco, San the high-resolution structures of a SWEET family of sugar transporter in Francisco, CA, USA, 2Departments of Physiology and Microbiology, three major conformational states, including outward-open, occluded Immunology & Molecular Genetics, Molecular Biology Inst, University of and inward-open conformations by protein engineering and crystallography. California Los Angeles, Los Angeles, CA, USA. Our structure of a glucose bound form provided insights into substrate The lactose permease of Escherichiacoli(LacY),amemberofthemajor recognition. Furthermore, through unbiased molecular dynamics simula- facilitator superfamily (MFS), catalyzes the symport of a galactopyrano- tions, we observed spontaneous transitions from an outward-open to sideandanHþ across the membrane by a mechanism in which the an inward-open state, as well as the accompanying substrate translocation sugar-binding site in the middle of the protein becomes alternately acces- events across the membrane. The unguided computational simu- sible to either side of the membrane through multiple conformational lations matched well with the experimental structures. These data, along changes. However, initial X-ray structures showed LacY in an inward- with functional studies and dynamic characterizations, suggest mechanisms facing conformation with a tightly sealed periplasmic side (1). Here utiliz- to explain allosteric coupling of gates and transitions between

BPJ 8636_8639 334a Monday, February 19, 2018 conformational states. These studies lead to atomic level insights into how implications both for homeostatic mitochondrial signaling determining sugar is translocated across the membrane through an alternating access health maintenance, and for pathological transformation, including transporter. abnormal cardiac rhythms.

1655-Pos Board B564 1657-Pos Board B566 Beyond the Structure: Deciphering the Molecular Mechanisms of Enzymatic Dynamics in Brief Maximal Exercise: a Skeletal Muscle Secondary Transport with Hydrogen-Deuterium Exchange Mass Glycogenolysis Metabolic Model Spectrometry Kevin M. Christmas, James B. Bassingthwaighte. Chloe P.V.J. Martens. Bioengineering, University of Washington, Seattle, WA, USA. Chemistry, King’s College London, London, United Kingdom. We extend a steady-state muscle glycogenolysis/glycolysis model (Vinna- The recent and spectacular progress of structural biology techniques is now kota Biophys J 91: 1264, 2006) that will simulate the metabolic dynamics providing a continuous stream of membrane proteins structures. However, and water and solute fluxes of maximal exercise over 1 minute leading to linking a protein’s static snapshots to its cellular function is a difficult 15% hemoconcentration. Using glucose/glycogen sources requires both task that requires a comprehensive understanding of complex molecular hexokinase and glycogen cycle enzymes. With glycolytic enzyme abun- mechanisms. Two key aspects underlying the molecular mechanism of dances from Maughan (2005) plus on- and off-kinetics for enzyme- membrane proteins are still poorly understood: their intrinsic conforma- substrate binding defines the systems capacitances, time constants, and tional dynamics and the role of their interactions with the lipid bilayer. fluxes, the model simulated exercise onset with 250-fold increased metabolic We address these challenges by studying the molecular mechanisms of three rate. Incorporating this model within a simplified recirculating whole body transporters from the Major Facilitator Superfamily (MFS); XylE, LacY and model composed of vasculature, interstitium and cells of other organs, the GlpT, which share a similar fold but perform different functions. In order to exercise decreased pH while increasing lactate and osmotic pressure in the identify the molecular details of specific functionality, we carried out con- active muscle cells, causing osmotic fluid shifts in all organs.Workingmus- servative mutations in conserved motifs, and subsequently monitored the cle volume can increase 10%, and blood concentrate by 20% as muscle changes in conformational dynamics using Hydrogen-Deuterium Exchange cells accumulate up to 2 liters in 1 minute in a 70 kg athlete. The energy mass spectrometry (HDX-MS). Our results show that despite differences source is primarily glycogenolysis during exercise. The lactate dehydroge- in function and energetics, there is a remarkable conservation in the confor- nase and GAPDH reactions maintain NADþ/NADH. ATP and PCr levels mational role of conserved motifs. The perturbation of conserved salt- are maintained by cytosolic glycolysis, while mitochondrial respiration is bridges on the cytoplasmic side systematically triggers opening of the extra- compromised by cellular hypoxia and reducing equivalents. This kinetically cellular side, indicating a conserved mechanism of alternating-access within detailed model, muscle þ body, characterizes the observed dramatic dy- the structural fold of MFS. Striking differences appear however when namics of maximal exercise. observing the conformational effect of transmembrane charged residues: while mutations cause the symporters LacY and XylE to close on the extra- cellular side, the antiporter GlpT presents an HDX pattern indicative of an 1658-Pos Board B567 extracellular opening. We are now building on these results to optimize the PM2.5 Exposure and ROS Production in NR8383 Rat Alveolar applicability of the HDX-MS workflow to more heterogenous membrane Macrophages systems. We reconstitute the transporters in nanodiscs of specific lipid com- Anthony Waterston, Joel Castillo, Micah Olivas, Alam Hasson, positions and use regions previously identified as conformational reporters Laurent Dejean. to understand the molecular role of lipid-proteins interactions. We surmise CSU, Fresno, Fresno, CA, USA. that the innovative methodology presented here is applicable to a variety Previous studies have established a link between adverse health effects and of membrane proteins. exposure to particulate matter that is less than 2.5 micrometers in diameter (PM2.5). The size of PM2.5 allows them to reach the distal ends of the res- piratory tract, where they induce an inflammatory response mediated by a Posters: Cellular Signaling and Metabolic high production of reactive oxygen species (ROS) in alveolar macrophages Networks (AM). Redox-active components of urban PM2.5, such as copper and qui- nones, are key suspects in PM2.5 toxicity, because these chemicals can generate ROS on the particles and inside a cellular context. However, the 1656-Pos Board B565 relative contribution of the above components to the AM global ROS Mitochondrial Chaos: Redox-Energetic Behavior at the Edge response remains to be determined. Our group has collected a number of Jackelyn M. Kembro1, Sonia Cortassa2, Steven J. Sollott2, Miguel A. Aon2. 1 ambient PM samples during the winter months of 2013 and summer Facultad de Ciencias Exactas, Fisicas y Naturales, Universidad Nacional de 2.5 2 months of 2012 in Fresno and Claremont. Samples were tested for Cordoba, Cordoba, Argentina, NIH/NIA/IRP, Baltimore, MD, USA. their potential to induce a ROS response in the rat NR8383 AM cell line Mitochondria serve a multiplicity of key cellular functions, including energy using the intracellular ROS-sensitive fluorescent probe 2’-7’-Dichlorodihy- generation, regulation of redox balance, thus making a major impact on drofluorescein diacetate (DCFH-DA). We first showed that the amount healthy and diseased states. Collectively, mitochondrial dynamics contribute þ of PM collected in these samples is sufficient to induce significant directly or indirectly to regulate the level of key metabolites (e.g., NAD , 2.5 ROS responses in NR8383 AM. Interestingly, the specific cellular ROS acetyl CoA, ATP, S-adenosyl methionine) at the cross-roads of response was depending on the sample origin; strongly suggesting that epigenome-genome-metabolic functional interactions arising from nutri- the chemical composition of PM was a major determinant of their effect tional status and lifestyle decisions. It is increasingly recognized that biolog- 2.5 on AM ROS metabolism. These same PM samples were also character- ical network stability/instability can play critical roles in determining health 2.5 ized for their chemical content using chromatography and mass spectrom- and disease. Herein, we investigate and characterize the conditions leading etry based techniques. Two types of quinones (1,2-Naphtaquinone, and from stability to mitochondrial chaotic dynamics. Using an experimentally þ 1,4-Naphtaquinone) and one transition metal (Cu2 ) were associated to validated bi-compartmental computational model of mitochondrial function, samples with high capacity to induce ROS production in NR8383 AM we show that complex oscillatory dynamics in key metabolic variables, cells. We are currently estimating the potential of each of these above arising at the ‘‘edge’’ between fully functional and pathological behavior, components to induce a cell ROS response separately using mock samples sets the stage for chaotic dynamics. Under those conditions, a mild, regular containing concentration ranges similar to that found in different PM sinusoidal redox perturbation, such as occurs naturally during each heart- 2.5 samples. beat, triggers chaotic dynamics with main signature traits such as sensitivity to initial conditions, positive dominant Lyapunov exponents, and strange at- tractors. The transition to mitochondrial chaos is exquisitely sensitive to the 1659-Pos Board B568 matrix SOD2 antioxidant capacity as well as to the amplitude and frequency Bisphosphonates Regulate Osteoblasts/Osteoclasts Proliferation Inducing of the extra-mitochondrial redox perturbation, shifting between low- (limit Bone Mineralization cycles without or with period doubling) and high-dimensional dynamic reg- Rosa Scala, Mariacristina Angelelli, Fatima Maqoud, Antonio Scilimati, imens (complex oscillations, chaos). Overall, this work describes the condi- Domenico Tricarico. tions leading to mitochondrial chaotic dynamics and their potential University of Bari, Bari, Italy.

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The most important class of anti-resorptive drugs used to treat bone dis- We carried out microsecond molecular dynamics simulations of the eases is represented by the bisphosphonates(BPs) however their actions open and closed CaM structural states with Met to Gln substitutions at mediating the therapeutic effects are poorly understood. In this study, M109 or M124 to gain an atomic-level understanding of (1) the role of we examined the in vitro and ex vivo effects of Zoledronic acid(ZOL), Ri- methionines in stabilizing CaM structure, and (2) how Met oxidation sedronate(RIS) and Alendronate(ALE) at increasing concentrations(50 might influence conformational equilibria and regulatory interactions. nM-100 mM) by using dehydrogenases activity CCK8 assay, cell viability We found that every CaM C-terminal Met participates in a Met- Crystal-Violet (CV) assay, and mineralization assay in tumor human cell aromatic interaction with nearby Phe residues with interaction energies lines PC3 (prostate cancer) and MG63 (osteosarcoma); pre-osteoblasts of 4-10 kcal/mol. M109 stably interacts with F92, but only in the murine cells MC3T3, pre-osteoclasts cells J774A.1 and RAW267.4 cells. open structure, and M124 stably interacts with F141, but only in the closed The rank order of efficacy as anti-proliferative compounds evaluated by structure. Met to Gln substitution at M109 or M124 alters interaction en- CV assay in PC3 cells was: ZOL (28.41%56.04) R ALE ergies with these nearby Phe and alters the stability of the open and closed (24.67%58.7) R RIS (21.31%56.78). In MG63 cells the rank order structures. We expect that these results will be applicable to understanding of efficacy as anti-proliferative compounds evaluated by CV assay was: the general mechanism through which Met oxidation orchestrates redox RIS (39.31%51,51) = ZOL (39.03%51.68) R ALE (35.20%5 cell signaling. 3.21). The rank order of efficacy as anti-proliferative compounds evalu- ated by CV assay in J774A.1 cells was: ZOL (53.18%52.45) > RIS 1662-Pos Board B571 (37.96%53.47) R ALE (33.71%53.72). In RAW 264.7 cells the Spatial Characterization of NADH Concentration and Diffusion in Cells rank order of efficacy as anti-proliferative compounds evaluated by CV and Tissue assay was: ZOL (55.04%52.98) R RIS (51.82%51.50) R ALE Rachel Cinco, Per Niklas Hedde, Michelle A. Digman, Enrico Gratton. (30.8%52.3). Furthermore, we observed mineralization at lower nano- Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. molar concentrations with ZOL in differentiated MC3T3 cells. Also in Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH play a bone marrow cells derived from tibia and femur of wild-type mice co- central role in bioenergetics as an enzymatic cofactor and reducing equivalent cultured with PC3 we observed a mineralization with ZOL. In conclusion, in cellular metabolic and redox reactions. The NADH/NADþ ratio has signif- in our experiments ZOL was the most effective compound within the BPs icant impact on energy production, cell survival, proliferation, longevity and under investigation showing anti-proliferative effects on osteoclasts at aging. Lifetime measurements of free/bound NADH autofluorescence has micromolar concentrations and inducing mineralization in cultured osteo- been demonstrated to approximate the NADH/NADþ ratio and has been estab- blasts stimulating mineralized bone nodule formation in vitro at nanomo- lished as a reliable means for estimating the mode of metabolism used by cells lar concentrations. and tissues. NADH microenvironment concentrations, diffusion speed, and po- tential sequestration areas may dictate NADH availability for biochemical re- 1660-Pos Board B569 actions and thereby modulate cell metabolism and signaling. To approximate A Novel Bacterial Cell to Cell Communication Mechanism the spatial distribution and diffusion of NADH in cells, we excited NADH at Arthur Prindle1, Jintao Liu2, Munehiro Asally3, Jordi Garcia-Ojalvo4, 740 nm 2-Photon excitation and performed raster image correlation spectros- Gurol Suel2. copy (RICS) and number and brightness (N&B). Application of RICS to deter- 1Northwestern University, Chicago, IL, USA, 2University of California, San mine NADH diffusion did not yield enough photons for a correlation to be Diego, La Jolla, CA, USA, 3University of Warwick, Coventry, United detected, since the NADH quantum yield is too low and the NADH concentra- Kingdom, 4Universitat Pompeu Fabra, Barcelona, Spain. tion too high in the cell to apply the RICS method. Application of N&B analysis Bacterial biofilms are organized communities containing billions of on NADH demonstrates a wide range of NADH levels distributed throughout densely packed cells. Such communities can exhibit fascinating macro- the cell, where the cytoplasm exhibits a larger range of NADH levels in com- scopic spatial coordination. However, it remains unclear how microscopic parison to the nucleus. This result is in accordance with previous observations bacteria could communicate effectively over such large distances. To demonstrating a large range of concentrations in the cytoplasm when investigate this question, we studied a Bacillus subtilis microbial commu- measuring the absolute concentration using phasor FLIM1. Taken together, nity that was recently reported to undergo metabolic oscillations triggered these observations support cellular NADH is heterogeneously distributed and by nutrient limitation. Using novel microfluidic techniques and fluorescent highlight the possibility NADH may be sequestered in nano-compartments reporter dyes, we discovered that these metabolic oscillations appear to be containing a variety NADH concentrations in cells. Supported by NIH coordinated by a previously unknown form of long-range electrical P50-GM076516 to R.C/P.N.H and P50GM076516 to E.G./M.A.D. 1. Ma, N., communication in biofilms. As predicted by a simple mathematical model, Digman, M. A., Malacrida, L. & Gratton, E. Biomed. Opt. Express, BOE 7, we further show that spatial signal propagation can be hindered by specific 2441-2452 (2016). genetic perturbations to a key ion channel. These findings reveal the spatial and temporal self-organization of metabolic states among cells 1663-Pos Board B572 via a novel bacterial cell to cell communication mechanism. These find- Diffusion as a Ruler: Modeling Kinesin Diffusion as a Lenth Sensor for ings further reveal an unexpected functional connection between microbi- Intraflagellar Transport Nathan L. Hendel1, Matt Thomson2, Wallace F. Marshall1. ology and neurobiology and thereby suggest new strategies for biofilm 1 control. University of California, San Francisco, San Francisco, CA, USA, 2California Institute of Technology, Pasadena, CA, USA. 1661-Pos Board B570 An important question in cell biology is how cells know how big to make their The Role of Calmodulin Methionine Oxidation in Regulating organelles. The eukaryotic flagellum is an ideal model for studying size con- Conformational Change trol because its linear geometry makes it essentially one-dimensional, greatly Daniel Walgenbach1, Jennifer C. Klein2, Andrew Gregory1. simplifying mathematical modeling. The assembly of flagella is regulated by 1University of Wisconsin, La Crosse, WI, USA, 2Biology, University of intraflagellar transport (IFT), in which trains of kinesin motors walk to the tip Wisconsin, La Crosse, WI, USA. of the flagellum and deposit the cargo necessary for the flagellum to grow. We have used molecular dynamics simulations to understand the role of The competing length control factor is a length-independent decay of the fla- calmodulin (CaM) methionine (Met) oxidation in disrupting calcium- gellum. In Chlamydomonas reinhardtii flagella, this process results in initial induced conformational changes associated with CaM’s regulation of its rapid growth followed by convergence to a steady-state length. Curiously, target proteins. Previous work suggests that C-terminal CaM methionines, the rate at which motors are recruited to begin transport is indirectly propor- particularly M109 and M124, are critical for functional interaction with tional to the length, implying some kind of communication between the base the major calcium release channel in muscle, the ryanodine receptor and the tip. We propose a model in which motors unbind after cargo delivery (RyR). Methionine to glutamine (Gln) substitutions at M109 or M124 de- and diffuse back to the base, and are reused in IFT. In this model, the diffu- signed to mimic oxidation of Met to Met sulfoxide block CaM activation sion time of the motors serves as a proxy for length measurement. To explore of RyR at low calcium levels and inhibition of RyR at high calcium levels the viability of this diffusion-based length control, we computationally built (Journal of Biological Chemistry 278(18) 2003). Spectroscopic distance this model in three different ways. First, we built an agent-based model in measurements across CaM’s lobes suggest that these same Met to Gln sub- which we used object-oriented programming to explicitly model flagella stitutions altered the equilibrium between key CaM conformations and motors, including time dynamics. Second, we modeled the number den- (Biochemical and biophysical research communications 456(2) 2015). sity along the flagellum as a vector, and built a stochastic matrix to simulate

BPJ 8636_8639 336a Monday, February 19, 2018 time dynamics and determine a steady-state. Third, we used differential equa- cesses like cell migration and tumor invasion. A basic challenge in studies of tions to directly solve for the steady-state length. In all three, we found that the binding properties of such proteins is that in-vitro assays, e.g. based on the diffusion model can achieve steady-state length and an inverse relation- surface plasmon resonance, can give misleading results, since the binding ship between length and recruitment rate. This is remarkable because this is substrates used in those techniques only approximate physiological mem- perhaps the simplest explanation of length control, giving it credence in light branes. Furthermore, non-lipid membrane components might influence the of evolution. binding properties in vivo. Therefore we established a live cell binding assay: We express the voltage 1664-Pos Board B573 controlled PI(3,4,5)P3-5-phosphatase Ci-VSP in CHO cells along with the Investigations into Idiosyncratic Drug-Induced Hepatotoxicity and protein of interest, which has been tagged with EGFP. Patch-clamp electro- Chronic Proliferation of Cancer Cells using a Label-Free Method physiology is used to control the activity of Ci-VSP, allowing for a tempo- Corina T. Bot1, Sonja Sto¨lzle-Feix2, Krisztina Juhasz2, Elena Dragicevic2, rally well controlled modulation of the PI(3,4)P2-content of the plasma 2 2 2 € 2 Leo Doerr , Matthias Beckler , Michael George , Andrea Bruggemann , membrane. This is done in parallel with the acquisition of time series of 1 2 Rodolfo J. Haedo , Niels Fertig . confocal images to asses the change in membrane association of the protein 1Nanion Technologies Inc, Livingston, NJ, USA, 2Nanion Technologies of interest. If the protein has substantial PI(3,4)P2 affinity in vivo, it will GMBH, Munich, Germany. translocate to the cell membrane in response to an increase in PI(3,4)P2 Hepatic toxicity has accounted for 15 of the 47 drugs withdrawn from the 1 concentration. market in the last two decades . More specifically, Drug Induced Liver We observed this behavior for several proteins, some of which had not yet been Injury (DILI) is the major cause of acute liver failure. DILI is classified described as PI(3,4)P2 binding, indicating their PI(3,4)P2 affinity at physiolog- as intrinsic (or dose-dependent) or as idiosyncratic2. A prominent example 2,3 ical membranes. of idiosyncrasy is acetaminophen , with a variable time of onset and This work was supported by UKGM grant 32/2011MR to C.R.H. and DFG not directly dependent on dose. We present a non-invasive DILI assay grant DFG Research Training Group GRK 2213 to D.O. approach based on impedance measurements in monocyte-derived hepato- cyte-like (MH) cells from MetaHepsÒ. MH cells were used on a 96-well screening system that monitors changes in impedance (or cell 1666-Pos Board B575 monolayer resistance). Cells are monitored under physiological conditions Bcl-2 or Bcl-xL Overexpresssion Affects Both Lactic Fermentation and Mitochondrial Metabolism in Growing Pro-Lymphocytes for temperature, humidity and CO2. We investigated hepatotoxic effects of paracetamol on MH cells when exposed for 24 and 48 hours. In agree- Catalina Olea, Rhaul Llanos, Krish Krishnan, Laurent Dejean. ment with other standard toxicity assays, such as the lactate dehydrogenase Department of Chemistry, CSU, Fresno, Fresno, CA, USA. release assay, low doses of paracetamol caused transient toxicity and It is now well established that shifts in energy metabolism are associated ‘adaptation’ was observed. At higher doses, hepatotoxic effects of paracet- with cancer development and progression. The most studied of these amol could be reversed upon washout after 24 hours, but continued exposure phenomena is the Warburg effect, which corresponds to an increase caused increased hepatotoxicity. In addition to hepatotoxicity, another of anaerobic glycolysis vs mitochondrial oxidative phosphorylation to pro- validation of the principle is shown for chronic proliferation of cancer cells. duce energy for cellular processes. However, the mechanisms related In this study, murine mammary carcinoma cells (H8N8 and H8N8 T3.2) to these metabolic switches are still a matter of debate. Bcl-2 family proteins were used and changes in impedance, were used as a measure of toxicity. contain both pro- (e.g. Bax), and anti-apoptotic (e.g. Bcl-2 and Intrinsic (dose-dependent) effects of the standard clinical regimen cyclo- Bcl-xL) members which are respectively encoded by tumor suppressors phosphamide, doxorubicin and 5-fluouracil could be identified consistent and proto-oncogenes. Up-regulation of the anti-apoptotic proteins Bcl-2 with other methods of live cell analysis systems. Therefore, the utilized and Bcl-xL have been associated with Non-Hodgkin’s lymphoma; 96-well impedance system in combination with murine mammary carcinoma and certain studies indicate that Bcl-2 family proteins play a role in the cells provides a novel tool for investigating therapy resistance of cancer regulation of energy metabolism. However, the metabolic phenotypes cells in vitro. References: 1. Stevens, J.L, 2008. Drug Disc.Tod. 2.Roth, associated directly with an overexpression of Bcl-2 or Bcl-xL (Bcl-2/ R.A, et al.2010, J Pharmacol Exp Ther. 3.McGill, M.R. et al 2013. Pharm xL) remain to be defined at the whole cell level. We recently applied 1 13 Res. 30(9). a H- and C-NMR-based approach on growing Bcl-2/xL-overexpressing pro-lymphocyte B cell lines to determine their respective metabolic 1665-Pos Board B574 trends. The comparison of 1H-NMR spectra obtained from whole cell Characterization of Posphatidylinositol-3,4-Bisphosphate Binding Proper- metabolites extractions indicate that significant metabolic changes are ties of Proteins in Living Cells Using the Voltage Sensing Phosphatase induced by Bcl-2/xL-overexpression; more particularly at the level of the CI-VSP intracellular fumarate pool. Also, both lactate production and glucose con- Tabitha Hees, Dominik Oliver, Christian R. Halaszovich. sumption fluxes are stimulated by Bcl-2/xL overexpression. This phenome- Neurophysiology, University Marburg, Marburg, Germany. non is associated with an increase of glycolytic/fermentative (LDH and Phosphoinositides (PIs) are constituents of cellular membranes. Changes in GAPDH enzyme activity) and mitochondrial (Citrate synthase activity) their concentration constitute signaling events that are conveyed by pro- markers; but only when Bcl-xL (and not Bcl-2) is overexpressed. These cesses that involve some mean of detecting these changes. Major sensors data suggest that Bcl-2 and Bcl-xL expression levels may play an to achieve this are PI binding proteins which will bind to the membrane active role in the stimulation of lactic fermentation commonly observed in in proportion to the membrane concentrations of PIs. This binding can be blood cancer cells; and that Bcl-2 and Bcl-xL-mediated stimulation of highly selective for specific PIs. For this study, we concentrate on the PI lactate production are the results of different molecular transduction Posphatidylinositol-3,4-bisphosphate (PI(3,4)P2), which plays a role in pro- mechanisms.

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Posters: Molecular Dynamics I a similar architecture of Zika viral envelope as that of the DENV and other fla- viviruses. On the other hand, while the infectivity of DENV reduced signifi- 1667-Pos Board B576 cantly at high temperatures, ZIKV was found to be insensitive up to 40 C. Does Cytosine Methylation Stabilize the BI Substate of DNA? To understand this differential temperature dependence, we have performed Jesse Garcia Castillo1, Jessica Romero2, Roxanne A. Fries3, coarse grained and all-atom Molecular Dynamics simulations of ZIKA and Georgia A. Macy3, Paul S. Nerenberg4. DENV envelopes at different temperatures. Results show that the ZIKV enve- 1Biological Sciences, California State University, Los Angeles, Los Angeles, lope remains intact at normal to high temperatures, but the envelope of DENV CA, USA, 2Physics & Astronomy, California State University, Los Angeles, exhibited pronounced instability breaking at the 5-fold axis and destabilizing Los Angeles, CA, USA, 3W.M. Keck Science Department, Claremont, CA, the inter-raft contacts. Also, the raft-raft interfaces in ZIKV showed larger polar USA, 4Physics & Astronomy and Biological Sciences, California State and H-bonding interactions than DENV. Further, we identified key residues University, Los Angeles, Los Angeles, CA, USA. playing crucial role in the stability of ZIKV, which upon mutation destabilized The local conformation of DNA plays a critical role in the recognition-and- the virus envelope. Our results provide useful insights to specifically target the binding process of transcription factors. In particular, the most common viral envelopes for novel antiviral therapeutics. form of the DNA double helix, B-DNA, exists as a conformational equilib- 1670-Pos Board B579 rium between BI and BII substates that are associated with changes in major Evolutionarily Conserved and Divergent Residue-Residue Contact and minor groove dimensions. It has been hypothesized that cytosine methyl- Dynamics Provide Insights into the Allosteric Regulation of Cyclophilins ation, the most common epigenetic modification and one that is often found in Phuoc J. Vu, Xin-Qiu Yao, Mohamed Momin, Donald Hamelberg. so-called CpG islands, modulates transcription factor binding affinity by Chemistry, Georgia State University, Atlanta, GA, USA. altering the BI-BII equilibrium in the neighborhood of methylation sites. Protein internal motions or dynamics are intimately coupled to molecular Indeed, previous molecular dynamics studies have suggested that cytosine function and its regulation. However, detailed mechanisms on how a protein methylation leads to a uniform stabilization of the BI substate in CpG islands. molecule harnesses thermal fluctuations for function remain elusive. An In this work we first benchmark the ability of the latest Amber DNA force effective way to understand a complex system is to introduce small varia- fields and a variety of three-point water models to reproduce BI-BII equilib- tions or perturbations to examine how the system responds. Practically, rium in the Dickerson dodecamer and reduce terminal base pair fraying. Using this approach has been implemented in laboratories to understand protein the best-performing combinations, we then re-examine the conformational ef- function via site-directed mutagenesis. On the other hand, nature has done fects of cytosine methylation in a prototypical CpG island, (GC) . We find 5 similar ‘‘experiments’’ in which natural proteins evolved to acquire diversity that the BI substate is stabilized for GpC steps in such sequences, but that through mutations and the subsequent natural selection or genetic drift. Dur- the BII substate is stabilized for the CpG steps. More interestingly, the Amber ing evolution, certain patterns of dynamics must be conserved to retain the ff99bsc0 DNA force field used in previous studies yields opposite conclu- core protein function, whereas variable dynamics in synergy with variable sions. These findings highlight the need for continued fixed-charge force field sequences underlie the functional specificity. Hence, a careful examination development for nucleic acids, as well as solution state experimental datasets of dynamics across extant homologous proteins provide unprecedented op- involving epigenetic modification that can serve as benchmarks for simulation portunity to uncover the complex structure-dynamics-function relationship. accuracy. Here we describe a comparative study of the conformational dynamics across 1668-Pos Board B577 the cyclophilin family, a group of enzymes catalyzing the peptidyl-prolyl Using Molecular Dynamics Simulations to Compare the Stability of cis-trans isomerization and model systems where dynamics determine func- Lysenin Structures Obtained through X-ray Crystallography and tion. With a combined approach of molecular dynamics simulations and Single-Particle Cryo-Electron Microscopy residue-residue contact analysis, we uncovered the allosteric mechanisms un- Vivek Govind Kumar. derlying the molecular recognition and catalysis in cyclophilins. Collec- Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. tively, our results provide a framework where both common and specific Single-particle cryo-electron microscopy is being used more frequently to functional mechanisms can be understood with an evolutionary analysis of determine the atomic structure of macromolecules. Recent advances in tech- contact dynamics. nology have greatly improved the quality of structures obtained with this method, which is now comparable to x-ray crystallography in terms of 1671-Pos Board B580 Trans resolution. Conformational Dynamics of the HIV-1 -Activation Response Lysenin is a member of the aerolysin family of pore-forming toxins. Recently, Element RNA Hairpin Bound to a Lab-Evolved Peptide both x-ray crystallography and cryo-EM structures of the lysenin pore have Chapin E. Cavender, Ivan A. Belashov, Joseph E. Wedekind, been published. We have used molecular dynamics simulations to analyze David H. Mathews. and compare both structures. In addition to the wild-type structures, we Biochemistry & Biophysics, University of Rochester, Rochester, NY, USA. also analyzed two different experimentally studied lysenin mutants. Both mu- We report microsecond-length classical molecular dynamics simulations of a lab-evolved peptide bound to the HIV-1 trans-activation response element tants showed a reduction in the fixed negative charge on the pore wall, result- 0 ing in reduced pore size and functional activity compared to the wild-type (TAR) RNA hairpin. An essential 5 -structure in all HIV-1 mRNAs, TAR in- pore. teracts with HIV-1 Tat to promote HIV-1 genome transcription, and is The simulation setup involved a nonameric lysenin pore, which was considered a promising therapeutic target. Using yeast display, a U1A- embedded in a membrane bilayer composed of POPC lipids. This protein- derived TAR binding protein (TBP) was selected that binds TAR with membrane complex was solvated in a water box and equilibrated to deter- subnanomolar affinity and extraordinarily high specificity over the native mine the stability and fluctuations of different regions of the protein. The U1A U1hpII target, thereby preventing Tat-dependent transcription in vitro. two structures obtained from different techniques were initially modeled A co-crystal structure of TBP in complex with TAR reveals the basis for its in their wild-type forms. The differential behavior of both structures, due unprecedented affinity and specificity. The complex exhibits a Watson-Crick to the mutation of functionally important residues, was then compared. C30-G34 base pair in the six-nucleotide apical loop. From chemical modifi- This research sheds light on the structure-function relationship in lysenin cation experiments and sequence conservation, this base pair has been and also investigates the relative reliability of the cryo-EM and crystal posited as a prerequisite for Tat binding. We investigated the role of a short structures. TBP peptide - responsible for the preponderance of TAR readout - in stabi- lizing the observed TAR structure using molecular dynamics simulations of 1669-Pos Board B578 TAR in the apo state and in complex with the full TBP protein and the short How Zika Sustains High Temperatures: Insights from Atomic Simulations TBP peptide. To define discrete conformational states, we performed prin- Pindi Chinmai, Venkat Reddy Chirasani, Mohammad Homaidur Rahman, cipal component analysis and clustered trajectories based on the resulting Mohd Ahsan, Prasanna Diddige Revanasiddappa, Sanjib Senapati. principal components. We observed that the apical loop C30-G34 base pair Biotechnology, Indian Institute of Technology, Chennai, India. and a major groove U23-A27-U38 base triple are stable features of Zika virus (ZIKV) is a mosquito-borne human pathogen belonging to the fam- peptide-bound TAR on the microsecond timescale. In agreement with ily of Flaviviridae. The other viruses in this family are Dengue (DENV), West NMR studies of free TAR, dissolution of the major groove base triple occurs Nile (WNV), Japanese encephalitis (JEV) etc. Due to the recent spread of Zika in trajectories of the apo state. The occupancy of contacts between the protein virus and its association with microcephaly in infants and Guillian-Barre syn- and the TAR major groove is greater for the arginine residues whose muta- drome in adults, the World Health Organization (WHO) has declared this tion to alanine has a greater impact on the binding affinity, as measured by epidemic ‘a public health emergency’. The recent cryo-EM structure revealed isothermal calorimetry.

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1672-Pos Board B581 N-methyl-D-aspartate (NMDA) receptors are ion channels expressed in neu- Conformational Transition Dynamics of a Potassium Channel Voltage rons. NMDA receptors (NMDARs) are heavily glycosylated in vivo, but the Sensor Domain role of glycans on NMDARs is insufficiently understood. We performed Tohru Terada. molecular dynamics simulations and Markov state modeling to study the Graduate School of Agricultural and Life Sciences, The University of Tokyo, structure and dynamics of glycans in the ligand-binding domain (LBD) of Tokyo, Japan. the GluN1 subunit of an NMDAR that contains three asparagine (N)-linked Voltage-gated ion channels are membrane proteins that conduct specific ions glycans. NMDAR simulations included homogenous oligomannose type upon activation with active membrane potentials. They sense the membrane po- (Man5) N-glycans, though the dynamics of glycans at different locations tential with the voltage sensor domains (VSDs). The active membrane potential on NMDAR were surprisingly different. The slowest-timescale motions in causes a conformational transition in the VSDs, which leads to opening of the some cases corresponded to a flipping of parts of glycans relative to each pore of the channel and to permeation of the ions through the pore. Molecular other, while in other cases reduced to a head-to-tail bending of a glycan. dynamics (MD) simulations have been performed to elucidate the mechanism Some of the conformational changes in the glycans correlated with the phys- of the conformational transition of VSDs. In most of the simulations, however, iologically important clamshell-like opening and closing of the GluN1 LBD higher voltages have been applied to accelerate the conformational transition. domain. We predict intramolecular interactions between glycans and poly- Therefore, the dynamics of the conformational transition under physiological peptide residues control the coagonist affinity, and studied glycan- conditions still remains unclear. polypeptide interactions with recombinant NMDAR and solution NMR In this study, we performed MD simulations of a VSD with physiological mem- spectroscopy. The receptor glycoprotein expressed in HEK293 cells was 13 brane potentials and constructed Markov state models (MSMs) that describe the monomeric and appropriately glycosylated. NMR spectra of [ CU]-glycans dynamics of the conformational transition. Specifically, the activated structure revealed glycan-polypeptide interactions between proximal N440- and of the VSD of a voltage-gated potassium channel was extracted from its crystal N771-glycan residues and the potential for interactions between distal structure and was embedded in a solvated lipid bilayer. The systems was equil- glycan residues and the poylpeptide. We conclude that the glycans attached ibrated with a restrained MD simulation. Then, a targeted MD simulation was to NMDARs are involved in location-specific interactions with the protein performed to cause the conformational transition to the resting structure. We part of the receptor, and thereby serve as an integral part of NMDA selected 128 snapshot structures from the trajectory of the targeted MD, and receptors. performed a 100-ns unrestrained MD simulation with active (0 V) or resting (100 mV) membrane potentials, starting from each of the selected structures. 1675-Pos Board B584 The aggregates of the MD trajectories were subjected to clustering, which Von Willebrand Factor, a Force-Selective Platelet Binder and Factor VIII yielded about 500 microstates. The numbers of transitions between the micro- Carrier states were counted for each trajectory and MSMs were constructed. Based on Klaus Bonazza1, Roxana Iacob2, Nathan Hudson1, John Engen2, Timothy Springer1. the models, we examined the voltage-dependent changes in the free-energy 1 2 landscape and in the dynamics of the conformational transition. Furthermore, Harvard / Boston Childrens Hospital, Boston, MA, USA, Northeastern we compared the models with that based on the experiment. University, Boston, MA, USA. The ultra-large blood plasma protein von Willebrand factor (VWF) responds 1673-Pos Board B582 to hydrodynamic forces of the blood flow in unique ways. It undergoes a Predicting Spectral Shift in the Teleost Rh2 Cone Opsins using Molecular change of its multimeric superstructure from a globular compacted to a Dynamics Simulations thread-like extended form by increasing flow. As a consequence of this Jagdish Suresh Patel1, Celeste J. Brown2, F. Marty Ytreberg3, large-scale deformation, tensile forces are transmitted to the functional do- Deborah L. Stenkamp2. mains of VWF inducing multiple local conformational changes, which in 1Center for Modeling Complex Interactions, University of Idaho, Moscow, turn account for functional regulations. The primary function of VWF is to ID, USA, 2Department of Biological Sciences, University of Idaho, Moscow, agglutinate platelets and so initiate blood clotting. Our fluorescence video mi- ID, USA, 3Department of Physics, University of Idaho, Moscow, ID, USA. croscopy had recently revealed that extension of VWF and activation of Many living organisms rely upon vision as the dominant sensory modality for platelet binding are two separate steps with different force requirements. foraging, predator avoidance, and social behaviors including mate selection. Here, we shed light onto the previously unclear mechanism of force dependent Vertebrate visual perception is initiated when light strikes rod and cone photo- binding of platelet receptor Glycoprotein Ib (GPIb). By means of Hydrogen receptors within the neural retina of the eye. Sensitivity to individual colors Deuterium Exchange Mass Spectrometry, kinetic binding measurements and i.e., spectral sensitivities of visual pigments are a function of the type of chromo- genetic mutation, we pinpointed the force dependent structural change, phore and the amino acid sequence of the associated opsin protein in the photo- responsible for GPIb binding, to a flexible glycosylated region that flanks receptors. Large differences in peak spectral sensitivities (39nm in lmax) can the VWFA1 domain, providing a mechanism of mechanical activation of result from minor differences in amino acid sequence of cone opsins (85% VWF. While GPIb and collagen binding is enhanced at increased flow, Factor identity). Little is known regarding how differences in protein structure result VIII shows an opposed force dependence. Tensile forces in the VWF chain in spectral differences. To address this problem, we selected a spectrally- lead to a release of FVIII, even in absence of further biochemical activation diverse group of 14 teleost Rh2 cone opsins for which sequences and lmax are pathways of FVIII, as shown here by Atomic Force Microscopy. The over- known, and carried out classical molecular dynamics simulations after embed- arching hypothesis is that the multifunctional protein VWF has two types of ding their homology models within explicit bilayers. These simulations revealed binders, immobile compounds of the vessel walls or platelets, like collagen structural features of visual pigments, particularly within the chromophore, or GPIb, which increase their affinity upon mechanical stress, and plasma sol- which contributed to spectral shift, as well as features that did not appear to be uble active compounds, such as FVIII (and hypothetically inflammation strongly predictive. Statistical tests performed on all the observed parameters markers), which decrease binding at higher forces. This would make VWF associated with the chromophore revealed that a two-term, first-order regression a mechanically operating drug deliverer, as VWF binds vessels and platelets model was sufficient to predict peak spectral sensitivities with considerable to become extended and release the drugs. accuracy over a lmax range of 452 - 528 nm. The modeling procedure was not only accurate, but efficient and simple in that site-by-site molecular modifica- 1676-Pos Board B585 tions, or complex quantum mechanics models, were not required to predict rela- Computational Study on Conformational Relaxation Dynamics of a Protein tive lmax. Furthermore, these studies identify structural features associated with the chromophore that explain diverged spectral shifts, and provide a platform for Sotaro Fuchigami. future, functionally predictive opsin modeling. To our knowledge this is the first Yokohama City University, Yokohama, Japan. report of a successful molecular modeling approach for predicting peak spectral Recent advances in various experimental and computational techniques have sensitivities of vertebrate cone opsins. revealed atomic details of protein conformational changes and their molecular mechanisms. One of interesting features is that pathways of a large conforma- 1674-Pos Board B583 tional change of a protein are not always identical but rather show variability Glycan-Protein Interactions in NMDA Receptors Revealed with because of the protein’s intrinsic flexibility. However, the underlying molecular Computational Modeling and NMR Spectroscopy details remain unclear. In the present study, we characterized conformational Anton V. Sinitskiy1, Ganesh P. Subedi2, Adam W. Barb2, Vijay S. Pande1. relaxation dynamics of a protein using molecular dynamics (MD) simulations. 1Chemistry, Stanford University, Stanford, CA, USA, 2Roy J. Carver As a target protein, we selected lysine-, arginine-, ornithine-binding protein Department of Biochemistry, Biophysics and Molecular Biology, Iowa State (LAO), which comprises two domains and undergoes slow and large- University of Science & Technology, Ames, IA, USA. amplitude domain motions. MD simulations of apo-LAO from the closed

BPJ 8640_8642 Monday, February 19, 2018 339a conformation in explicit water were repeated many times starting from the actual function. A modified variant of this problem arises at telomeres, where same initial coordinates but with different initial random velocities using hundreds of target sites for the two telomeric dsDNA-binding proteins - TRF1 MARBLE and the CHARMM22/CMAP force field parameters. Almost all tra- and TRF2 - are clustered together within a short region of chromosomal DNA. jectories showed conformational relaxation from the closed form to the open Hence, telomeric proteins slowly diffuse along the telomeric tracts, pairing form, and their relaxation times were obviously different; some relaxations with each other to assemble the shelterin, the protein complex involved in telo- occur immediately, others slowly. We will analyze a variety of pathways of mere maintenance. While the problem of sequence search is relatively well conformational changes using the time-structure based independent component studied with coarse grained models at low spatial resolution, a comprehensive analysis (tICA) and a Markov state model (MSM). atomic-level picture of dynamic sequence interrogation and DNA binding is still lacking. To address this issue, we used extensive (>0.5 ms in total) MD 1677-Pos Board B586 simulations to study the dynamical aspects of sequence-specific binding of NMR Relaxation and Molecular Dynamics Simulations of Side Chain TRF1 at both telomeric and non-cognate DNA. For the first time, we describe Dynamics in Proteins the spontaneous formation of a sequence-specific native protein-DNA complex Falk Hoffmann1, Mengjun Xue2, Frans Mulder2, Lars Sch€afer1. 1 2 in atomistic detail, and study the mechanism of DNA sequence interrogation at Ruhr-University Bochum, Bochum, Germany, University of Aarhus, different stages of complex formation. We quantify the contribution of indi- Aarhus, Denmark. vidual amino acids to the sequence specificity to show how proteins avoid Molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) off-target binding while retaining high affinity for target sites. Our calculated spin relaxation experiments have become increasingly powerful to study pro- free energy landscapes reproduce the thermodynamics of sequence-specific tein dynamics at atomic resolution due to steady improvements in physical binding in unprecedented detail, while statistical approaches allow for a models and computation power. Good agreement between generalized 2 comprehensive description of intermediates of the binding process. We further Lipari-Szabo (S NH) order parameters derived from experiment and simulation explore the possibility of studying protein diffusion along both single- and is now observed for the backbone dynamics of a number of proteins. Unfortu- 2 double-stranded DNA with atomistic resolution using contact map-based nately, the agreement for side chains, as e.g. probed by S CH3 for methyl- Markov state models. containing side chains, is much poorer. In this work we discuss several issues with methyl side chains that need to be addressed to close the gap between NMR and MD. Accounting for protein tumbling is one very important factor 1680-Pos Board B589 to obtain a good agreement. In our hands, the application of improved water Atomic-Level Characterization of the HIV-1 Capsid and Host-Pathogen force fields with an appropriate way of including anisotropic overall protein Interactions from Molecular Dynamics Simulations tumbling improves the prediction of experimentally measured dynamic observ- Juan R. Perilla. ables by MD simulations. We demonstrate these aspects for T4 lysozyme as a Chemistry and Biochemistry, University of Delaware, Newark, DE, USA. representative example. Our results guide the way for extracting from the NMR Virus capsids are protein shells that package the viral genome. Although their relaxation data the most accurate parameters that describe protein side chain morphology and biological functions can vary markedly, capsids often play crit- dynamics and report on conformational entropy. Additionally, we reparame- ical roles in regulating viral infection pathways. Therefore, a detailed knowledge trized side chain dihedral angle energy barriers for methyl rotation in the of the HIV-1 capsid, including its dynamic structure, interactions with cellular Amber99SB*-ILDN force field. We demonstrate that this leads to a much factors, and the specific roles it plays in the replication cycle, is imperative for more realistic dynamics of methyl groups and is another important feature to the development of antiviral therapeutics. In this poster, I present an emerging close the gap between spectral densities from NMR relaxation and MD area of computational biology that focuses on the structure and dynamics of vi- simulation. rus capsids. State-of-the-art computer simulations reveal the role of ions in the stability of the HIV-1 capsid. In addition, molecular dynamics reveal the ener- 1678-Pos Board B587 getics associated with the permeation of nucleotides through various pores of The Effects of Linker Histone Isoforms on the Structure and Dynamics of the HIV-1 capsid thus unveiling the molecular mechanism associated with the Chromatosome nucleotide translocation. Finally, emergent properties of the entire HIV-1 capsid Dustin C. Woods1, Jeff Wereszczynski2. like acoustic motions and effective electrostatics reveal large-scale cooperativ- 1Chemistry, Illinois Institute of Technology, Chicago, IL, USA, 2Physics, ity between capsomers spanning over large regions of the capsid. Illinois Institute of Technology, Chicago, IL, USA. The nucleosome core particle (NCP) is the fundamental packaging unit of DNA 1681-Pos Board B590 in eukaryotes and the building block of chromatin fibers. The NCP consists of Molecular Dynamics Simulation of Toll-Like Receptor 4 (TLR4) an octameric protein core wrapped with DNA. As an extension, the chromato- Ectodomain some consists of the NCP in complex with an external linker histone. Experi- Alireza Tafazzol, Yong Duan. ments have shown that this linker histone can bind to DNA in either an on- or Department of Biomedical Engineering and Genome Center, University of off-dyad orientation and its presence can have cascading effects that may affect California Davis, Davis, CA, USA. greater chromatin dynamics. However, the molecular mechanisms that drive Toll-like receptors (TLRs) have a central role in both the innate and adaptive the binding mode, how these modifications are influenced by specific linker immune systems. These proteins recognize pathogen-associated molecular pat- histone residues, and the major biological implications of these variations in terns and induce the release of the effector molecules of the immune system. chemical composition and conformation, remains poorly understood. Here, The dysregulation of the TLR system may cause various autoimmune diseases we report on the chemical-physical properties of the chromatosome from a and septic shock. TLR4 was identified as the signaling receptor for lipopolysac- series of molecular dynamics simulations of three different linker histone iso- charide which is responsible for the endotoxic shock, a severe inflammatory forms bound in on- and off-dyad binding modes. Initial results show that the disease that leads rapidly to multi organ failure and death. Moreover, several flexibility of the linker DNA is reduced upon linker histone binding, producing studies suggest a possible role for TLR4 in cancer, cardiovascular disease, more compact chromatosome conformations. Additionally, an analysis of Alzheimer’s, obesity, and diabetes. Recent advances in crystallography and relative binding energies suggests that Globular Histone H5 (GH5) exhibits a in silico techniques provide promising opportunities for TLR4 investigations stronger binding affinity to the NCP than the other linker histones, Globular and drug design. Recently the X-ray structures of the extracellular domain Histone H1 (GH1) and the Globular Histone H5 penta-mutant (GH5-pMut), (ECD) of mouse-TLR4 in ligand-bound (PDB 5IJC and 5IJD) and unbound and prefers the on-dyad binding mode. Overall, these atomistic simulations pro- (PDB 5IJB) states have been resolved. The TLR4 ECD contains leucine rich vide insight into the physical and chemical effects of linker histone isoforms on repeats that folds into a characteristic horseshoe-like structure and with the the structure and dynamics of the chromatosome. aid of its co-receptor (MD-2) is responsible for ligand recognition. We used multiple long-timescale (1.2 ms) molecular dynamics simulations on these 1679-Pos Board B588 structures to investigate TLR4 ECD conformational changes and interaction How Proteins Bind to DNA. Target Discrimination and Dynamic Sequence sites for its potential roles in signaling mechanism. All simulations were Interrogation on Telomeres done in an explicit-water model with AMBER force field on graphics process- Milosz Wieczor1, Jacek Czub2. ing units (GPUs). Compared to earlier simulations done by others using a 1Dept. of Physical Chemistry, Gdansk University of Technology, Gdansk, different force field that showed a highly dynamic TLR4 with an overall Poland, 2Dept. of Physical Chemistry, Gdansk University of Technology, RMSD of 10 A (de Aguiar et al, Proteins: Struct., Funct., Bioinf. 2015), our Gdansk, Poland. simulations showed that TLR4 structure was well maintained. Further analysis In the nucleus of a human cell, sequence-specific binding of DNA by nuclear showed that the interface was also stable. Comparison of the ligand-bound and proteins requires efficient and accurate sampling of billions of potential binding unbound structures shows the function of the MD-2 co-receptor and ligands in sites in order to find the sparsely located targets where the protein performs its stabilizing the structure of TLR4 ECD.

BPJ 8640_8642 340a Monday, February 19, 2018

1682-Pos Board B591 ing pocket. Three independent Molecular Dynamics simulation runs indi- Characterizing the Motions of N2-N3 Domains of IsdH cated general stability of donepezil, galantamine and rivastigmine in their Joseph Clayton, Jeff Wereszczynski. respective enzyme binding pocket as well as the tendency to form hydrogen Physics, Illinois Institute of Technology, Chicago, IL, USA. bonds with the water molecules. The binding of rivastigmine in the Torpedo Iron is an important resource for metabolism, but is not always readily available California AChE binding pocket is interesting as it eventually undergoes car- inside organisms. In response to this, many pathogens have developed a mech- bamylation and breaks apart according to the X-ray structure of the complex. anism to acquire iron from their host. Staphylococcus aureus meets this Similarity search in the ZINC database and targeted docking on the gorge re- resource need through the iron-regulated surface determinant (Isd) protein gion of the AChE enzyme have identified new inhibitor molecules with high pathway, which involves removing heme from hemoglobin and passing the predicted binding affinity. We will present the findings as how computational group through the bacterial membrane. In this pathway, the N2 and N3 domains and experimental biophysical techniques can lead to identify potent mole- of the protein IsdH are responsible for the extraction of heme and bind directly cules in a fatal disease. to hemoglobin. The structure of N2 and N3 bound to hemoglobin has been determined, but the process of heme transfer remains unknown. In addition, 1685-Pos Board B594 an NMR study on unbound IsdH revealed that the N2 domain has a range of Oligomerization of Nicotinic Acetylcholine Receptors in Domain-Forming motions and does not have a stable positioning relative to the N3 domain. Membranes Here, we use molecular dynamics to simulate the unbound N2 and N3 domains Kristen N. Woods, Liam M. Sharp, Grace Brannigan. to identify configurations prior to hemoglobin binding. In these simulations we Computational and Integrative Biology, Rutgers University, Camden, NJ, calculate the free energy of several unbound configurations and specify a USA. compact state and an extended state–supporting the found range of motion of The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric N2 and suggesting that IsdH follows a conformational selection mechanism ligand gated ion channel found throughout the nervous system. At the neuro- in binding to hemoglobin. We also use these simulations to identify pathways muscular junction, nAChRs cluster at a high density in order to transmit a between these states in order to ascertain preferred motions that may be fast and robust signal to the muscle. While nAChR clusters are stabilized by required to bind to hemoglobin. Knowing these pathways and configurations the dimerization of the intracellular protein, rapsyn, this dimerization process of unbounded IsdH allows for future comparison to similar motions of bound is extremely sensitive to changes in lipid environment. Neuronal membranes IsdH, potentially providing insight to the heme extraction mechanism. are rich in polyunsaturated lipids (PUFAs), cholesterol, and PE headgroups, with an overall composition similar to that of the Torpedo Californica electric 1683-Pos Board B592 organ. One PUFA in particular, Docosahexaenoic acid (DHA), is prevalent in Covalent Complex Model of DNA Topoisomerase and DNA for Molecular both neuronal and Torpedo membranes, and is associated with domain forma- Dynamics Simulation tion in membranes. Through our preliminary simulations, we observed single Purushottam Tiwari1, Prem Chapagain2, Yuk-Ching Tse-Dinh2, nAChRs partitioning into the liquid-disordered (ld) domains, while remaining Aykut Uren1. close to liquid-ordered (cholesterol-rich) domains. While researchers have 1Georgetown University, Washington, DC, USA, 2Florida International speculated that nAChR clustering is facilitated by the formation of lipid do- University, Miami, FL, USA. mains, experiments investigating such lipid-protein interactions have been DNA topoisomerase is responsible for regulation of DNA topological state. The inconclusive. relaxation of supercoiled DNA is catalyzed by DNA topoisomerase. Upon In the present study, we use coarse-grained molecular dynamics simulations interaction, DNA topoisomerase form a transient covalent linkage with to investigate the partitioning and clustering behavior of multiple nAChRs DNA. This transient covalent complex can be trapped using topoisomerase in- in a range of membrane environments with various amounts of intrinsic hibitors and these inhibitors have a potential to be developed as a new class of domain formation. For quasi-native membranes, nAChRs consistently parti- drugs targeting topoisomerase. In Escherichia coli (E. coli), accumulation of tion into liquid-disordered phases, exhibiting a strong affinity for the topoisomerase I (topoI)-DNA complex leads to bacterial cell death. We have domain interface and even dimerizing along well-defined interfaces. Oligo- designed a model of the complex to be investigated using molecular dynamics merization is not observed in membranes lacking domains. Boundary lipids (MD) simulations. This model allowed us to study topoisomerase-DNA cova- are rich in polyunsaturated acyl chains regardless of the number of nAChR lent system using computer simulations incorporating structural flexibility as molecules, but preventing domain formation also reduces the likelihood of well as effect of small molecule in the topoisomerase-DNA interaction. As these acyl chains aggregating around nAChR. Taken together, these results an example, we used E. coli topoisomerase I (EctopoI), which is the most imply that nAChR organization, oligomerization, and local environment is extensively studied bacterial type IA topoI and a target for antibacterial drug highly sensitive to membrane organization, with long-tailed PUFAs playing discovery. Utilizing our model, we first simulated the EctopoI-DNA covalent a critical role in both phase separation and protein clustering in neuronal complex and then performed screening of a library of compounds against the membranes. minimized EctopoI target, using virtual screening, to identify potential EctopoI inhibitors. Finally, taking one of the identified inhibitors, we investigated the 1686-Pos Board B595 dynamics of ternary complex of EctopoI, inhibitor, and DNA. Our simulation Experimental and Computational Studies of Structural Differences results show that both the EctopoI-DNA binary complex as well as EctopoI- Between Alternative Exon Skipped Repairs for Duchenne Muscular inhibitor-DNA ternary complex were stable for 50 ns of MD simulations. Dystrophy Our results further imply that the identified inhibitor prevents relegation of Manyuan Ma, Nick Menhart, Jeff Wereszczynski. the cleaved DNA, which leads to accumulation of the EctopoI-inhibitor- Illinois Institute of Technology, Chicago, IL, USA. DNA ternary complex. Exon skipping is a novel therapy for the devastating genetic disease, Duchenne Muscular Dystrophy, DMD. It aims to therapeutically skip an additional exon 1684-Pos Board B593 adjacent to the patient’s original defect to correct the reading frame and restore Molecular Dynamics and Docking Studies on Acetylcholinesterase (AChE) dystrophin protein expression. However, the restored protein is edited relative Inhibitors to the wild type, missing both the original deletion as well as the therapeutically Rejwan Ali1, Mostafa Sadoqi1, Simon Moller2, Allal Boutajangout3, skipped region. In many cases, this edit can be done in different ways, with Mihaly Mezei4. possibly clinically relevant differences. However, the functionality of such edi- 1Physics, St John’s University, Queens, NY, USA, 2Biological Sciences, St ted proteins is unknown. To study the effects of exon skipping on the structures John’s University, Queens, NY, USA, 3Department of Neurology and and stabilities of exon skipped proteins, we expressed various edits that are Neuroscience & Physiology and Psychiatry, New York University Langone alternative repairs of the same underlying defect, and assessed them for differ- Medical Center, New York, NY, USA, 4Department of Pharmacological ences by a variety of spectroscopic and biochemical tools. Significant differ- Sciences, Icahn School of Medicine at Mount Sinai, Queens, NY, USA. ences appeared between alternative repairs, especially the folded structure Recently we have performed molecular dynamics and docking studies on ratio, thermal stability, and resistance to protease challenge. Since no experi- several inhibitors on acetylcholinesterase (AChE), an important target in Alz- mental 3D structures are available for most of dystrophin, the details of these heimer disease. We have assessed by docking and molecular dynamics the differences at the molecular level are unknown. To decipher that, we applied binding mechanisms of three FDA-approved Alzheimer drugs: donepezil, homology modeling using Robetta to produce atomic level model structures galantamine and rivastigmine. Dockings by the softwares Autodock-Vina, of each edit, and then examined these by molecular dynamic simulations to PatchDock and Plant reproduced the docked conformations of the compare with the experimental results. Good agreement with the experimental inhibitor-enzyme complexes within 2A˚ of RMSD of the X-ray structure. results was seen in relative alpha-helicity of equilibrated structure in implicit Free-energy scores show strong affinity of the inhibitors for the enzyme bind- solvent, and thermal denaturation conducted by replica exchange MD. This

BPJ 8640_8642 Monday, February 19, 2018 341a agreement provides confidence in our modeled structures as well as revealing 1690-Pos Board B599 the molecular details of their differences, and suggests that this approach may Novel Insights to the Design of Apolipoprotein AI Mimetic Peptides be useful in assessing alternative exon skipping strategies for any given person Mohsen Pourmousa, Richard W. Pastor. in a less time-consuming way. National Institutes of Health, Bethesda, MD, USA. Peptides mimicking the major protein of high-density lipoprotein, apolipopro- 1687-Pos Board B596 tein AI (APOA1), are promising therapeutics for cardiovascular diseases Molecular Dynamics Simulation Studies of Polymyxin B Derivatives in because they impede the development of atherosclerotic lesions. Similar to Homogeneous E. coli K12 Bilayers APOA1, their atheroprotective characteristic is attributed to the ability to Seonghoon Kim1, Marcos Pires2, Wonpil Im1. form discoidal bilayer when lipid transporters pleat lipids and cholesterols 1Biological Sciences, Lehigh University, Bethlehem, PA, USA, 2Chemistry, from macrophages, a process called cholesterol efflux. Understanding the struc- Lehigh University, Bethlehem, PA, USA. ture of nanodiscs generated by APOA1 mimetics is crucial to the rational The outer leaflet of the outer membrane of Gram-negative bacteria is composed design of such therapeutics. However, high-density lipoproteins are resistant of lipopolysaccharides (LPS) that are essential to structural integrity and pro- to traditional high-resolution approaches, such as NMR or X-ray crystallog- tection of the bacteria. One of natural product antibiotics, polymyxin B raphy. Herein, all-atom Molecular Dynamics simulations on Anton-2 super- (PMB) has a high binding affinity with lipid A of LPS, and thus the PMB de- computer determine the structure of nanodiscs and contrast behaviors of two rivatives with antigenic epitopes, connected by the spacers, can be used to re- peptides that were experimentally shown to be potent and inactive in choles- cruit antibodies to kill bacteria. Recently, Pires and collaborator experimentally terol efflux. The peptides are a-helical, amphipathic, have 18 residues, and showed that the spacer length played an important role in displaying epitopes solely contain Glu, Leu, and Lys. A 3-ms simulation of a nanodisc with potent above the bacterial cell wall. To explore the influences of the spacer length peptides shows that they migrate from the disc surface to the edge to cover acyl on the epitopes’ distributions on E. coli K12 LPS surface, we modeled three- chains of lipids. In contrast to APOA1 which takes a ‘‘belt’’ configuration, the different spacer lengths of PMB derivativesin homogeneous E. coli K12 bilayer peptides form a ‘‘picket-fence’’ arrangement whereby they orient along the disc by placing the same kind of two PMB derivatives (one above and the other normal. Each peptide interacts with its two neighbors through Lys-Glu salt- below the K12 bilayer). Presented and discussed are the structure and dynamics bridges mostly in an antiparallel fashion, hence a peptide oligomer. A 2-ms of the PMB derivatives and their dependence on the spacer length on E. coli simulation of a nanodisc with inactive peptides, in contrast, demonstrates their K12 bilayers. incapability to stabilize nanodiscs. Starting from an ideal antiparallel picket- fence on the disc edge, the peptides dimerize but dimers migrate from the 1688-Pos Board B597 edge to the surface leaving acyl chains exposed to water. This is due to the Molecular Dynamics Simulations for Conformational Changes on a repulsion of negatively charged sidechains of adjacent dimers. Therefore, Reaction Step of SR-Ca2D-ATPase the simulations establish that APOA1 mimetics should oligomerize to effec- Chigusa Kobayashi1, Yasuhiro Matsunaga1,2, Jaewoon Jung1,3, tively promote cholesterol efflux and that their dimerization is necessary but Yuji Sugita1,3. not sufficient. This insight informs the future design of these peptides. 1Computational Biophysics Research Team, Riken Aics, Kobe, Hyogo, Japan, 2JST Presto, Kawaguchi, Saitama, Japan, 3Theoretical Molecular 1691-Pos Board B600 Science Laboratory, Riken, Wako, Saitama, Japan. Results Regarding the Resolving of Membrane-Protein Complexes using Sarco(endo)plasmic reticulum Ca2þ-ATPase is a representative protein of Neutron Reflection in Molecular Dynamics P-type ATPases, which transports Ca2þ across membrane against a large Bradley W. Treece1, Arvind Ramanathan2, Frank Heinrich3, concentration gradient by utilizing ATP hydrolysis. Structural and biochem- Mathias Lo¨sche1. ical studies have suggested functional roles of coupled motions between the 1Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2Oak Ridge cytoplasmic domains and the transmembrane helices in individual reaction National Laboratory, Oak Ridge, TN, USA, 3National Institute of Standards steps. It is, however, difficult to investigate the coupled motion by simulating and Technology, Gaithersburg, MD, USA. the reaction step due to its slow time-scale. To overcome this difficulty, we Detailed characterization of membrane-associated proteins on fluid lipid bila- have developed a high-performance MD simulation package, GENESIS, yers is a challenge despite the availability of many biophysical techniques. which is available as free software. (The website is http://www.aics.riken. Neutron reflectivity (NR) is a method that provides sub-nanometer resolution jp/labs/cbrt/). of such structures in biommetic environments. Analysis of NR data provides In this study, we applied a rare event sampling method (string method) imple- density distribution of protein along the membrane normal and coupling this mented in GENESIS to the conformational changes of Ca2þ-ATPase. on a data with complementing structural information from crystallography or reaction step. We discuss rearrangements of the transmembrane helices NMR can reveal a full atomistic description of the protein-membrane complex. coupled with large-scale cytoplasmic domain movements. For systems in which this information is not available, because of disordered regions or ensembles of conformational states, additional work is required. Mo- 1689-Pos Board B598 lecular dynamics (MD) simulations can provide the necessary structural infor- Influenza Viral Envelope Simulation Reveals Novel Druggable Pockets on mation, but long equilibration times and sampling is an issue. Surface Glycoproteins Here we show a method to incorporate NR data into MD simulations to steer Sarah E. Kochanek1, Jacob D. Durrant2, Rommie E. Amaro1. simulations toward configurations which reproduce experimental results. 1Chemistry and Biochemistry, University of California, San Diego, La Jolla, Biasing potentials are calculated from the one-dimensional densities derived CA, USA, 2Biological Sciences, University of Pittsburgh, Pittsburgh, PA, in NR and evolve as the MD trajectory progresses. The bias directs the mole- USA. cule toward the configurations that NR suggests and grows weaker as these con- Influenza virus infection continues to be a major healthcare issue, with formations are adopted. Instead of rigidly confining the structure, the system is 3-5 million cases of severe disease reported and 300,000-500,000 deaths permitted to fluctuate about these configurations. We demonstrate applications worldwide each year. Variation in the sequences and structures of the two to peptides and larger proteins with disordered regions. major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), from strain to strain complicate prophylactic and therapeutic approaches. 1692-Pos Board B601 Integrative modeling techniques combining cryo-electron tomography with Modeling the Rotational Dynamics of Novel Hetero-FRET Probes as x-ray crystallography, homology modeling, and protein-protein docking Measured using Time-Resolved Anisotropy have led to the construction of a whole-virion model of influenza. Using Ryan E. Leighton1, Hannah Leopold1, Jacob Schwarz1, Arnold J. Boersma2, the petascale computing machine Blue Waters, we have performed molecu- Erin D. Sheets1, Ahmed A. Heikal1. lar dynamics (MD) simulations of this virion model. Principal component 1Chemistry and Biochemistry, University of Minnesota Duluth, Duluth, MN, analysis reveals that simulation of the complete virion allows exploration USA, 2Biochemistry, University of Groningen, Groningen, Netherlands. of greater glycoprotein conformational space as compared to single glyco- Macromolecular crowding is prevalent in all living cells due to the presence protein simulations. Further, Markov state models constructed from the of proteins, nucleic acids, and organelles. Cellular crowding is heterogeneous trajectories of the glycoproteins reveal novel druggable pockets in both and is known to influence biomolecular transport, biochemical reactions, and HA and NA that can be targeted for the development of novel treatments protein assembly/folding. Emerging evidence suggests that some cell pathol- for influenza virus infection. The identification of potentially druggable ogies may be correlated with compartmentalized crowding. As a result, there pockets previously unseen in simulations of individual glycoproteins demon- is a need for robust biosensors to quantify crowding both spatially and strates the utility of modeling in the sub-cellular scale and beyond to inform temporally in living cells. Importantly, those biosensors should be compatible drug discovery. with noninvasive and quantitative methods. We have developed a model to

BPJ 8640_8642 342a Monday, February 19, 2018 describe the rotational dynamics of hetero-FRET biosensors using time- Zurich, Switzerland, 5Swiss Institute of Bioinformatics, Quartier Sorge, resolved anisotropy. The model was tested on wavelength-dependent exper- Baˆtiment Genopode, Lausanne, Switzerland, 6RCSB Protein Data Bank, iments of hetero-FRET probes (mCerulean-linker-mCitrine) with linkers Institute for Quantitative Biomedicine, Rutgers, The State University of New of varying length and flexibility in buffer at room temperature. This Jersey, Piscataway, NJ, USA, 7Protein Data Bank in Europe (PDBe), model guided our experimental design towards the optimum wavelength- European Molecular Biology Laboratory, European Bioinformatics Institute dependent time-resolved anisotropy that provides the most sensitivity to (EMBL-EBI), Wellcome Genome Campus, Hinxton, United Kingdom. macromolecular crowding. Using this model to fit our experimental results, Advances in experimental techniques are providing access to ever more com- we are able to determine the energy transfer rate constant and the energy plex and larger macromolecular data sets. Definition and extraction of substruc- transfer efficiency of these hetero-FRET biosensors. Our findings are in gen- tures represents a crucial step required for both analysis and visualization of eral agreement with the standard fluorescence lifetime-based FRET analysis. these data. A common language for defining macromolecular substructures However, wavelength-dependence of time-resolved anisotropy and its sensi- would streamline this step, and enable communication among different compu- tivity to the energy transfer efficiency are rather distinct from time-resolved tational and visualization tools. However, software tools usually define their fluorescence approaches. Currently, we are using the developed model to own query language or support a language that is not sufficiently general to investigate the energy transfer efficiency of selected FRET sensors in homo- describe complex spatial and other relationships within macromolecular geneous and heterogeneous environments. Our results represent an important structures. step towards the application of quantitative and non-invasive time-resolved Herein, we introduce the Molecular Query Language (MolQL), a declarative anisotropy of hetero-FRET probes to investigate macromolecular crowding language for describing substructures within molecular data. The design of and protein-protein interactions in living cells. the language has three main goals: 1) Make it easy for tool developers to imple- ment the specification; 2) Be expressive enough to describe a wide range of molecular substructures; and 3) Be readily extensible. We describe the initial Posters: Computational Methods and specification of the language, and provide its reference implementation that can execute MolQL expressions on any Protein Data Bank archival entry Bioinformatics I (pdb.org) and immediately show the result in a web browser. Moreover, we demonstrate the versatility of the language by translating Jmol, PyMOL, and 1693-Pos Board B602 VMD selection expressions into MolQL. A Data Dictionary and Prototype Deposition System for Archiving Inte- Find out more about MolQL and try it online at http://molql.org/. grative/Hybrid Models 1 2 1 2 Brinda Vallat , Benjamin Webb , John Westbrook , Andrej Sali , 1695-Pos Board B604 Helen Berman1. 1 Comparative Protein Dynamics with Droids 1.0 - A Gui-Based Pipeline for Research Collaboratory for Structural Bioinformatics, Center for Integrative Functional Evolutionary Protein Analysis and Visualization Proteomics Research, Rutgers, The State University of New Jersey, 1 2 2 2 Gregory A. Babbitt , Jamie S. Mortensen , Erin E. Coppola , Piscataway, NJ, USA, Department of Bioengineering and Therapeutic Lily E. Adams1, Justin K. Liao2. Sciences, Department of Pharmaceutical Chemistry, University of California 1Life Sciences, Rochester Inst Technol, Rochester, NY, USA, 2Biomedical San Francisco, San Francisco, CA, USA. Engineering, Rochester Inst Technol, Rochester, NY, USA. Structural characterization of complex macromolecular assemblies is increas- Traditional bioinformatics in comparative genomics and molecular evolution ingly being carried out using Integrative/Hybrid (I/H) methods. Traditional work only with static representations of biomolecules (i.e. sequence and struc- structure determination methods such as X-ray crystallography and NMR ture), thereby ignoring molecular motion over time. Yet, molecular dynamics spectroscopy are generally inadequate for tackling such complex assemblies. often underpins crucial aspects that define molecular functioning in the cell Recently, I/H methods have been developed that combine spatial restraints potentially connecting one of the longest time scale processes in the universe derived from a variety of complementary experimental techniques, including (i.e. molecular evolution) to one of the shortest (i.e. molecular bond motion). cryo-electron microscopy, small angle scattering, chemical crosslinking, Here, we leverage advances in GPU-accelerated molecular dynamics simula- mass spectrometry and other proteomics and bioinformatics methods. An tion software to develop a comparative method of analysis and visualization important feature of integrative modeling is that it allows for multi-scale, that can be applied to any two homologous PDB structures. Our open source multi-state, and time-ordered ensembles, which are very different from the software, DROIDS (Detecting Relative Outlier Impacts in Dynamic Simula- mono-scale atomistic models currently archived in the Protein Data Bank. tions) is designed to work in conjunction with AMBER16 molecular dynamic Our goal is to facilitate the archiving of I/H models so that they can be avail- simulation, CPPTRAJ, and UCSF Chimera 1.11 molecular modeling software. able to the broader biological research community. We have created a data DROIDS implements multiple test corrected Kolmogorov-Smirnov statistics dictionary that captures the details of I/H models including a variety of on atom bond fluctuations and/or correlations in atom vector trajectories to experimentally-derived spatial restraints and the modeling of multi-scale, identify significant differences affecting the molecular dynamics of proteins, multi-state, time-ordered ensembles. The dictionary is an extension of the or their physical interactions with other biomolecules. Thus, DROIDS provides PDBX/mmCIF dictionary used by the Protein Data Bank to archive macro- a framework to address functional or evolutionary questions surrounding molecular structures. The dictionary and supporting documentation are biophysically-encoded information resulting in energy changes affecting mo- publicly accessible via a GitHub repository (https://github.com/ihmwg/ lecular motion. DROIDS is simple to use, incorporating separate GUI controls IHM-dictionary). A prototype deposition system called PDB-Development for MD simulation, vector trajectory analysis and the final statistical and visual (PDB-Dev, https://pdb-dev.wwpdb.org) has been created to accept I/H representations. As case examples, we demonstrate a null comparison of two models that comply with the new dictionary. PDB-Dev currently consists independent runs on ubiquitin, comparative measures of thermostability in of three example I/H models obtained from the Integrative Modeling Plat- several well-known thermophilic enzymes and an analysis of the biophysical form (IMP) software: the Nup84 sub-complex of the nuclear pore complex, impacts of sickle cell mutation on hemoglobin. DROIDS can also be utilized the exosome complex and the mediator complex. Preliminary visualization to visually investigate significant quantitative changes in molecular dynamics of these I/H models can be carried out using the ChimeraX visualization soft- due to binding interactions of pharmaceuticals, toxins, or other biomolecules. ware. Building an archive for I/H models is vital to the evolving structural Visit our code repository at https://github.com/gbabbitt/DROIDS-1.0 and for biology community. The I/H methods data dictionary and prototype deposi- examples, visit our YouTube channel at https://www.youtube.com/channel/ tion system are critical steps in this direction. This work is supported by NSF UCJTBqGq01pBCMDQikn566Kw. EAGER award number 1519158. 1696-Pos Board B605 1694-Pos Board B603 Channelsdb and Moleonline - Database and Tool for Analysis of Molql: Towards a Common General Purpose Molecular Query Language Biomacromolecular Tunnels and Pores Alexander S. Rose1, David Sehnal2,3, Spencer Bliven4,5, Luka´s Pravda1, David Sehnal1, Karel Berka2, Veronika Navra´tilova´2, Stephen K. Burley1,6, Sameer Velankar7. Dominik Tousek2,3,Va´clav Bazgier2, Radka Svobodova´Varekova´1, 1RCSB Protein Data Bank, San Diego Supercomputer Center, University of Michal Otyepka2, Jaroslav Koca1. California, San Diego, CA, USA, 2CEITEC - Central European Institute of 1Ceitec, Masaryk University, Brno, Czech Republic, 2Physical Chemistry, Technology, Masaryk University Brno, Brno-Bohunice, Czech Republic, Palacky University, Olomouc, Czech Republic, 3Ceitec, Masaryk University, 3National Centre for Biomolecular Research, Faculty of Science, Brno- Brno, Czech Republic. Bohunice, Czech Republic, 4Institute of Applied Simulation, School of Life Channels (tunnels and pores) are important structural features of bio- Sciences und Facility Management, Zurich University of Applied Sciences, macromolecules intimately connected with their biological function.

BPJ 8640_8642 Monday, February 19, 2018 343a

ChannelsDB (http://ncbr.muni.cz/ChannelsDB) is a database providing infor- 1699-Pos Board B608 mation about the positions, geometry and physicochemical properties of chan- Novel Tools for Analyzing the Three-Dimensional Cellular Shape Space nels found within structures deposited in the Protein Data Bank. Channels were C. David Williams1, Julie A. Theriot2,1, Molly M. Maleckar1. deposited from two sources; from literature using manual deposition and from The Allen Inst for Cell Science1. aweb server tool MOLEonline (http://mole.upol.cz) detecting tunnels leading 1The Allen Inst for Cell Science, Seattle, WA, USA, 2Stanford University to the enzymatic active sites and selected cofactors, and transmembrane pores School of Medicine, Stanford, CA, USA. automatically within any given structure. Both database and tool gather infor- Understanding cellular organization and how it produces characteristic pheno- mation about geometrical features (e.g. length and radius profile along a chan- types and changes as cells progress through the cell cycle, differentiate, and nel) and physicochemical properties involving polarity, hydrophobicity, otherwise change state offers key insight into cells’ function. While assessment hydropathy, charge and mutability of detected channels. The stored data are in- of cell and nuclear morphology can serve as a first-line assay for overall cell terlinked with available UniProt annotation data mapping known mutation ef- organization and physiological state, existing work has primarily focused on fects to channel lining residues. All structures with channels are displayed in a the development of methods for identification of unique cell populations or clear interactive manner, further facilitating data manipulation and interpreta- comparison across tens to hundreds of 2D cell image segmentations. Enabled tion. As such, ChannelsDB and MOLEonline provide invaluable resources by a unique high-quality and high replicate 3D multichannel fluorescent micro- for research related to deciphering the biological function of bio- scopy dataset of human induced pluripotent stem cells (hiPSC), we developed a macromolecular channels. set of cell membrane and nuclear position and shape dimensionality reduction methods to understand the variation in our population. We quantified their 1697-Pos Board B606 reconstruction error and visualized population variation across a set of more Design Principles for Functionalized Surfaces than 15,000 human iPSC 3D image stacks. Each method is a two-step process, Tamara C. Bidone1, Aravind Rammohan2, Matt McKenzie3, consisting of 1) encoding the voxel segmentation as a representation (binary Gregory A. Voth1. volume, continuous convolution across such volume, or trace along segmenta- 1University of Chicago, Chicago, IL, USA, 2Corning Incorporated, Corning, 3 tion surface) followed by 2) a representation via a lower dimensional basis (us- NY, USA, Corning Research and Development Corporation, Corning, NY, ing PCA, ICA, Fourier decomposition, or latent space representation via a USA. convolutional neural net). Variation across these lower dimensional represen- When cultured on a surface, cells form micrometer-sized adhesions that con- tations of cell and nuclear position and shape is then used to explore variation nect their cytoskeleton to the external surface. Cell adhesions contain trans- in this data set, which is too large to display directly. For example, we demon- membrane integrin receptors, that directly bind ligands. Integrin binding strate interactive morphology interpolation, rendered as a real-time web depends on the density of ligands and their spatial organization, as well as browser tool, for outside user exploration of cell morphology, and can prospec- the rigidity of the surface to which the ligands are attached. Synthetic ma- tively map nuclear position and shape to cell cycle stage. These decompositions terials decorated with adhesive peptides are widely used to promote adhe- both allow exploration of the previously unknown morphology space these sions and to induce specific cellular response that depend integrin/ligand cells inhabit and permit identification of new cell classes. binding. The design of these surfaces depends on the type of used ligands and their specificity. However, how ligands with different properties inter- 1700-Pos Board B609 play with surface rigidity to determine integrin binding and adhesion forma- Exploring Deep Neural Network Architectures for Automated Electron tion is still largely unknown. Here, we developed a coarse-grained Micrograph Segmentation computational model in order to characterize regimes of integrin binding Matthew D. Guay1, Zeyad A. Emam2, Adam B. Anderson3, in response to both ligands properties and mechanics of the adhesive surface. Richard D. Leapman1. We used the model to systematically investigate how density of ligands, 1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2AMSC, their length and specificity interplay with surface rigidity in the assembly University of Maryland College Park, College Park, MD, USA, 3CMSC, of integrin adhesions. Our results allowed defining of a ‘‘phase-diagram’’ University of Maryland College Park, College Park, MD, USA. of nascent versus mature adhesions, depending on the distance separation Modern serial block face electron microscopy (EM) and related techniques are between ligand-bound integrins. Taken together, our modeling results can capable of producing teravoxel 3D images of biological structures at nanometer help in the design of functionalized surfaces for directed cell adhesion resolution, but a full analysis of these images is made impossible by the and growth. massive time requirements for manual image segmentation. Segmentation par- titions an image into labeled regions corresponding to image content, and is 1698-Pos Board B607 fundamental to many data analysis workflows in biological EM. Fast, auto- Interactive Exploration of Non-Covalent Interactions with the NGL mated segmentation algorithms would dramatically enhance biomedical re- Viewer searchers’ analytic capabilities, but practical solutions meeting stringent Alexander S. Rose1, Stephen K. Burley2,1. accuracy requirements have historically been out of reach. In recent years, 1RCSB Protein Data Bank, San Diego Supercomputer Center, University of 2 deep learning algorithms have exhibited significant performance gains for auto- California, San Diego, CA, USA, RCSB Protein Data Bank, Institute for mated segmentation problems, but it remains difficult to create high- Quantitative Biomedicine, Rutgers, The State University of New Jersey, performing deep neural networks for complex biological segmentation tasks. Piscataway, NJ, USA. To overcome this challenge our lab is developing genenet, a Python package Non-covalent interactions and intermolecular forces represent an important that simplifies the creation, training, and deployment of deep neural networks class of contacts within macromolecules, between macromolecules, and with for segmentation in TensorFlow on multi-GPU and distributed systems. At the small molecules. They are critical for folding/stability of macromolecules, Laboratory of Cellular Imaging and Macromolecular Biophysics, we use gen- stability of macromolecular assemblies, and ligand binding. They are involved enet to train the neural networks, which underlie our segmentation algorithms. in many dynamic, transitory, and enzymatic molecular processes in molecular A sufficiently accurate algorithm can be used to quickly "bootstrap" the full biology and biological chemistry. Knowledge of these interactions is crucial for segmentation of a large EM volume by first manually segmenting a smaller re- our understanding of ligand binding and is integral to structure-guided drug gion. Bootstrapping dramatically accelerates the segmentation process, allow- discovery. ing a scientist to segment much larger cell and tissue volumes within a fixed Herein, we extended the NGL Viewer (http://nglviewer.org/), our web-based time budget. Our approach to segmentation using deep learning promises to tool for molecular graphics and structure analysis, to calculate and visualize enable nanoscale imaging of biological structures across much larger spatial a wide array of non-covalent interactions, including hydrogen bonds, hydro- volumes than has previously been possible. phobic contacts, metal complexation, halogen bonds, salt bridges, cation-pi stacking, and pi-pi stacking. The calculations are performed in real-time 1701-Pos Board B610 within the web-browser and can be controlled by a set of criteria that define PerMM: Web Server and Database for Prediction of Membrane what exactly constitutes an interaction (e.g., with structures determined at Permeability and Translocation Pathways of Molecules different resolution limits). The implementation can use structural data that in- Irina D. Pogozheva, Henry I. Mosberg, Andrei Lomize. cludes chemical descriptors at different levels of completeness. For standard College of Pharmacy, University of Michigan, Ann Arbor, MI, USA. protein and DNA/RNA residues typically found in the Protein Data Bank The thermodynamics-based PerMM (Permeability of Molecules across Mem- (www.pdb.org), tables with chemical descriptors are employed. For chemi- branes) method, web server, and database were developed for quantification cally modified residues and ligands, information pertaining to bond order, of passive permeability of structurally diverse molecules across membranes. aromaticity, formal charges, and explicit hydrogen atoms is prefered whenever This method combines the universal solvation model, the atomic representation available. of solute molecules, and a description of the lipid bilayer by transmembrane

BPJ 8640_8642 344a Monday, February 19, 2018 profiles of several polarity parameters. The optimal translocation pathway of a native-like interfaces. The enhanced discrimination rendered by the new score solute was defined by calculating the lowest energy rotational orientation of the function results in an 8-fold increase in near-native docked structures after the molecule in every point of the transmembrane trajectory. The web server uses first, coarse-grained stage as compared to RosettaDock 3.2. These structures are coordinates of the molecule in PDB format as input and produces: (a) the then refined in the full-atom stage using a well-tested score function. The optimal position of a molecule in the lipid bilayer and the energy of membrane method automatically modulates backbone sampling rates to adapt to the diver- binding; (b) the free energy profile of a molecule along the membrane normal; sity and size of the ensemble. Moreover, the method is now amenable to (c) the log of the permeability coefficient to make a conclusion about the ability scaling-up; when increasing the ensemble sizes from 1 receptor and 10 ligand of a molecule to passively cross the membrane; (d) coordinates of a molecule in conformations to 100 conformations each, it takes just 20--80% longer. When different spatial positions across the membrane; and (e) interactive 3D images testing a benchmark set of 88 proteins, with statistical resampling of docked of a molecule within the lipid bilayer visualized by GLMol. This method was candidate structures, our expected success rate is 77% for rigid complexes, tested on 500 molecules ranging from small organic compounds to cyclic 49% for moderately flexible complexes, and 31% for highly flexible com- peptides, which were included in the recently created PerMM database plexes. Overall, RosettaDock 4.0 is successful for 32% more complexes than (http://permm.phar.umich.edu/) to facilitate their comparative analysis. The RosettaDock 3.2 while being up to 12-fold faster for large ensembles. Lastly, best correlation between experimental and calculated permeability coefficients for highly flexible proteins, we demonstrate that if a suitable conformer gener- was obtained for translocation across black lipid bilayers (R2=0.87 for 57 com- ation method exists, RosettaDock 4.0 can dock the complex successfully. pounds). The new method was applied to evaluate the permeability coefficients of 41 opioid peptidomimetics with high in vitro mu-agonist/delta-antagonist 1704-Pos Board B613 activity. The calculated passive permeability coefficients of these compounds Charmm-GUI Membrane Builder with Glycolipids and Lipopolysaccha- were between values for morphine and meperedine. However, only 19 com- rides 1 2 3 1 pounds demonstrated the antinociceptiveactivity invivo upon parenteral admin- Jumin Lee ,Go¨ran Widmalm , Jeffery B. Klauda , Wonpil Im . 1Lehigh University, Bethlehem, PA, USA, 2Stockholm University, istration. Other molecules were BBB-inactive, most likely due to their efflux by 3 P-glycoproteins. Stockholm, Sweden, University of Maryland, College Park, MD, USA. Glycoconjugates, such as glycolipids and lipopolysaccharides (LPS), play 1702-Pos Board B611 crucial roles in various cellular functions, so characterizing their structure Fast, Accurate pH Dependent Alchemical Free Energy Calculations and dynamics at the molecular level is essential to understand their biological Towards Rational Drug Design roles. However, systematic generation of glycolipid and LPS structures is Daniel J. Mermelstein. challenging because there are great variations in lipid acyl chains and glycan Chemistry and Biochemistry, UCSD, San Diego, CA, USA. sequences (i.e., carbohydrate types and their linkages). To facilitate the gener- Alchemical free energy calcualtions (AFE) using molecular dynamics (MD) ation of all-atom structures of various glycolipid and LPS structures, we have simulations are excellent tool to enable our understanding of a wide variety developed Glycolipid Modeler and LPS Modeler in CHARMM-GUI (http:// of biological processes and to expedite the drug design process. However, www.charmm-gui.org), a web-based interface to facilitate a building of com- computing power and theory have long been insufficient. One of the major dif- plex biological simulation systems. In addition, we have recently incorporated ficulties in performing AFE calculations is the length of time required for cal- these modules into CHARMM-GUI Membrane Builder, so that one can easily culations to converge to the accurate ensemble average. CPU implementations build a complex biological membrane system with glycolipids or a Gram- of MD based free energy algorithms have only been capable of reaching tens of negative bacterial outer membrane with LPS molecules. nanoseconds per day for systems on the order of 50,000 atoms, even running on 1705-Pos Board B614 hundreds of cores. Therefore, free energy calculations of long time scale events Gamer 2.0: Software Toolkit for Adaptive Mesh Generation from are untenable, preventing researchers from gaining crucial insight into molec- Structural Biological Datasets ular recognition and potential druggability. Graphics cards (GPUs) can change Christopher T. Lee1, John Moody2, Michael J. Holst1, this. We present here a GPU implementation of thermodynamic integration J. Andrew McCammon1, Rommie E. Amaro1. capable of reaching speeds of 120 ns/day for a 45,000 atom system, with accu- 1University of California, San Diego, La Jolla, CA, USA, 2ViaSat, San Diego, racy and precision identical to the existing CPU implementation in AMBER. CA, USA. While speed is the key component missing from the common use of alchemical Computer-aided mathematical simulation of signaling cascades and other bio- free energy, biological and chemical accuracy is missing in certain respects. logical phenomena have become increasingly popular. While some initial pH, a well-known moderator of enzymatic activity, is often neglected as a ther- models have been built using simplified geometries, the use of high-fidelity modynamic variable in molecular dynamics simulations. However, work in the geometric models from imaging data has the promise to improve simulation ac- McCammon lab has demonstrated that failure to account for protonation state curacy. GAMer (Geometry-Preserving Adaptive MeshER), an open-source can result in errors of up to 8 kcal/mol. This is significant when considering that software, was developed to generate high-fidelity mesh models from segmented the expected free energy of binding of a lead compound before optimization image data. The original implementation suffered from lack of cross-platform (when AFE would be applied) is only 12 kcal/mol. We have applied a com- support and instability. We introduce a substantial update to GAMer featuring a bined method for the high-throughput calculation of the binding free energy of complete redesign to support cross-platform use across major operating sys- two inhibitors of beta-secretase 1, the primary target in rational drug design in tems. This version also features the automated resolution of some common the fight against Alzheimer’s disease. mesh defects. We hope that GAMer 2.0 will enable faster and easier develop- ment of new models from imaging data. 1703-Pos Board B612 Efficient Flexible-Backbone Docking of Challenging Protein Complexes 1706-Pos Board B615 Shourya S. Roy Burman1, Nicholas A. Marze1, William Sheffler2,3, ‘Martinizing’ the Variational Implicit Solvent Method (VISM): Solvation Jeffrey J. Gray1,4. Free Energy for Coarse-Grained Proteins 1Chemical and Biomolecular Engineering, Johns Hopkins University, Clarisse Gravina Ricci1,BoLi2, Li-Tien Cheng3, Joachim Dzubiella4, Baltimore, MD, USA, 2Department of Biochemistry, University of J. Andrew McCammon1. Washington, Seattle, WA, USA, 3Institute for Protein Design, University of 1Pharmacology, UCSD, San Diego, CA, USA, 2Department of Mathematics, Washington, Seattle, WA, USA, 4Program in Molecular Biophysics, Johns UCSD, San Diego, CA, USA, 3Mathematics, UCSD, San Diego, CA, USA, Hopkins University, Baltimore, MD, USA. 4Institut fur€ Physik, UCSD, Humboldt-Universit€at zu Berli, CA, USA. Computational prediction of protein-protein complex structures facilitates a Solvation is a fundamental driving force in many biological processes fundamental understanding of biological mechanisms and enables drug design. including biomolecular recognition and self-assembly, not to mention protein Binding-induced backbone variations challenge all current computational folding, dynamics, and function. Many computational methods attempt to pro- docking algorithms by exponentially increasing the degrees of freedom. A vide an accurate description of the solvation effects underlying such processes. promising approach to explore relevant backbone space is to simulate The Variational Implicit Solvent Method (VISM) is currently developed to conformer selection during the formation of the encounter complex. Previous estimate solvation free energies for systems of very complex topology, such methods employing this approach have found success by sampling a small as biomolecules. VISM’s theoretical framework makes it unique because it number of conformations with limited interface deviations. For highly flexible couples hydrophobic, van der Waals and electrostatic interactions as a func- proteins, a docking protocol must accommodate large ensembles and efficiently tional of the solvation interface. By minimizing this functional, VISM produces identify near-bound conformations. In this study, we present RosettaDock 4.0, the solvation interface as an output of the theory, and is able to capture subtle a computational docking method that rapidly samples hundreds of backbone though important behavior deriving from heterogeneous solvation patterns - conformations and uses a novel, coarse-grained score function to select such as polymodal hydration and dewetting. In this work, we adapted VISM

BPJ 8640_8642 Monday, February 19, 2018 345a to work with MARTINI, a well-established coarse-grained force field for pro- (SCFT) is used to design the system by incorporating a Wormlike Chain model teins and other biomolecules, whose application is particularly interesting to to model the polymer chain rigidity. This field theoric model properly accounts study the interaction between very large biomolecular systems. We applied for the highly non-additive coupling of molecular interactions among all the the MARTINI-VISM method to investigate the nature of dry-wet transitions species at different biological solution environments. The structural, thermody- in the encounter of two binding systems which differ greatly in terms of elec- namic and electrostatic properties of the polyelectrolyte, drug and solvent are trostatic/hydrophobic character: barstar-barnase and p53-MDM2. Our results taken into account at the molecular level. Polyelectrolyte-drug binding is demonstrate the suitability of the MARTINI-VISM method to study important accommodated with a ligand-receptor binding mechanism. This model explic- solvation effects mediating protein-protein binding, which cannot be captured itly captures the change in structure and charge regulation properties of the by more traditional implicit solvent methods. polyelectrolyte chains with varying pH, salt concentration, grafting density and length of the polyelectrolyte chains. Results of the theory suggests a domi- 1707-Pos Board B616 nance of electrostatic charge regulation-dysregulation on the extent of drug Fast Implicit Potentials for Accurate Prediction and Design of Membrane binding to the polyelectrolyte. Possible improvements in the system can be Protein Structures made by the addition of other polyelectrolytes with certain desired properties. 1 2 2 1 Rebecca F. Alford , Patrick Fleming , Karen G. Fleming , Jeffrey J. Gray . This model can be used as a generic tool to design and improve polymer medi- 1Chemical and Biomolecular Engineering, Johns Hopkins University, 2 ated biomedical methodologies by providing valuable insights towards the vari- Baltimore, MD, USA, Biophysics, Johns Hopkins University, Baltimore, ation of system parameters. MD, USA. Membrane proteins partner with the lipid environment to perform key functions 1710-Pos Board B619 including signaling, transport, and catalysis. They constitute 30% of all proteins Detrending: How to Correct Images for Bleaching and are targets for over 50% of pharmaceuticals on the market. However, our Rory Nolan, Luis Alvarez, Sergi Padilla-Parra. ability to predict and design membrane protein structures has been hindered by Wellcome Centre Human Genetics, University of Oxford, Oxford, United experimental difficulties and limited computational resources. Specifically, Kingdom. computing pair potentials between atoms in the protein and the chemically Image series is affected by bleaching must be corrected by ’detrending’ prior diverse lipid bilayer is computationally expensive in both molecular dynamics to the performance of quantitative analysis. There are several detrending and Monte Carlo approaches. In this work, we developed a fast implicit poten- methods (polynomial detrend, running average, exponential smoothing). tial that accounts for the geometry and continuum properties of the membrane. We assess which methods are best, using examples from fluorescence First, we developed a strategy to fit the implicit membrane thickness and fluctuation spectroscopy. We outline mistakes that are often made when de- aqueous pore given the current protein conformation. Next, we incorporated trending. We provide a free, user-friendly software for correctly detrending new terms based on experimental data for water-to-bilayer partitioning energy images. (Moon & Fleming), electrostatics (MD Simulation), solvation, and pH effects. Finally, we tested the method using a large benchmark set which includes three energy landscape sampling tests and eight validation tests with experimental data. Preliminary testing suggests that our method outperforms current implicit Posters: Optical Microscopy and Superresolution membrane models in Rosetta. Imaging: Applications to Cellular Molecules I 1708-Pos Board B617 Simulation of Brownian Dynamics on a Curved Surface 1711-Pos Board B620 Kenji Kimura, Yasuhiro Inoue. Counting Single Molecules with Localization Microscopy Inst. Front. Life Med. Sci., Kyoto Univ., Kyoto, Japan. Joshua Milstein. In this study, we developed a particle-based simulation to reproduce the Chemical and Physical Sciences, University of Toronto, Mississauga, ON, Brownian dynamics of particles on a curved surface, and applied the method Canada. to investigate their diffusion on a biological membrane. The mean squared Cell biology is becoming increasingly quantitative with advances in light mi- displacement of Brownian particles in a Euclidean space increases with croscopy strongly driving this trend. Beyond imaging structure, significant time, but characterizing diffusion in a non-Euclidean space, such as on a effort has gone into developing microscopy based approaches to determining curved surface, has remained elusive. In recent years, theoretical studies the abundance of proteins and nucleic acids in cells. Molecular counting exper- have shown that the time evolution of the mean squared geodesic distance iments can yield additional insight into cellular structure and define the stoichi- of a particle diffusing due to Brownian motion on a curved surface depends ometry of interacting protein complexes. Moreover, since microscopy provides on its Gaussian curvature. This suggests that the shape of a cellular membrane information at the single-cell level, it may be used to study stochastic variation may change the molecular diffusion kinetics occurring on it. Therefore, in this within a population due to varying levels of mRNA and protein copy number, study, we developed a particle-based simulation of Brownian dynamics in which is inaccessible to bulk techniques. Single-molecule localization micro- which diffusion depends on the surface’s Gaussian curvature, and investigated scopy (SMLM) has the potential to serve as an accurate, single-cell technique the molecular diffusion dynamics on a biomembrane with a complicated for counting the abundance of intracellular molecules. However, the stochastic shape. In this method, during each computational time step, a particle is blinking of single fluorophores can introduce large uncertainties into the final considered to move by projecting its coordinates onto the nearest curved sur- count. Here we provide a theoretical foundation for applying SMLM to the face represented by triangular meshes, a key step enabling the simulation. problem of molecular counting based on the distribution of blinking events Model results using this method agree well with theoretical predictions. We from a single fluorophore. then applied this method to demonstrate how molecular diffusion on a curved surface is affected by cellular shape in terms of the first passage time of the 1712-Pos Board B621 molecules. Multicolor Spatial Intensity Distribution Analysis of Laser Scanning Microscopy Images to Study Dopamine Receptor Dynamics 1709-Pos Board B618 Daniel J. Foust1, Antoine G. Godin2, Alessandro Ustione1, Study of Polyelectrolyte-Small Molecule Drug Binding with Wormlike Paul W. Wiseman3, David W. Piston1. Chain Model 1Cell Biology and Physiology, Washington University School of Medicine, Merina Jahan, Mark J. Uline. St. Louis, MO, USA, 2Physics, McGill University, Montreal, QC, Canada, Chemical Engineering, University of South Carolina, Columbia, SC, USA. 3Physics and Chemistry, McGill University, Montreal, QC, Canada. Rational design of polymeric materials has created new horizon in the field of Fluorescence fluctuation spectroscopy (FFS) encompasses a set of statistical drug discovery and many other biomedical applications. Fundamental under- tools for determining the physical characteristics of emitters underlying fluores- standing of the structure and properties of these materials by using theories cent signals measured by photodetectors. In biology, FFS is commonly used to of statistical thermodynamics is crucial for novel drug design. Understanding measure concentration, aggregation, and dynamics of fluorescently labeled of the underlying physics of polymer behavior in different biological environ- molecules. Molecular brightnesses measured by FFS can be used to detect olig- ments has the potential to tremendously help experimental researchers to syn- omerization of fluorescent species when the brightnesses of monomeric con- thesize novel materials with specific physiochemical trait. This study stems trols are known. Membrane proteins are especially challenging for study by from the urgency to design a sustained and controlled drug delivery system FFS because their slow diffusion can make it difficult to observe a statistically for diseases that demand frequent dosing of drugs in high concentration. Poly- significant number of fluctuations before photobleaching perturbs the sample. electrolytes end-tethered to a nano-particle surface are used as carrier for a One strategy for studying slow processes is to use imaging approaches rather small molecule drug in this system. A Self Consistent Mean Field Theory than measurements from a fixed point to avoid local photobleaching effects

BPJ 8640_8642 346a Monday, February 19, 2018 and observe more fluctuations in the same time interval. Spatial intensity dis- mouse model of emphysema, the use of OPT enables quantitative imaging of tribution analysis (SpIDA) was developed to adapt analysis of the photon relevant proliferative cell lineage in cleared, large murine lung samples. The counting histogram (PCH) for laser scanning microscopy images to determine volume and distribution of alveolar cell clusters which have been labelled by molecular brightnesses. We have extended SpIDA for multicolor analysis that immunofluorescence are measured in the intact lung of a mouse and visualized allows for sensitive detection of heteromeric fluorescent species. Optical sys- in 3D. Our design is easy to implement and is completely open-source, tems in FFS typically encode spectral information by using dichroic filters to providing opportunities for further open microscopy developments for large direct emitted light onto two or more photodetectors. In contrast, our imaging organ imaging in model organisms. system (Zeiss LSM 880) uses a diffraction grating to disperse emitted light onto an array of 32 PMTs. The increased spectral resolution afforded by this geom- 1715-Pos Board B624 etry has the potential to be powerful in FFS, however new challenges are intro- Flat-Field Illumination Microscopy for Large Field-of-View Quantitative duced including intrinsic crosstalk between adjacent detectors due to their Imaging physical proximity. We introduce an approach for addressing these non-ideal Ian Khaw, Benjamin Croop, Kyu Young Han. detector effects. Multicolor SpIDA with non-ideal detector corrections is CREOL, University of Central Florida, Orlando, FL, USA. employed to study the interactions of D2-like dopamine receptors with Fluorescence imaging is a powerful tool for understanding of molecular mech- signaling partners including G-proteins and G-protein coupled inwardly recti- anisms of cellular processes and functions. It is desirable to have a robust fying Kþ (GIRK) channels. imaging method that enables us to obtain quantitative information. However, in laser-based fluorescence imaging, the nonuniform illumination distribution 1713-Pos Board B622 by the Gaussian-shaped beam results in severe problems for quantitative Spatially Selective Dissection of Signal Transduction in Neurons Grown on analysis of images. Additionally, it limits the field-of-view and leads to rapid NETRIN-1 Printed Nanoarrays via Segmented Fluorescence Fluctuation photobleaching at the center of the beam. The solution for the uneven distribu- Analysis tion is to have an even illumination beam. The method proposed is the reshaping Angelica A. Gopal1, Sebastien G. Ricoult2, Stephanie N. Harris3, of the Gaussian illumination profile into a Flattop illumination profile by using David Juncker2, Timothy E. Kennedy3, Paul W. Wiseman1,4. refractive optics. Our results showed that the flat-field illumination yielded far 1Department of Chemistry, McGill University, Montreal, QC, Canada, more leveled intensities and much wider field-of-view compared to Gaussian 2Department of Biomedical Engineering, Genome Quebec Innovation profile. We demonstrated epi- and TIRF illumination with multiple wavelengths. Centre, McGill University, Montreal, QC, Canada, 3Department of Our method is likely to combine with high-throughput imaging as well. Neurology and Neurosurgery, Montreal Neurological Institute, McGill 4 1716-Pos Board B625 University, Montreal, QC, Canada, Department of Physics, McGill Multicolor Two-Photon Fluorescence Lifetimes Microscopy by Wave- University, Montreal, QC, Canada. lenght Mixing for Efficient and Simultaneus NADH and FAD Imaging Axonal growth cones extend during neural development in response to precise Reveals Metabolic Shifts Associated to Cellular Differentiation and Oxida- distributions of extracellular cues. Deleted in colorectal cancer (DCC), a recep- tive Stress in Living Tissues tor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, Chiara Stringari, Emmanuel Beaurepaire. and is essential for mammalian neural development. To elucidate how the Ecole Polytechnique, Laboratory for Optics and Biosciences, Palaiseau, extracellular distribution of netrin-1 influences the distribution of DCC and France. F-actin within axonal growth cones, we patterned nanoarrays of substrate Multiphoton imaging of metabolic coenzymes nicotinamide adenine dinucleo- bound netrin-1 using lift-off nanocontact printing. The distribution of DCC tide (NADH) and flavin adenine dinucleotide (FAD) is usually hampered by and F-actin in embryonic rat cortical neuron growth cones was then imaged us- sequential dual wavelength excitation resulting in motion artifacts in dynamic ing total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluc- tissues. tuation analysis via image cross-correlation spectroscopy (ICCS) was applied Here we implement multicolor two-photon Fluorescence Lifetime microscopy to extract the molecular density and aggregation state of DCC and F-actin, by combining wavelength mixing and efficient Fluorescence Lifetime micro- identifying the fraction of DCC and F-actin colocalizing with the patterned scopy (FLIM) to simultaneously and efficiently image NADH and FAD. Pulse netrin-1 substrate. ICCS measurement of spatially segmented images based trains from two laser beams are synchronized to give rise to two-color two- on the substrate nanodot patterns revealed distinct molecular distributions of photon excited fluorescence through a virtual wavelength. We demonstrate F-actin and DCC in regions directly overlying the nanodots compared to simultaneously co-registered NADH and FAD images of comparable intensity, over the reference surface surrounding the nanodots. Quantifiable variations one shot ratiometric redox (FAD/(NADHþFAD)) imaging free of motion arti- between the populations of DCC and F-actin on and off the nanodots reveal facts and simultaneous two-photon FLIM of NADH and FAD. specific responses to the printed protein substrate. We report that nanodots of In living tissues we measure distinctive NADH and FAD lifetime gradients substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin associated with cellular differentiation states and an increase in NADH and organization. These effects were blocked by tetanus toxin, consistent with FAD lifetime associated to oxidative stress induced by UVA light exposure. netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2- We show that cellular heterogeneity in the ratiometric redox ratio (FAD/ dependent mechanism. Our findings demonstrate the utility of segmented (NADHþFAD)) and FAD lifetime is different with respect to NADH lifetime ICCS image analysis, combined with precisely patterned immobilized ligands, distribution demonstrating that that these three parameters provide complemen- to reveal local receptor distribution and signaling within specialized subcellular tary information on the metabolic pathways and allow to identify unique compartments. metabolic cellular phenotypes. 1714-Pos Board B623 1717-Pos Board B626 Open-Source Optical Projection Tomography of Large Organ Samples Long-Term Super-Resolution Imaging of Amyloid Structures using Tran- Pedro P. Vallejo Ramirez1, Joseph Zammit2, Bogdan Spiridon3, sient Binding of Standard Amyloid Probes Fergus Riche4, Florian Stroehl1, Romain F. Laine1, Clemens F. Kaminski1. Kevin Spehar1, Tianben Ding2, Yuanzi Sun1, Jin Lu2, George R. Nahass1, 1Chemical Engineering and Biotechnology, University of Cambridge, Matthew D. Lew2, Jan Bieschke1. Cambridge, United Kingdom, 2Computer Laboratory, University of 1Biomedical Engineering, Washington University, Saint Louis, MO, USA, Cambridge, Cambridge, United Kingdom, 3Center for Advanced Photonics 2Department of Electrical and Systems Engineering, Washington University, and Electronics, University of Cambridge, Cambridge, United Kingdom, Saint Louis, MO, USA. 4Department of Engineering, University of Cambridge, Cambridge, United Oligomeric amyloid structures are crucial therapeutic and diagnostic targets in Kingdom. Alzheimer’s disease and other amyloid diseases. However, these oligomers are The study of biological mesoscopic samples (those with characteristic dimen- too small to be resolved by conventional light microscopy. We have developed sions of 1-10 mm) benefits from techniques that can analyse and depict anatom- a new tool to image amyloid structures on a nanometer scale using standard am- ical information on a whole organism level. Optical Projection Tomography yloid dyes such as Thioflavin T (ThT), without the need for covalent labeling of (OPT) uses visible light to generate 3D imaging data of transparent mesoscopic the amyloid protein or staining via fluorescently labeled antibodies. Transient objects (e.g. cleared mouse embryos) at micron-level resolution in both fluores- amyloid binding (TAB) imaging is compatible with epifluorescence and cence and transmission. We present a low-cost OPT solution to image large TIRF microscopies and uses 488 nm cw laser excitation to excite ThT mole- transparent specimens (up to 17 x 14 mm) at a lateral resolution of 23 um cules that are bound to amyloid structures. Dynamic binding and unbinding and provide an extensive open-source library for image calibration, reconstruc- of ThT molecules generate photon bursts (‘blinking’) that are used for single tion, and noise-reduction. In the development of treatment which induces fluorophore localization at nanometer resolution. Thus, photobleaching cannot alveolar cell proliferation and repair in the porcine pancreatic elastase (PPE) degrade either the number or brightness of blinking events, which enables us to

BPJ 8640_8642 Monday, February 19, 2018 347a image the same structure for multiple days without loss of image quality. As 1720-Pos Board B629 proof-of-principle experiments, we imaged oligomeric and fibrillar structures A Liquid Tunable Microscope as a New Paradigm in Optical Microscopy formed during different stages of amyloid-b aggregation as well as the struc- to Paint 4D Chromatin Organisation in the Cell Nucleus tural remodeling of amyloid fibrils by the anti-amyloid compound epi- Alberto Diaspro1,2, Isotta Cainero1,2, Luca Lanzano`1, Paolo Bianchini1, gallocatechin gallate (EGCG). TAB promises to directly image native amyloid Giuseppe Vicidomini1, Francesca Cella Zanacchi1, Luca Pesce1,2, in cells and tissues using standard probes at nanometer resolution and at the Simone Pelicci1,2, Michele Oneto1, Melody Di Bona1,2, Mario Faretta3, same time record amyloid dynamics over time scales of minutes to days. Paola Barboro4, Aymeric Le Gratiet1. 1IIT - Italian Institute of Technology, Genoa, Italy, 2Department of Physics, 1718-Pos Board B627 University of Genoa, Genoa, Italy, 3Department of Experimental Oncology, Quantitative Phase Imaging Biological Applications using Quadriwave European Institute of Oncology, Milan, Italy, 4IRCCS Azienda Ospedaliera Lateral Shearing Interferometry Universitaria San Martino, Genoa, Italy. Sherazade Aknoun1, Antoine Federici1, Flor A. Medina2, Pierre Bon3, The possibility of integrating different light-matter interaction ways for produc- Julien Savatier3, Benoit Wattellier1, Serge Monneret3. 1 2 ing images in optical microscopy is the starting point for the design and reali- PHASICS, Saint Aubin, France, PHASICS, San Francisco, CA, USA, zation of a brand new optical microscope that has been named ‘‘liquid tunable 3 Institut Fresnel, Aix-Marseille Universite, Marseille, France. microscope’’. This represents a new paradigm in data collection and image for- Quantitative phase imaging (QPI) techniques are now commonly used in mi- mation with a potential high impact in biophysics. It is designed with the aim of croscopy for imaging of semi-transparent samples as they provide their optical bringing a contribute to answer a key question in cellular and molecular path difference (OPD). In addition, those techniques are non-invasive imaging biology: what are the local and global 4D (x,y,z,t) chromatin structures in modalities and ensure a fast approach. We propose the use of a commercial the nucleus that rule the compaction and function of the human genome in quadri-wave lateral shearing interferometer (QWLSI) device (SID4Bio, Pha- the interphase cells and mitotic chromosomes? We aim to achieve: 1) the real- sics) directly plugged onto a lateral video port of a classical inverted micro- ization of a liquid microscope in the sense of allowing multiple contrasts avail- scope using a standard illumination source. Single-shot measurements with able in the image formation process including a label-free one; 2) the painting, sub-nanometric OPD precision is achieved along with a diffraction-limited at single cell level, of a 4D quantitative vision of those organizational motifs lateral resolution and a true video rate permitting intracellular components that today are not completely demonstrated about chromatin organization; 3) detection and dynamic follow-up. We will introduce several advanced applica- an optimal strategy for correlating images produced by different approaches tions of our technique that take advantage of its high OPD resolution, its ach- from multiphoton to confocal microscopy, from expansion to light sheet micro- romaticity and its easy implementation on conventional setups. Firstly, we will scopy and from super-resolved fluorescence to label-free approaches. The show that the high-contrast and artifacts-free images produced by our QWLSI S(1,4) element of the Mueller matrix, named CIDS (Circular Intensity Differ- device enable a precise and automatic cellular segmentation from which we can ential Scattering), will provide a label-free scouting of nuclear regions with monitor cell cycle over an entire population with a cellular resolution as well as different chromatin-DNA organizational motifs. Our interest is related to imag- a quantitative determination of the cell dry mass. ing the physiological chromatin organization and potentially pathological Then, we will present multimodal imaging, combining phase measurements changes induced by DNA damage, DNA damage response and repair focusing with fluorescence and polarization signals. While QWLSI detects cells on: i) the possible interference of transcription and replication processes during morphology and can lead to refractive index measurement, fluorescence signal oncogene-induced replicative stress, ii) the expression and activity of PARP1 in is related to specific components such as cells skeleton or DNA for example. In prostate cancer and iii) the Hutchinson-Gilford progeria syndrome linked mu- the other hand, QWLSI can be also used for applications such as ordered fibrous tations in the LMNA. structures imaging in biological samples like cytoskeleton fibers or collagen us- ing polarized light. Anisotropic specificity is thus detected and leads to 1721-Pos Board B630 contrast-enhanced images. New Insights into the Antimicrobial Mechanism of Silver Ions Revealed by Finally, three-dimensional samples reconstruction with intracellular resolution Super-Resolution Fluorescence Microscopy combining QWLSI and low-spatially coherent illumination will also be pre- Prabhat Khadka, Venkata Rao Krishnamurthi, Meaad Alqahtany, sented as well as temperature mapping around nano- and microsources of Yong Wang. heat, like gold nanoparticles, via the measurement of the thermal-induced vari- Dept of Physics, University of Arkansas at Fayetteville, Fayetteville, AR, ation of the refractive index. USA. Silver compounds have been found to show antimicrobial activities against 1719-Pos Board B628 bacteria, making them a promising alternative to antibiotics. However, sCMOS Noise Correction Algorithm for Microscopy Images although exciting progresses have been made, the molecular mechanism of Sheng Liu, Michael J. Mlodzianoski, Zhenhua Hu, Yuan Ren, the antimicrobial effect of silver ions (Agþ) remains a subject of debate. In Kristi McElmurry, David A. Miller, Karl F. Ziegler, Paula-Marie Ivey, this work, we tested a hypothesis that was suggested by TEM imaging of bac- Donghan Ma, Daniel M. Suter, Fang Huang. teria – Agþ causes DNA condensation – using super-resolution fluorescence Purdue University, West Lafayette, IN, USA. microscopy in combination with biochemical assays. By examining, in vitro, Scientific CMOS (sCMOS) cameras are rapidly gaining popularity in life sci- the size of bacterial genomic DNA in the absence and presence of Agþ,we ence for its significant improvement in imaging speed, field of view and sensi- found that exposure to Agþ does not directly result in DNA condensation. tivity over traditional cameras such as CCD and EMCCD. However, sCMOS Instead, spatial reorganization of histone-like nucleoid structuring (H-NS) pro- camera introduces pixel-dependent noise that generates image artifacts and tein was observed in vivo using super-resolution fluorescence microscopy, biases in quantification. Previously by considering the pixel dependent statis- which is expected to cause DNA compaction and condensation. It was also tics, precise and accurate localization of single molecules was achieved. How- found that the expression of H-NS was enhanced in the presence of Agþ.In ever, this correction works exclusively on images with point objects such as in addition, we varied the treatment time and concentration of Agþ, from which single molecule studies. No general algorithm that works on arbitrary micro- we obtained the quantitative dose- and time-dependence. Our data showed that scopy images exists. To address this issue, we developed an algorithm that uti- the previous hypothesis is incorrect, or at least incomplete. Our results suggest lizes a common property of microscopy images, the optical transfer function a new mechanism for Agþ’s antimicrobial activities. (OTF), and dramatically reduces sCMOS noise from microscopy images with arbitrary structures. We show that the new algorithm allows the camera’s 1722-Pos Board B631 performance approaching that of an ideal camera. We demonstrated the perfor- Direct Visualization of Lipoprotein Mediated Cholesterol Transport at the mance of the new algorithm in fluorescence imaging of peroxisome membrane Phospholipid Bilayer Interface protein and end-binding protein that were tagged with tdEos, and F-actin Birgit Plochberger1, Markus Axmann2, Erdinc Sezgin3, Johannes Preiner1, tagged with SiR-actin. We notice that the high variance pixels, the hallmark Andreas Karner1, Clemens Ro¨hrl2, Michael D. Brodesser1, feature of sCMOS images, are absent throughout the entire field of view. Christian Eggeling3, Gerhard J. Schutz€ 4, Herbert Stangl2. The developed algorithm can generally be applied to sCMOS-based detection 1Medical Engineering, University of Applied Sciences Upper Austria, Linz, and quantitative analysis in a broad spectrum of microscopy techniques, for Austria, 2Institute of Medical Chemistry, Medical University Vienna, Vienna, example, light-sheet microscopy (LSM), total internal reflection fluorescence Austria, 3Institute of Molecular Medicine, University of Oxford, Oxford, microscopy (TIRF), fluorescence resonance energy transfer microscopy United Kingdom, 4Institute of Applied Physics, TU Wien, Vienna, Austria. (FRET), and speckle microscopy. We hope that these fields can now benefit It is still unclear how lipids are removed from the circulation and transferred from the increased quantum efficiency, field of view and imaging speed of from lipoproteins– the main carriers of cholesterol in the blood stream – to sCMOS camera without compromising its quantitative detection. cells. Low density Lipoprotein (LDL)-uptake is facilitated mainly via

BPJ 8640_8642 348a Monday, February 19, 2018 receptor-mediated endocytosis in clathrin-coated pits but can also deliver its The simple optical setup needs no spectrometers, beam splitters or channel lipid cargo by selective transfer without degradation of the LDL particle. registration. Briefly, antibodies are pre-conjugated to a ‘protector’ strand of Whereas, High Density Lipoprotein (HDL) particles are captured from the ssDNA. After cellular labeling and before imaging, a complementary ‘tem- blood stream by the corresponding receptor - the subsequent lipid-transfer pro- plate’ strand, which is labeled with the dye, is added to the sample to bind cess, however, is highly speculative. and label the protector. After imaging, a third ‘invader’ ssDNA is introduced Here we used high-end techniques, namely combined atomic force (AFM) and that gains a toe-hold on the template, and competes it off the antibody- single molecule fluorescence microscopy together with high speed AFM (HS- protector complex. Orthogonal sets of protector\template\invader are used to AFM) and fluorescence cross correlation spectroscopy (FCCS), to address the label different cellular structures. All antibody labeling is done at once, before different transfer behaviour of lipids out of an LDL or HDL particle into a sup- imaging. Both the template binding and invader action only require about 1 ported lipid membrane and their interaction kinetics. minute, allowing quick progression through the different structures. We Combined force and single molecule fluorescence microscopy allowed us to demonstrate the concept by performing dSTORM imaging on several different directly monitor the transfer process of amphiphilic fluorescent probe lipids proteins in a single cell. The concept is easily extended to >10 proteins and from lipoprotein particles to the lipid bilayer upon contact and their integration could be used for other SR techniques such as STED and SIM. into the lipid bilayer was seen. FCCS experiments indicate that Cholesterol was freely mobile in the GUV membrane, additionally two color FCS revealed the 1725-Pos Board B634 absence of co-diffusion of the two species, indicating that cargo was transferred Nanoscopy with Multiple Off-States 1,2 2 2 2 and separated from the particle. With the aid of HS-AFM we are able to achieve Johann G. Danzl , Sven Sidenstein , Carola Gregor , Nicolai Urban , Peter Ilgen2, Stefan Jakobs2, Stefan W. Hell2. nanoscale spatial resolution together with real-time temporal resolution. Inter- 1 2 estingly, on mica-supported DOPC membrane LDL in contrast to HDL was IST Austria, Klosterneuburg, Austria, MPI for Biophysical Chemistry, strongly interacting with the surface beneath by repetitive fusion and release. Goettingen, Germany. Taken together, in synthetic membranes, particle contact and incorporation Coordinate-targeted super-resolution techniques distinguish fluorophores into the lipid bilayer are sufficient requirements for cargo delivery. Whereby, within a diffraction-limited zone by differing molecular states (typically on the interaction kinetics and the integration of hydrophobic lipoprotein moieties vs. off) that are imprinted with light. In STED microscopy, state contrast is in the core of the lipid bilayer strongly depends on the lipoprotein species. generated via stimulated emission. For strong spatial confinement, this requires high STED light intensities. Here we show that enhanced state contrast can be 1723-Pos Board B632 achieved by combining multiple off-state transitions, specifically by combining SPLIT-STED Imaging of Nuclear Structures stimulated emission with reversible photoswitching in ‘‘protected STED’’. This Luca Lanzano’1, Maria J. Sarmento1, Lorenzo Scipioni1, Michele Oneto1, directly translated into improved resolution and contrast while fluorophores Simone Pelicci1,2, Melody Di Bona1,2, Luca Pesce1,2, Mario Faretta3, were protected from photobleaching by high STED light intensities by virtue Laura Furia3, Gaetano I. Dellino3, Pier G. Pelicci3, Paolo Bianchini1, of an additional off-state (J. G. Danzl, S. C. Sidenstein et al., Nature Photonics Alberto Diaspro1,2. 10, 122 (2016)). It enabled repeated imaging and 3D-diffraction unlimited im- 1Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of aging of living cells and tissues with a genetically encoded marker. 3 Physics, University of Genoa, Genoa, Italy, Department of Experimental 1726-Pos Board B635 Oncology, European Institute of Oncology, Milan, Italy. Multicolor Imaging Based on Interferometric Information in 4Pi Single- The fact that the size of many molecular complexes is in the range of 10 to Molecule Switching Nanoscopy 100nm has generated a growing interest for the development of super- Kevin Hu1, Yongdeng Zhang2, Joerg Bewersdorf1. resolved fluorescence microscopy techniques. Optical super-resolution is 1Department of Biomedical Engineering, Department of Cell Biology, Yale generally achieved by switching on-off the fluorophores by stochastic or tar- University, New Haven, CT, USA, 2Department of Cell Biology, Yale geted approaches [1]. For instance, in stimulated emission depletion (STED), University, New Haven, CT, USA. excited fluorophores located on the periphery of the Point Spread Function By utilizing a dual-objective setup in single-molecule switching nanoscopy (PSF) are completely switched off by a doughnut-shaped beam, producing an (SMSN; e.g. PALM, STORM or FPALM), it is possible to obtain three- effective PSF of smaller size. Recently, we have introduced an alternative strat- dimensional (3D) images at 10 to 20 nm resolution. We have recently expanded egy for super-resolution imaging, called separation of photons by lifetime tun- this technology to the imaging of thick cells through the addition of deformable ing (SPLIT), based on the separation of the position-dependent fluorophore mirrors in each arm of the 4Pi-interferometric cavity and new image processing dynamics rather than fluorophore switching [2]. We demonstrated that the anal- algorithms, significantly increasing the application range. ysis of lifetime variations generated in a CW-STED microscope produces a Here we present a novel scheme for simultaneous multicolor imaging in whole- tunable, subdiffraction PSF that can be used for imaging [2] and for measuring cell 4Pi-SMSN (W-4PiSMSN). This filter-free approach extracts the color- molecular diffusion [3]. information from the interference pattern of the single-molecule fluorescence Here, we apply the SPLIT method to perform nanoscale imaging of nuclear by taking advantage of the wavelength-dependence in the interference inten- structures for their structural and functional characterization. We show that sities. We demonstrate that this approach can be used to separate two or SPLIT offers some advantages compared to other super-resolution methods. more emission spectra in the phase space, allowing for probe identification First, the analysis of dynamics provides a direct, calibration-free estimation without any modifications to the beam path. With the example of Alexa Fluor of the improvement of resolution provided by the STED microscope. Second, 647 and CF 680, both of which are compatible with SMSN, we show a quan- the phasor plot provides a simple graphical tool to predict the effective spatial titative analysis of the dependences between obtainable spatial resolution, resolution achievable in the SPLIT image, also in presence of noise. Finally, the cross-talk and coherence length. tunable PSF allows collecting images of nuclear foci at variable spatial resolu- Our new approach represents a novel path to extract color-information from tion, which ultimately leads to a robust estimation of their size. As an example SMS super-resolution data. of this concept, we use the tunable PSF of SPLIT to measure the size of tran- scription foci in MCF10A cells. 1727-Pos Board B636 [1] Hell, Science (2007). Super-Resolution Fluorescence Microscopy of Protein Association and [2] Lanzano` et al, Nat Commun (2015). Higher-Order Structure [3] Lanzano` et al, Nat Commun (2017). Adriano Vissa, Maximiliano Giuliani, William S. Trimble, Peter K. Kim, [Work partly funded by AIRC-Cariplo TRIDEO#17215]. Christopher M. Yip. University of Toronto, Toronto, ON, Canada. 1724-Pos Board B633 The interaction and spatial organization of proteins within cells fuels a myriad Multi-Structure Super-Resolution Imaging using DNA Strand of biological processes. Protein modification, transport, signaling, polymeriza- Displacement tion and membrane remodeling are just a few examples. In cellular pathways, Diane S. Lidke1, Cheyenne Martin1, Farzin Farzam2, Jeremy S. Edwards2, such as the endocytic membrane transport pathway, the recruitment of specific Matthew Lakin3, Sandeep Pallikkuth2, Keith A. Lidke2. proteins and protein complexes to the membrane are hallmarks of the matura- 1Pathology, University of New Mexico, Albuquerque, NM, USA, 2Physics tion and progression of the process. Interaction of membrane proteins from and Astronomy, University of New Mexico, Albuquerque, NM, USA, different organelles is of great importance as well. Recent discoveries have 3Computer Science, University of New Mexico, Albuquerque, NM, USA. suggested that organelles may communicate with each other and exchange We describe a simple new method for super-resolution of imaging of many lipids and metabolites through the association of tethering proteins. Their pro- different structures/proteins in the same cell using a sequential imaging strategy liferation may also be directly dependent on inter-organelle cooperativity such where one of the best dSTORM dyes (Alex647), is used to image all structures. as the interaction of the endoplasmic reticulum with mitochondria and

BPJ 8640_8642 Monday, February 19, 2018 349a peroxisomes. Protein polymerization allows the creation of large macromolec- reduces the fluorescence of quenched imager strands to negligible levels. We ular foundations that are mediators of diffusion and compartmentalization and characterise the use of Quencher-Exchange-PAINT with synthetic, cellular give the cell structure through scaffolding. The organizational motifs by which and thick tissue samples. Quencher-Exchange-PAINT opens the way for effi- these assemblies are built are of interest for structure-function relationships. cient multiplexed imaging of complex nanostructures and in thick tissues The interaction and association of protein complexes as described above takes without the need for washing steps. place at the nanoscale, below the optical diffraction limit. Although the coloc- alization of proteins can been visualized by traditional forms of fluorescence 1730-Pos Board B639 microscopy, many biological questions require a finer level of resolution. Imaging of Human Subcutaneous Adipose Tissue Reveals Insulin Super-resolution fluorescence microscopy has revolutionized optical imaging Refractive and Responsive Populations by harnessing advances in hardware, software and the modulation of light. Var- Chad D. McCormick, Ludmila Bezrukov, Hang Waters, iations in illumination and emission can be used to break the diffraction limit Ginikanwa Oneyekaba, Jordan Levine, Shahzaib Khan, Paul Blank, and observe a new level of detail in the cell. Structured Illumination Micro- Andrew Demidowich, Jack Yanovski, Joshua Zimmerberg. scopy and Single-Molecule Localization Microscopy are used to probe ques- NICHD, NIH, Bethesda, MD, USA. tions of nanoscale protein interactions in the endomembrane system and Insulin resistance, the lack of a response in tissues like adipose to an insulin during peroxisomal division, and the supramolecular arrangement of cytoskel- challenge, is a precursor for prediabetes, metabolic syndrome, and type 2 dia- etal septin proteins. betes. Traditional methods of assessing insulin resistance (such as glucose up- take assays in adipose tissue) generate a tissue average. We hypothesize that 1728-Pos Board B637 such assays may not reflect the cell-to-cell variability found within adipose Sub-Micron to Nanoscale Chemical Characterization of Biological tissue, and that there exists cellular mosaicity of responsive and refractory Systems using Laser and AFM Based IR Spectroscopy cells rather than all cells becoming equally less responsive to insulin as a Eoghan Dillon1, Anirban Roy1, Curtis Marcott2, Craig Prater1. consequence of insulin resistance. Using subcutaneous adipose tissue biopsy 1Anasys Instruments, Santa Barbara, CA, USA, 2Light Light Solutions, sample from subjects recruited to the NIH Clinical Center, we tested if Athens, GA, USA. AKT (Thr308) phosphorylation (pAKT), a signaling node of the insulin- Infrared microspectroscopy is a powerful technique for obtaining unique chem- signaling pathway and membrane composition probe (PtIns(4,5)P2 and more ical information from a variety of biological systems. In general, spatial reso- notably PIP3), is a biomarker reflecting the number and distribution of lutions are limited by the Abbe diffraction laws to l/2 (3-10 mm). In recent insulin-responsive or refractory regions in fixed tissue. Tissues imaged by years, the development of the AFM-IR technique has allowed for overcoming multiplexed, tiled, confocal immunofluorescence revealed two regional popu- these diffraction limitations by using an AFM probe as the detector for IR ab- lations: low pAKT, primarily seen in the absence of insulin stimulation, and sorption. Using a quantum cascade IR laser (QCL), the molecular vibrations in high pAKT, primarily seen after insulin stimulation in tissue from healthy a sample are excited, leading to thermal expansion of the surface, which is subjects, as fit by a mixture model (basal-like and insulin-responsive). In monitored using an AFM cantilever. This technique reduced the spatial resolu- response to maximal 360 nM insulin stimulation we found the fraction of tion to < 10 nm, however, in certain cases this high resolution is not required membranes with high levels of pAKT was significantly correlated with the and the size of an image in AFM-IR is limited by the piezo stage to sub 100 mm. fraction of tissue that demonstrated translocation of GLUT4, the insulin- A new technique has been developed that fills the gap between IR microspec- stimulated glucose transporter, to the adipocyte plasma membrane. Addition- troscopy and AFM-IR. This is an optical based technique that still breaks the ally, whole-body insulin-sensitivity (determined clinically) was positively diffraction limits associated with conventional IR microspectroscopy, and pro- associated with the fraction of insulin-responsive regions that was visualized, vides sub-micron spatial resolution. Taking advantage of the same fundamental in agreement with previous work from isolated adipocytes. Understanding the principles that govern AFM-IR, the thermal expansion of a sample surface spatial nature of insulin-resistance in native tissue will lead to a mechanistic induced by a QCL IR laser is monitored using an additional laser, instead of understanding of why some regions respond to insulin in prediabetic subjects an AFM probe. This unique technique provides transmission like IR spectra and how to modulate signaling to increase the number of insulin-responsive while operating in a reflective regime. The spatial resolution of this technique regions in this population. is limited by the diffraction limit of the visible probe laser, while also being in- dependent of the IR wavelength used. In this talk we will focus on the combi- 1731-Pos Board B640 nation of this new optical probe based IR technique with AFM-IR to fully Understanding Boundary Effects and Confocal Optics Enables Quantita- characterize a variety of biological samples, including cells, bones and pharma- tive FRAP Analysis in the Confined Geometries of Animal, Plant and cological materials, from the micro scale to the nanoscale using the same QCL Fungal Cells 1 2 1 1 laser source. James L. Kingsley , Jeffrey P. Bibeau , Sayed I. Mousavi , Cem Unsal , Zhilu Chen3, Xinming Huang3, Luis Vidali2, Erkan Tuzel1. 1729-Pos Board B638 1Department of Physics, Worcester Polytechnic Institute, Worcester, MA, Versatile Multiplexed Super-Resolution Imaging of Nanostructures by USA, 2Department of Biology and Biotechnology, Worcester Polytechnic Quencher-Exchange-Paint Institute, Worcester, MA, USA, 3Department of Electrical and Computer Tobias Lutz1, Alexander H. Clowsley1, Ruisheng Lin1, Stefano Pagliara1, Engineering, Worcester Polytechnic Institute, Worcester, MA, USA. Lorenzo di Michele2, Christian Soeller1. Fluorescence Recovery After Photobleaching (FRAP) is an important tool to 1Living Systems Institute, University of Exeter, Exeter, United Kingdom, study the diffusion and binding kinetics of vesicles, proteins, and other mole- 2Cavendish Laboratory, University of Cambridge, Cambridge, United cules in the cytoplasm, nucleus or cell membrane. While many FRAP models Kingdom. have been developed over the past decades, the influence of the complex The optical super-resolution technique DNA-PAINT (Point Accumulation Im- boundaries of three-dimensional cellular geometries on the recovery curves, aging in Nanoscale-Topography) provides a flexible way to achieve imaging of in conjunction with ROI and optical effects (imaging, photobleaching, photo- nanoscale structures at 10 nanometre resolution. In DNA-PAINT, fluores- switching, and scanning), has not been well studied. Here, we developed a cently labelled DNA ‘imager’ strands bind transiently and with high specificity three-dimensional computational model of the FRAP process that incorporates to complementary target ‘docking’ strands anchored to the structure of interest. particle diffusion, cell boundary effects, and the optical properties of the scan- The localisation of single binding events allows the assembly of a super- ning confocal microscope, and validated this model using the tip- growing cells resolution image and the approach effectively circumvents photobleaching. of Physcomitrella patens. We then show how these cell boundary and optical The solution exchange of imager strands is the basis of Exchange-PAINT effects confound the interpretation of FRAP recovery curves, including the which enables multiplexed imaging that avoids chromatic aberrations. Fluid number of dynamic states of a given fluorescent protein, in a wide range of exchange while imaging typically requires specialised chambers or washes cellular geometries, namely nuclei, filopodia, and lamellipodia of mammalian which can disturb the sample. Additionally, diffusional wash-out of imager cells, and in cell types such as the budding yeast, S. pombe, and tip-growing strands is slow in thick samples such as biological tissue sections. Here, we plant cells. We explored the performance of existing analytical and algorithmic introduce Quencher-Exchange-PAINT, a new approach for Exchange-PAINT FRAP models in these various cellular geometries, and determined that in regular open-top imaging chambers that overcomes the comparatively the VCell VirtualFRAP tool provides the best accuracy to measure diffusion slow imager strand switching by diffusional imager wash-out. Quencher-Ex- coefficients. Our computational model is not limited only to these cells types, change-PAINT uses oligonucleotides which inhibit the imager from binding but can easily be extended to other cellular geometries via the graphical Java- to the targets, ‘quencher’ strands, to rapidly reduce unwanted single-stranded based application we also provide. This particle- based simulation—called imager concentrations to negligible levels and decoupled from absolute imager the Digital Confocal Microscopy Suite, DCMS—can also perform fluorescence concentration. The quencher strands contain an efficient dye quencher which dynamics assays, such as number and Brightness (N&B), Fluorescence

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Correlation Spectroscopy (FCS), Raster Image Correlation Spectroscopy monomer, a trimer should be three times brighter. This was shown to hold true (RICS), and could help shape the way these techniques are interpreted. for selected fluorescent proteins in two-photon excitation. However, with widely used one-photon excitation a reduced fluorescence probability of 1732-Pos Board B641 fluorescence proteins in higher-order oligomers was observed. Molecular Fast Fluorescence Lifetime Imaging for Longitudinal Studies of Protein brightness measurements of homo-oligomers allow us to estimate their fluores- Aggregation in Living C. Elegans cence probability. We propose to use the obtained value to correct molecular 1 2 1 3 Tessa Sinnige , Romain F. Laine , Kai Yu Ma , Amanda J. Haack , brightness data measured with one-photon excitation. This procedure allows 2 2 1 4 Peter Gaida , Nathan Curry , Michele Perni , Ellen A.A. Nollen , us to precisely quantify Influenza virus NEP (Nuclear Export Protein) monomer 1 1 Christopher M. Dobson , Michele Vendruscolo , Gabriele S. Kaminski and oligomer fractions within the cytoplasm and nucleus in an infection rele- 3 2 Schierle , Clemens F. Kaminski . vant cell-line using FCS. As a key-regulator NEP participates in both modu- 1Centre for Misfolding Diseases, Department of Chemistry, University of 2 lating transcription and replication activity of viral polymerases and nuclear Cambridge, Cambridge, United Kingdom, Laser Analytics Group, Chemical export of newly assembled viral genome segments (vRNP). In our study, we Engineering and Biotechnology, University of Cambridge, Cambridge, 3 observe concentration-independent but phosphorylation-dependent dimeriza- United Kingdom, Molecular Neuroscience Group, Chemical Engineering tion of NEP in the cytoplasm, whereas in the nucleus a lower dimer but higher and Biotechnology, University of Cambridge, Cambridge, United Kingdom, 4 monomer fraction is observed. Here we predict a regulation mechanism for European Research Institute for the Biology of Aging, University Medical NEP shuttling between cytoplasm and nucleus based on shielding and exposure Centre Groningen, Groningen, Netherlands. of its two nuclear export signals (NES) through dimerization, a mechanism Protein aggregation into amyloid fibrils is a hallmark of a spectrum of neuro- commonly used by proteins transporting cargos between these two compart- degenerative diseases, yet it is still unclear how this process relates to toxicity ments. Eventually, we will not only investigate the influence of different viral (1). The nematode worm Caenorhabditis elegans is a useful model organism components such as membrane-bound Hemagglutinin (HA), viral RNAs or for imaging studies of protein misfolding diseases, because of its ease of whole vRNPs on dimerization and shuttling of NEP, but will also relate its culturing, short lifespan of 2-3 weeks, and transparency. shuttling dynamics between nucleus and cytoplasm to its monomeric and Previously, work from our laboratories has shown that the fluorescence lifetime dimeric state. of a fluorophore is sensitive to the aggregation state of the protein it is fused to (2). However, so far this method was not compatible with high-throughput 1735-Pos Board B644 applications to screen larger numbers of nematode worms across various con- Probing Asymmetric Behavior of a Cell Cycle Regupatory Protein in Live ditions. Here we present a method for fast time-gated fluorescence lifetime im- Caulobacter using Single-Molecule Imaging aging (TG-FLIM) that allows for very rapid acquisition of fluorescent lifetime Jiarui Wang1, Lucy Shapiro2, W.E. Moerner1. data, reporting on the aggregation state of the protein inside living C. elegans. 1Chemistry, Stanford University, Stanford, CA, USA, 2Developmental Using this approach, we performed longitudinal studies of ageing nematodes Biology, Stanford University, Stanford, CA, USA. expressing Parkinson’s and Huntington’s disease-related proteins. We Asymmetric cell division is a fundamental event in developmental programs observed differences in the nature of the protein inclusions and the kinetics where two daughter cells are produced and exhibit distinct characteristics of protein aggregation between alpha-synuclein and polyglutamine, which and fates. Remarkably, asymmetric cell division is also observed in the bacte- are expressed in the respective strains. rium Caulobacter crescentus (3mm x 0.5mm). Caulobacter regulates cell cy- Whereas conventional imaging studies require the worms to be paralysed, we cle events through its master regulator CtrA, which is specifically degraded at furthermore show that we can image freely moving C. elegans in agarose one cell pole (stalked) during the swarmer-to-stalk (G1-to-S) transition and in micro-chambers, using fast TG-FLIM combined with motion correction during late predivisional cell to enable replication onset and the transcription of over data processing. 90 genes. Surprisingly, the CtrA degradation adaptor protein, PopA, localizes Altogether, our method paves the way for high-throughput imaging studies to symmetrically to both cell. PopA binds a secondary messenger molecule, identify novel modifiers of protein aggregation and toxicity. Furthermore, our c-di-GMP (cdG). cdG accumulates asymmetrically at only one of the two TG-FLIM approach can be extended to other applications, e.g. to study protein- daughter cells (stalked). (Christen et al. 2010, Science) We hypothesized that protein interactions or biosensors in living C. elegans. the differential distribution of cdG could imply that PopA proteins at the two 1. Knowles, T. P. J., Vendruscolo, M. & Dobson, C. M. Nat. Rev. Mol. Cell cell poles are intrinsically different. We report the first single-molecule Biol. 15, 384-96 (2014). tracking studies in live Caulobacter of wild type (WT) and mutant PopA that 2. Kaminski Schierle, G. S. et al.. ChemPhysChem 12, 673-680 (2011). lacks cdG binding capability. By quantifying our single-molecule trajectories, 1733-Pos Board B642 we found that PopA that cannot bind cdG diffuses 1.28x faster than the WT. Fluorescence Lifetime Trajectory of the Mouse Pre-Implantation Embryo The difference in the diffusion coefficients is consistent with the possibility Predicts its Viability that WT, when cdG is bound, forms a complex twice the size of mutant that Ning Ma. does not bind cdG. We also obtained evidence for preferential dimerization BME, UCI, Irvine, CA, USA. of PopA at the stalked pole, the site of cdG synthesis, using bimolecular fluo- The first child conceived after in vitro fertilization (IVF) was born in the UK in rescence complementation. Furthermore, the different diffusion coefficients of 1978. Since then IVF has been performed for nearly 40 years. Given the up- PopA at the two poles, as well as the distinct impact on the diffusion coefficient surge in infertility rates, same sex couples ready to start a family, and readily when cdG cannot bind, suggest different localization mechanisms at the two available IVF treatments we need to improve in IVF success and safety. A poles. We propose that by forming different oligomeric states, PopA can grand challenge in IVF is the availability of non-invasive screening methods perform multiple functions at the two poles and contribute to asymmetric for selecting healthy and viable embryos for implantation. Currently there cell division. are few technologies in this area that have been developed to achieve high suc- cessful pregnancies which are largely due to morphologically abnormalities 1736-Pos Board B645 from the embryos themselves. Here, we propose to use a state-of-the-art micro- Deciphering the Role of Bacterial Electrophysiology in Mechanosensation scopy technique called the phasor approach to fluorescence lifetime imaging Giancarlo N. Bruni, Benjamin Dodd, Anjali Rao, Bradley Prythero, microscopy (Phasor-FLIM) to follow changes in the metabolic state in pre- Andrew Weekley, Joel Kralj. implantation mouse embryo and identify their developmental potential. MCDB, University of Colorado Boulder, Boulder, CO, USA. The flow of ions has long been appreciated as a means of cellular signaling. 1734-Pos Board B643 While well characterized in electrically excitable eukaryotic cells, electrophys- Oligomerization and Nuclear Shuttling Dynamics of Viral Proteins iological studies in bacteria were limited by a lack of tools that function in Studied by Quantitative Molecular Brightness Analysis using Fluorescence living cells. Bacterial electrophysiology influences metabolism, division, and Correlation Spectroscopy persistence through antibiotic treatment, but little is known about its role in Madlen Luckner1, Valentin Dunsing2, Salvatore Chiantia2, signaling. The development of novel fluorescent voltage indicators revealed Andreas Herrmann1. that bacteria contain rapid membrane depolarization and repolarization events. 1Humboldt-Univerist€at zu Berlin, Berlin, Germany, 2Universit€at Potsdam, My project strives to uncover the physiological role of these voltage transients Potsdam, Germany. in E. coli. Using a combination of fluorescent voltage and calcium biosensors, Virus genesis is strongly associated with oligomerization of viral proteins in we found bacteria are similar to electrically excitable eukaryotic cells; voltage host cells at various steps of the infection cycle. The analysis of the molecular depolarization induces calcium influx, hinting at a potential signaling mecha- brightness is a powerful tool in fluorescence microscopy to investigate the olig- nism. We discovered that cytoplasmic calcium levels and transients increased omeric state of protein complexes. Theoretically a dimer is twice as bright as its upon mechanical stimulation with a hydrogel, and single cells altered protein

BPJ 8640_8642 Monday, February 19, 2018 351a concentrations dependent upon the same stimulus. Blocking electrophysiology measurable effect on the oligomerization and mobility of fluorescently labeled flux altered mechanically induced changes in protein concentration, while proteins in the NE. This work has been supported by a grant from the NIH (R01 inducing calcium flux reproduced these changes. This evidence suggests that GM64589). voltage and calcium relay a bacterial sense of touch, and alter cellular lifestyle. In order to better understand what roles electrophysiology plays in lifestyle 1739-Pos Board B648 changes, we now seek to find small molecules that alter electrophysiology in Glucose Response of Trans-Differentiated Alpha to Beta Cells in Pancre- E. coli. These chemical tools will allow us to further explore this novel biology, atic Islets 1 1 2 and determine if we can use this potential signaling mechanism to selectively Michael DiGruccio , Zeno Lavagnino , Talitha van der Meulen , Mark Huising2, Dave Piston1. alter bacterial lifestyle. For example, mechanosensation in S. typhimurium 1 2 activates virulence. By discovering small molecules that alter electrophysi- Washington University St. Louis, St. Louis, MO, USA, University of ology, we could potentially alter virulence in species where this signaling California Davis, Davis, CA, USA. mechanism mediates mechanosensation. These data also provide evidence Type 1 Diabetes is defined by insulin deficiency resulting from beta cell that dynamic voltage and calcium exists as a signaling modality in single celled destruction caused by auto-immune attack. Considerable efforts have focused organisms, and therefore studying electrophysiology beyond canonical electri- on the beta cell in an attempt to cure diabetes. The objective of this study is cally excitable cells could yield exciting new findings. to identify and understand the function of beta cells that are continuously gener- ated during adulthood at a specialized region within the islet we named the 1737-Pos Board B646 ‘neogenic niche’. In this region at the islet periphery, new beta cells born Extending Live-Cell Fluorescence Imaging to Anaerobes of the GUT from alpha cells arise throughout adulthood. We can identify and isolate these Microbiome cells by genetic labeling. A red calcium sensor RCamP1h driven by the rat Hannah E. Chia, Matthew H. Foley, Neil G. Marsh, Nicole M. Koropatkin, glucagon promotor reports the Ca2þ response in alpha cells where as EGFP un- Julie S. Biteen. der the control of the mouse insulin promoter labels beta cells. Transdifferen- University of Michigan, Ann Arbor, MI, USA. tiated beta cells expressing both insulin and glucagon are positive for both The gut microbiome supplements human metabolism and immunity by EGFP and the RCamP1h Ca2þ sensor. Using live cell imaging which captures catabolizing host-indigestible glycans. The microbiome operates as a commu- the glucose mediated Ca2þ response necessary for islet cell secretory events, nity of trillions of bacteria, and we are beginning to understand the contribution the functional properties of beta cells arising via trans-differentiation alpha of individual species to glycan digestion. This work extends wide-field and cells are studied. During this process, alpha cells that are transitioning to super-resolution fluorescence imaging to the complexity of the human gut mi- beta cells acquire a switch in glucose response. Instead of responding to low crobiome. Past in vitro studies have been limited by mono-culture approaches, glucose they now respond to high glucose like a fully mature beta cell with a and previous live-cell fluorescence microscopy has been limited by the oxygen- threshold at or above 8 mM. The Ca2þ responses of transdifferentiated beta dependence of traditional fluorescent proteins like GFP. To address these cells as compared to alpha cells are captured using diSPIM (dual view inverted limitations, we have implemented anaerobic live-cell imaging based on the Selective Plane Illumination light-sheet fluorescence microscopy) which al- fatty-acid binding protein, UnaG, in the gut bacterium Bacteroides thetaiotao- lows for rapid three-dimensional whole islet Ca2þ response acquisition with micron (B. theta). We have also extended our previous work on B. theta starch isotropic spatial resolution. This work determines whether alpha cells that utilization system (Sus) enzymes to mixed cultures of B. theta and Ruminococ- transdifferentiate into beta cells are capable of mounting a glucose response cus bromii, a key species in resistant starch degradation, to understand the rela- that would be indicative of a functionally mature beta cell. tionship between the two species in a microbiome. Finally, we are studying the xyloglucan locus in Bacteroidesovatus to compare polysaccharide utilization 1740-Pos Board B649 loci across species. Pair Correlation Analysis of Ku Dynamics upon DNA Damage Michelle A. Digman1, Francesco Palomba2, Xiangduo Kong3, 1738-Pos Board B647 Kyoko Yokomori3, Enrico Gratton1. Harnessing Spatial and Temporal Fluorescence Fluctuations to Differen- 1Biomedical Engineering, University of California, Irvine, Irvine, CA, USA, tiate Luminal and Membrane-Bound Proteins in the Nuclear Envelope 2Chemistry, Alma Mater Studiorum-University of Bologna, Bologna, Italy, Jared Hennen, Kwang-Ho Hur, G.W. Gant Luxton, Joachim D. Mueller. 3Biological Chemistry, University of California, Irvine, Irvine, CA, USA. University of Minnesota, Minneapolis, MN, USA. We used the pair correlation function (pCF) analysis developed for fast frame The nuclear envelope (NE) separates the nucleoplasmic and cytoplasmic com- acquisition to measure the changes in molecular connectivity of the Ku protein partments of the cell and serves as a node for cellular signaling events. Consist- following double strand DNA damage. Ku protein binds to the ends of double- ing of two membranes separated by the approximately 40 nm thick perinuclear strand breaks and is required in DNA-repair for non-homologous end joining space or lumen, the structure of the NE differs significantly from other regions (NHEJ). Before damage, Ku diffuses freely with low anisotropy in the entire of the cell. The differential localization of proteins to the nuclear membranes or nucleus. After damage, we observe the formation of barriers of Ku at the dam- perinuclear space plays an important role in their function and this localization age site. In the rest of the nucleus the diffusion coefficient of Ku increases that may be dynamic for proteins such as the AAAþ ATPase torsinA. It is imper- persists four hours post damage. The diffusion anisotropy tensor of Ku in the ative to characterize this unique environment as a prerequisite for the quantita- nucleus was measured and an increase in anisotropy compared to the control tive interpretation of fluorescence fluctuation studies of NE proteins. The cells without damage was observed. Given that DNA-PKcs play a critical mobility of proteins occupying the perinuclear space is likely influenced by hy- role in NHEJ, loss of their activity can lead to a reduced repair rate and would drodynamic coupling with the membranes. We determined the diffusion coef- alter Ku motility at the damage sites. To test this, we used three DNA-PKcs in- ficient of concatenated fluorescent protein molecules within the perinuclear hibitors which resulted in an increase in diffusion anisotropy. Interestingly after space and compared the results with the Stokes-Einstein relation and other DNA damage, we observed the formation of foci in the nucleus indicating Ku theoretical models. We further conduct equivalent experiments with accumulation. Interestingly the effect of using the DNA-PKcs inhibitors re- membrane-bound fluorescent proteins. We use the results of these studies to sulted in an increase of number of foci. We used fast line scanning to determine explore strategies for distinguishing luminal proteins that are associated with the presence of diffusion barriers across these foci and found that the foci repre- membranes from proteins that are diffusing through the perinuclear space. sent regions of very slow molecular exchange with the free Ku protein diffusion An additional factor complicating experiments is the presence of temporal fluc- around the foci. Stable HeLa cells expressing Ku-eGFP were used. Images tuations in the separation between the nuclear membranes, which were were acquired using the fast Airy scan option on the Zeiss LSM880 with a observed recently. Here we explore both the spatial and temporal correlations frame size of 128x128 and frame rate of 37.4msec. The pCF was calculated associated with this process and use this information as an additional tool to at a distance of 5-10 pixels using the SimFCS software developed at the distinguish membrane-bound from proteins residing in the perinuclear space. LFD. This work is supported by NIH grant P41 GM103540 and NSF MCB- Finally, we investigate whether the oxidizing environment of the NE has a 1615701.

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Posters: Force Spectroscopy and Scanning Probe 1743-Pos Board B652 Pinpointing Unlabeled RNA Sequences Within a Protein-RNA Complex Microscopy with Atomic Force Microscopy Youngkyu Kim1, Zhenghan Gao2, Duckhoe Kim1, Wei Shen Aik1, 1741-Pos Board B650 Liang Tong1, Ozgur Sahin1,2. Nanomechanics of DNA-Binders to DNA by Magnetic Tweezers 1Biological Sciences, Columbia University, New York, NY, USA, 2Physics, Ying Wang, Dennis Kreft, Andy Sischka, Volker Walhorn, Katja Toensing, Columbia University, New York, NY, USA. Dario Anselmetti. Structural information of biomolecules is crucial to understand their working Experimental Biophysics, Faculty of Physics, Bielefeld University, Bielefeld, mechanisms and to develop drugs. However, determining biological structures Germany. at sub-nanometer resolution often demands tedious sample preparation and vast DNA-binding agents are broadly used in many biotechnological or biomed- instrumentational resources. In addition, although super-resolution microscopy ical applications for detecting DNA in cells and gels or as drugs in techniques have enabled tracking fluorescent molecules at the nanometer scale, cytotoxic oncologic cancer treatments. Their binding alters the structural the requisite of labeling fluorophores remains a hurdle in studying the innate and nanomechanical properties of DNA and affects the associated biological structure of biological complexes. We have developed a multicolor microscopy processes. Although interaction modes like intercalation and minor that shows chemical identities on top of topography at sub-nanometer resolu- grove binding already have been identified, associated mechanic effects tion based on atomic force microscopy. We record torsional deflection of like DNA strand elongation as well as unwinding and softening of T-shaped cantilevers to observe single-molecule interaction force on the micro- the dsDNA strand often remain obscure. We used single-molecule second timescale, and by modifying the AFM tip with a probe molecule that magnetic tweezers force experiments to quantitatively investigate the impact generates a distinct force-distance profile for each interaction with multiple tar- of four DNA-dyes (YOYO-1, DAPI, DRAQ5 and PicoGreen) as well as the gets, multicolor imaging is made possible. Here we present label-free imaging anti-cancer drug mitoxantrone at room temperature in a concentration depen- of a histone mRNA-protein complex using a specifically-designed probe DNA dent manner. By extending and overwinding individual, torsionally con- tip. Histone mRNAs have a conserved stem-loop (SL) structure which forms a strained, nick-free dsDNA molecules, we determined the contour lengths, ternary complex with stem-loop binding protein and 3’-5’ exoribonuclease. In persistence lengths and molecular forces, which allow estimation of several the crystal structure, SL is sandwiched in between the two protein domains and thermodynamic and nanomechanical binding parameters. Whereas for both of the 5’- and 3’-flanking sequences of the SL are exposed. We have de- YOYO-1 and DAPI the binding mechanisms can be assigned to bis- signed a probe DNA that partially hybridizes to each flank sequence for the spe- intercalation and minor groove binding, respectively, DRAQ5 and Pico- cific recognition, and the rupture of the transiently formed duplex marks the Green exhibit both binding modes in a concentration dependent manner. position of the flank sequence. Superimposing consecutive images of the Similarly, the chemotherapeutic drug mitoxantrone is found to interact same complex has shown that the detection signals for each sequence were with dsDNA depending on the concentration as a groove binder and separately clustered on the complex. This imaging platform can be used to an intercalator. Our experiments show that single-molecule nanomechanical study structures of biological complexes with minimal sample preparation in experiments can be used for elucidating the binding mechanics of the physiologically relevant conditions. DNA binders and can contribute to the registration process in drug regulation. 1744-Pos Board B653 Bio-Functionalized Core-Shell Microparticles for High Force Optical Trapping 1742-Pos Board B651 Dana N. Reinemann1, Juan Carlos Cordova1, Rizia Bardhan1, Composition-Dependent Alterations in Thickness and Physical Properties Matthew J. Lang1,2. of Lipid Bilayer FILM Revealed by Frequency Modulation Atomic Force 1Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, Microscopy TN, USA, 2Molecular Physiology and Biophysics, Vanderbilt University 1 2 2,3 Akinori Kogre , Yoshikazu Takahashi , Hideo Shindou , Medical Center, Nashville, TN, USA. 4 2,4 Fuyuki Tokumasu , Takao Shimizu . Optical traps are widely used to study the mechanics of biomolecules and bio- 1AMC Department, Shimadzu Techno-Research, INC, Hadano, 2 polymers at the single-molecule level due to their piconewton and nanometer Kanagawa, Japan, Lipid Signaling, National Center for Global Health and resolution. Recently, Jannasch et al. developed core-shell particles with optical Medicine (NCGM), Tokyo, Japan, 3Dept. of Lipid Sciences, The University 4 properties that enhance trapping ability and are promising candidates for high- of Tokyo, Tokyo, Japan, Dept. of Lipidomics, The University of Tokyo, force experiments. To fully harness these particles in biophysical assays, a Tokyo, Japan. straightforward method for functionalizing these particles with biocompatible Assembly of microdomains and membrane viscosity due to these physical handles is required. We developed a robust synthetic strategy for producing properties of membrane have been highlighted as important factors support- titania microparticles with an anatase core that can be covalently functionalized ing proper functions of membrane proteins. Although advanced imaging with proteins and nucleic acids. This was accomplished by adding a silane-thiol technologies including scanning probe microscopy and super-resolution chemical group to the shell surface. These particles display up to a four-fold optical microscopy have revealed dynamics of microdomains and visco- increase in trap stiffness compared to conventional plastic beads featured in op- elastic properties of cell membranes, it is still difficult to link modifications tical tweezers experiments. The core-shell beads were also utilized in inher- of nanoscale membrane structure and specific diseases. In this study, ently high force biophysical assays such as amyloid fiber tethering and actin we aimed to obtain high-resolution structural information of supported rupturing to demonstrate their use and range of applications. We anticipate planner membrane with different fatty acid lengths using liquid dynamic- that our synthetic approach will permit wider use of these improved beads in mode AFM and FM-AFM (Frequency Modulation Atomic Force Micro- optical trapping applications requiring currently inaccessible high forces, scope). In FM-AFM, the cantilever is moved in a non-contact state, main- D such as with stable protein dynamics, as well as in combined coincident optical taining the cantilever frequency shift ( f) is constant. This enables a trapping and fluorescence assays where low laser power but strong trapping highly sensitive force detection, 20 times better S/N than with existing ability is imperative. methods, thereby markedly improving the image resolution. We used sup- ported phosphatidylcholine membranes with fatty acids ranged from PC 1745-Pos Board B654 12:0 to PC 22:6. For saturated PCs, membrane thickness increased along Effect of Graphene Oxide Packing on Bacterial Adhesion using Single Cell with the number of carbon from 4.32 nm (PC12:0) to 6.37nm (PC18:0), Force Spectroscopy but diarachidoylphosphatidylcholine (PC20:0) showed reduced membrane Elise Linna1, Sara BinAhmed2, Benjamin L. Stottrup3, thickness: PC20:0 thickness had 6.09 nm, that is z0.3 nm thinner than Santiago Romero-Vargas Castrill¢n2. PC18:0. Unsaturation of fatty acid further reduced membrane thickness to 1Augsburg University, Minneapolis, MN, USA, 2Department of Civil, 4.12 nm for PC20:4, suggesting that structural arrangement of PCs with Environmental, and Geo-Engineering, University of Minnesota, fatty acid chain length and unsaturation level occurred. These data demon- Minneapolis, MN, USA, 3Physics, Augsburg University, Minneapolis, MN, strated that our imaging modality can detect membrane thickness with min- USA. imal membrane compression by scanning probe while maintaining high Many studies have investigated the biocidal properties of Graphene Oxide detection sensitivity. We will also present modulations of mechanical prop- (GO) resulting in different possible explanations behind these effects.Possi- erties of membrane in the functions of carbon chain length and the level of bly, the orientation of GO sheets can result in more exposed carboxyl groups unsaturation. or the edges of the sheets acting as sharp corners. In this study, we use

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Langmuir-Blodgett trough to deposit GO on Si wafers at varied packing sults support a physiological role for titin in active muscle contraction and force densities to obtain different GO morphologies on solid silicon substrates. enhancement. Roughness and morphology is characterized with AFM. Then, AFM is used to study the interaction of bacterial cells with these GO substrates at sin- 1748-Pos Board B657 gd gle cell level. T Cell Inhibit Growth and Metastasis of Breast Cancer Cells by Altering Cellular Biophysical Properties and Cancer Cell Metabolism 1746-Pos Board B655 Yi Hu, Yangzhe Wu. Neural Network Approach for the Analysis of AFM Force-Distance Curves Jinan University, Guangzhou, China. for Brain Cancer Diagnosis gd T cell, different form cytotoxic T cells and natural killer cells in many ways, Eleonora Minelli1, Gabriele Ciasca1, Tanya Enny Sassun2, such as its MHC independent targeting of cancer cells and easy in-vitro expan- Manila Antonelli3, Massimiliano Papi1, Valentina Palmieri1, sion, has becoming a superior candidate for T cell based cancer Giuseppe Maulucci1, Antonio Santoro2, Felice Giangaspero3, immunotherapy. Roberto Delfini2, Gaetano Campi4, Marco De Spirito1. Nevertheless, to achieve specific and effective killing of target cancer 1Physics Institute, Catholic University of Sacred Heart, Rome, Italy, cells by gd T cell in clinical settings, its killing mechanism needs to be 2Department of Neurology and Psychiatry, Policlinico Umberto I, Rome, elucidated. In the past, molecular mechanism has been proposed for gd T Italy, 3Department of Radiological, Oncological and Anatomo Pathological cell mediated cancer cell killing, mainly through NKG2D and MICA recog- Sciences, Sapienza University, Rome, Italy, 4Institute of Crystallography, nition and subsequently perforin and granzyme release induced cancer cell CNR, Monterotondo, Italy. lysis. The extracellular matrix (ECM), which is the principal extracellular component However, metabolism and biophysical properties variations in the cancer cell of tissues, provides the scaffold and the physical support to cells and regulates after gd T cell treatment have rarely been explored, which are essential to biochemical and biomechanical signalling. understand the killing mechanism and design more effective gd T cell During tumor onset and progression, the mechanical properties modifications immunotherapy. of cells and their microenvironment may profoundly affect tissue homeostasis. Here, we demonstrate that there are alterations of metabolism and biophys- The capability of the atomic force microscopy (AFM) to probe the nanome- ical properties in breast cancer cells after gd T cell treatment. Using AFM, chanical response of biological systems has triggered a growing interest in we characterize membrane morphology and cytoskeleton structure destruc- the translational process of AFM in clinical practice. tion of cancer cell after gd T cell treatment. Meanwhile, biomechanics mea- In this work we report a comprehensive study on the nano-mechanical proper- surements indicate decrease in cancer cell stiffness and surface adhesion ties of human brain tumor tissues, glioblastoma multiforme (GBM) and menin- force, two parameters that might correlate with gd T cell sensitivity against gothelial meningioma (MM), obtained after surgical resection and analyzed by cancer cells. We also demonstrate that glycolysis in triple negative breast AFM in the force spectroscopy mode. cancer (TNBC) cell line MDA-MB-231 is inhibited by gd T cell addition. Normal brain ECM, which is mainly composed by hyaluronan and proteogly- Whereas glycolysis in non-TNBC cell lines MCF-7 and SkBr3 is not cans, is very soft with an average Young’s modulus of 1-2 kPa, while both distinctly altered, indicating less sensitive metabolic response of the latter GBM and MM microenvironment are characterized by high rigidity, 10 kPa two. and 15 kPa, respectively. This large difference in the mechanical response sug- Together, these results indicate biophysical properties and metabolism evalua- gests the AFM as a potential diagnostic tool. tion in breast cancer cell after gd T cell treatment might enable us to assess the One of the factors still hindering the use of AFM in clinical practice is related efficacy of gd T cell immunotherapy on certain types of cancer patients, espe- to the complexity of the data analysis. We demonstrate, on the clinically rele- cially those with poor prognoses. vant problem of discriminating between brain tumor and healthy tissue, an operator-independent neural network (NN) approach for the analysis and clas- 1749-Pos Board B658 sification of AFM data. The NN algorithm permits us to classify tumor and Probing Structural Features of Biomolecular Ensembles with Atomic healthy tissue, and to highlight the presence of infiltrating tumor cells with Force Microscopy high accuracy. Alexander Lushnikov, Alexey Krasnoslobodtsev. Nanoimaging Core Facility, University of Nebraska Medical Center, Omaha, 1747-Pos Board B656 NE, USA. Calcium Dependent Interaction Between N2A-Halo and F-Actin: A Single Atomic Force Microscopy (AFM) is a powerful tool for resolving structural Molecule Study aspects of molecular topology. It is indispensable for imaging of biomole- Samrat Dutta1, Brent Nelson1, Matthew Gage2, Kiisa Nishikawa1. cules, membranes and polymers which could be arranged in dry sample as 1Center for Bioengineering Innovation, Northern Arizona University, well as in biologically relevant conditions. Furthermore, capabilities of Flagstaff, AZ, USA, 2Department of Chemistry, University of Massachusetts AFM spread well beyond topological aspects of biomolecular assemblies. Lowell, Lowell, MA, USA. It could be combined with time-lapse imaging, force spectroscopy measure- In the last two decades, interactions between titin’s I-band region and actin ments and mechanical characterization of bioassemblies at specific locations. have been widely investigated for skeletal and cardiac isoforms. Previous Here, we demonstrate that this technique is helpful to resolve structural studies suggested a calcium-dependent interaction between titin and actin. peculiarities of biomolecular assemblies spanning a large range of sizes: nu- However, previous in vitro motility studies did not quantify the strength of cleic acids (RNA nanostructures)[1], DNA/proteins complexes (such as interaction between titin’s N2A region and actin in the presence of calcium. DNA/SfiI endonuclease)[2], exosomal nanoparticles, and self-assembling Here, we used AFM single molecule force spectroscopy (SMFS) to probe the polyamine-based ‘‘hybrid’’ peptide amphiphiles (PPAs)[3]. Structural char- interaction strength between a recombinant N2A-HALO construct and actin fil- acteristics and the efficiency of biomolecular assembly can be obtained aments (F-actin), in the presence (pCa = 4.0) and absence of calcium. The N2A- from topographical AFM images. We discuss aspects of topographical reso- Halo construct consisted of the N2A region of titin (Ig80-IS-Ig81-Ig82-Ig83, lution and estimate the size of assemblies using RNA nanostructures of where IS is the insertion sequence) with a C-terminal Halo-tag. The construct various shapes. Obtaining structural features of a bioassembly and their cor- was immobilized on an AFM cantilever tip via its N-terminal cysteines. The relation to biological function is discussed using an example of DNA/SfiI interaction strength was determined by probing the molecule during multiple complexes. Elastic properties sometimes play a critical role in determining approach-retraction cycles at physiological pulling rates (300-1000 nm/sec) behavior of biological objects. Here, we compare elastic properties of exo- over various locations on a substrate functionalized with F-actin stabilized somes from various cell lines and correlate Young moduli with membrane on a lipid bilayer with ATP. Preliminary data show that both the yield and composition. While AFM allows to obtain topographical, structural, and strength of N2A-Halo interactions with F-actin nearly doubled in the presence mechanical properties separately, a combination of these features defines of Ca2þ, with multiple peaks in the rupture force distribution at 50 5 25 pN, 75 important properties of assemblies for biological applications. As an 5 10 pN, and 100 5 25 pN. The force extension curves also showed different example, we discuss full characterization of self-assembling polyamine- rupture lengths, indicating multiple actin binding sites. No significant interac- based ‘‘hybrid’’ peptide amphiphiles. Structure, dynamics of formation, tions occurred between N2A constructs and the charged lipid bi-layer surface in elastic properties, and adhesive forces determine the success of PPA nanofib- the absence of F-actin. These experiments show that calcium increases the ers as nanocarriers and scaffolds for tissue engineering applications [3]. interaction probability and binding strength between N2A-Halo and F-actin 1. Halman, J.R. et al. Nucleic Acids Res 2017, 45, 2210-20 and that the binding strength between N2A titin and F-actin is nearly 4 times 2. Lushnikov, A. et al. Biochemistry 2006, 45, 152-8 greater than previously reported for PEVK titin - F-actin interactions. These re- 3. Samad, M.B. et al., Macromol Biosci 2017, doi: 10.1002/mabi.201700096

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1750-Pos Board B659 ment. Revealing this structural arrangement may assist in understanding the New Obligate Folding Intermediate of an RNA Pseudoknot Observed remarkable resilience of viral capsids against harsh environmental exposures. Using Atomic Force Microscopy Based Force Spectroscopy with 10 ms Resolution 1753-Pos Board B662 Robert Walder1, Ty W. Miller1, William J. Van Patten1, Biomechanical Characterization of Protein-Based Hydrogels using a Thomas T. Perkins2,3. Force-Clamp Rheometer 1JILA, University of Colorado at Boulder, Boulder, CO, USA, 2JILA, NIST, Luai R. Khoury, Joel Nowitzke, Kirill Shmilovich, Ionel Popa. Boulder, CO, USA, 3Molecular, Cellular, and Developmental Biology, Physics, University of Wisconsin-Milwaukee, Milwaukee, WI, USA. University of Colorado at Boulder, Boulder, CO, USA. Here we introduce a new force spectroscopy method to study the mechanics of Programmed 1 frameshifting (1 PRF) is an important biological process for protein-based hydrogels under controlled force conditions. Our technique relies the modification of gene expression enabled by the presence of RNA secondary on exposing cylindrical hydrogels to mechanical force by tethering them be- and tertiary structures. However, the mechanism for this process is still under tween a voice coil motor and a force sensor. Our unique approach uses an active study. Here, we investigate the role of mechanical force in 1 PRF by analog PID controller to continuously adjust the hydrogel extension, such characterizing mechanically induced folding and unfolding of RNA pseudo- that the experienced force follows predefined protocols. We demonstrate this knots using an enhanced atomic force microscopy (AFM) based single- technique by using Bovine Serum Albumin (BSA) hydrogels ranging from molecule force spectroscopy (SMFS) assay featuring 10 ms resolution. The 0.7mM to 5mM, crosslinked via a photoactivated reaction. When exposed to pioneering SMFS study of RNA psuedoknots associated with 1 PRF used a linear controlled-stress/relaxation, BSA-based hydrogels show a hysteresis custom-built optical trap. Unexpectedly, this study indicated PRF efficiency associated to unfolding/ refolding of protein domains. This hysteresis decreases was correlated with the presence of alternative folding pathways (rather than with BSA concentration while the measured Young’s moduli had an opposite with average unfolding force), indicating a complex role of RNA psuedoknots behavior in Tris/glycerol solution. Furthermore, in the presence of glycerol so- in 1 PRF involving folding dynamics. Here, we found a new folding interme- lution, hydrogels stiffen, while when immersed in guanidinium chloride diate in an RNA pseudoknot associated with the sugarcane yellow leaf virus (GuHCl) 6M, hydrogels soften and lose their hysteresis. Under constant force (ScYLV) that was not observed in the original optical-trapping based assay. mode, the length of BSA-based hydrogels changes continuously with time due We speculate that the shorter linkers and stiffer force probe in our AFM assay to the viscoelastic effects resulting from unfolding/refolding of protein do- (relative to an optical trap) are the primary reasons for this enhanced state mains. These constant force measurements enable the direct separation be- resolution. Our initial measurements of contour length and folding kinetics tween elastic and viscoelastic behaviors, represented by gel network indicate this folding intermediate is an RNA hairpin that is part of the overall response and protein unfolding/refolding events, respectively. In constant force pseudoknot structure. We observed this intermediate every time the pseudoknot mode, BSA-based hydrogels also showed smaller extension in glycerol 50% folds, indicating that this intermediate is obligate for folding. Overall, our re- and a higher extension in GuHCl 6M than in Tris solution. Additionally, sults indicate that the folding dynamics of RNA pseudoknots are significantly BSA-based hydrogels show a purely elastic behavior in GuHCl 6M. In conclu- more complex than previously observed. Because of the role of the folding dy- sion, this new method is a critical step toward establishing a new class of ex- namics of RNA pseudoknots in 1 PRF, we expect these new insights into periments to study protein nanomechanics in an ensemble mode and will RNA pseudoknot folding dynamics will provide a deeper understanding into open new routes for smart biomaterials discovery. the mechanisms of 1 PRF. 1754-Pos Board B663 Probing Elastic Properties of Mouse Articular Cartilage across Tissue 1751-Pos Board B660 Thickness Measuring the Effect of Antimicrobial Peptides on the Biophysical Proper- Emilios K. Dimitriadis1, Preethi L. Chandran2, Edward Mertz3, ties of Bacteria using Atomic Force Microscopy Ferenc Horkay3. 1 1 1 2 Catherine Volle , Kanesha Overton , Helen Greer , Megan Ferguson , 1BEPS, NIBIB, NIH, Bethesda, MD, USA, 2Chemical Engineering, Howard Eileen Spain3, Megan Nunez4. 3 1 2 University, Washington, DC, USA, NICHD, NIH, Bethesda, MD, USA. Biology, Cottey College, Nevada, MO, USA, Chemistry, SUNY New Paltz, Cartilage matrix is composed of a dense collagen matrix and of the highly- New Paltz, NY, USA, 3Chemistry, Occidental College, Los Angeles, CA, 4 charged glycoprotein, aggrecan that is responsible for tissue osmotic swelling. USA, Chemistry, Wellesley College, Wellesley, MA, USA. The interplay of the two is presumably critical for the cushioning of bones at the With the increase in bacteria developing resistance to traditional antibiotics, joints. The relationship between osmotic and mechanical properties and there is pressing need for new antibiotics. Antimicrobial peptides are currently composition is poorly understood and mapping of the related parameters being explored as just such an option. These peptides are found as part of the through tissue depth is complicated by several issues relating to the need to sec- immune system of many animals, and are typically small peptides that kill tion the tissue to gain access. Tissue damage and intrinsic roughness at the bacteria in one of two mechanisms. In the first mechanism, multiple peptide sectioned surface, and aggrecan diffusional loss from the section are some of subunits join together to form pores in the bacterial membranes. In the second, those issues. In our experiments, aggrecan loss was minimized by mild fixation the peptide crosses the membrane and binds to some molecule in the cell, pre- which, however, did not prevent thickness changes of the cut sections. We per- venting its action. Here, we use atomic force microscopy to characterize the formed high-resolution (1um) elasticity mapping of the cartilage matrix of biophysical changes that occur in the cell when Escherichia coli are exposed newborn mouse across tissue depth. The indentations were performed on thin to antimicrobial peptides. (12um) cartilage sections cut parallel to the bone longitudinal axis. We opti- mized the size of the spherical indentation probe (5um) and applied relatively 1752-Pos Board B661 deep indentations. We developed a novel data analysis method that determines Temperature-Dependent Nanomechanics and Topography of Bacterio- a virtual contact point, and a fitting protocol that ensures that we are probing the phage T7  bulk matrix properties and are minimizing the effects of surface roughness, tis- Zsuzsanna Vo¨ro¨s, Gergely Sevcsik, Gabriella Csı´k, Levente Herenyi, sue inhomogeneity and corrected for the finite sample thickness. We demon- Miklo´s S. Kellermayer. strated the robustness and effectiveness of our method and we compared its Department of Biophysics and Radiation Biology, Semmelweis University, performance to the standard methods used in the field. Thickness changes Budapest, Hungary. upon sectioning, and the high cell density, characteristic of mouse cartilage, Viruses are nanoscale infectious microorganisms which may be inactivated by that leaves narrow matrix septa between cells are two factors that may cause heat treatment. Although heat inactivation is thought to be caused by the release underestimation of the measured elastic moduli. Fixation is possibly causing of genetic material from the capsid, the thermally-induced structural changes in over-estimation. We discuss the implications of these uncertainties and future viruses are little known. In this work we measured the heat-induced changes in directions to address them. the properties of T7 bacteriophage particles. T7 is a non-enveloped, short-tailed icosahedral E.coli phage that contains a 40 kbp genomic DNA. T7 samples 1755-Pos Board B664 were exposed to two-stage (65 C and 80 C) thermal effect, and the structural Micropipette Geometry-Induced Electrostatic Trapping of Nanoparticles changes were investigates by using AFM-based nanomechanical and Yazgan Tuna1, Ji-Tae Kim2, Hsuan-Wei Liu1, Vahid Sandoghdar1. topographical measurements. The structural features of the T7 capsids were 1Max-Planck-Institute for the Science of Light, Erlangen, Germany, extracted from the AFM images with particle analysis. We found that exposure 2Department of Mechanical Engineering, The University of Hong Kong, to 65 C causes the release of genomic DNA due to the loss of the capsid Hong Kong, China. tail which leads to a destabilization of the T7 particles. Further heating to Trapping and manipulation of small objects have been of great interest for a 80 C surprisingly leads to an increase in mechanical stability due to partial range of applications. Here we report on an electrostatic trapping of charged denaturation of the capsomeric proteins kept within the global capsid arrange- nanoparticle between the aperture of a nanopipette and a glass substrate in

BPJ 8643_8645 Monday, February 19, 2018 355a an aqueous medium without the need for external potentials. We also demon- observe the folding dynamics of single PrP molecules held by optical twee- strate of a single gold nanoparticle trapped by a scanning aperture can be used zers. We compared the behavior of hamster PrP (HaPrP) to that of rabbit PrP as a nano-antenna for plasmonic enhancement of fluorescence in fluidic envi- (RbPrP) and bank vole PrP (bvPrP): hamsters are disease-susceptible, rabbits ronment. Our trapping approach with appropriately tailored geometry might are quite resistant, and bank voles are amongst the most susceptible of all allow us to trap and levitate single proteins or macromolecules even in high species. Unfolding and refolding trajectories were measured while ramping salt concentrations which could open doors to well-controlled studies at the the applied force up and down. The resulting force-extension curves single-molecule level. (FECs) revealed the existence of any on-pathway intermediates or misfolded (off-pathway) states, reflecting also their energetics and kinetics. In contrast 1756-Pos Board B665 to HaPrP, which previously exhibited two-state folding, the folding of RbPrP Mechanical Unfolding of the High Potential Iron-Sulfur Protein Probed by involved multiple on-pathway intermediates; in both cases, native folding Single Molecule Atomic Force Microscopy was rapid and misfolded states were not detected in FECs. For bvPrP, Jiayu Li, Hongbin Li. FECs included zero or at most one on-pathway intermediate, but the folding University of British Columbia, Vancouver, BC, Canada. kinetics were much slower. Notably, metastable misfolded states with life- High potential iron-sulfur protein (HiPIP) is an important category of metallo- times on the order of seconds were observed for bvPrP. By relating the protein with a 4Fe-4S cluster center coordinated by four cysteines. Different contour-length changes observed in FECs to structural features of the pro- from other iron-sulfur proteins, the cluster in HiPIP has a high reduction poten- teins, we propose possible intermediates in the native folding pathways for tial, making it an essential electron carrier in bacterial photosynthesis. Here we RbPrP and bvPrP. These results show that the subtle sequence differences used single molecule force spectroscopy to investigate the mechanical unfold- between PrP from different species produce important differences in the ing of HiPIP and the rupture of the ferric-thiolate bond under different redox folding dynamics. potential. We found that the unfolding of HiPIP could be triggered by the rupture of the ferric-thiolate bonds at forces of 150 pN, which was lower 1759-Pos Board B668 than those for other iron-sulfur proteins, such as rubredoxin and ferredoxin. Single Particle Virus Isoelectric Point Determination with Chemical Force Both one-step and two-step unfolding processes were observed in our experi- Microscopy ments, indicative of multiple mechanical unfolding pathways for HiPIP. In Xue Mi, Caryn L. Heldt. addition, the oxidation states of the metal center have little effect on the unfold- Chemical Engineering, Michigan Technological University, Houghton, MI, ing behavior of HiPiP. Further mutational studies will allow us to elucidate the USA. complete rupture mechanism of the iron-sulfur center and the mechanical un- Virus surface characteristics plays a key role in virus sorption processes, but folding pathways of HiPIP. there is very limited information in the literature on viral surface properties. 1757-Pos Board B666 One important surface characteristics is the isoelectric point (pI), which corre- High Precision AFM-Based SMFS of Mechanically Labile T3SS Effectors sponds to the pH where the net charge on the virus particle is zero. The surface Marc-Andre LeBlanc1, Robert Walder2, Devin Edwards2, Thomas Perkins3, charge of virus particles is pH dependent, which dictates the mobility and con- Marcelo Sousa1. trols the colloidal behavior in virus sorption processes. While traditional char- 1Biochemistry, University of Colorado Boulder, Boulder, CO, USA, acterization methods, including zeta potential and viral adsorption to a charged 2Physics, University of Colorado Boulder, Boulder, CO, USA, 3University of surface chemistry, have been used to study viral surface charges and determine Colorado Boulder, Boulder, CO, USA. the pI of virus, they are bulk measurements that are limited by virus purification The Type III Secretion System (T3SS) is a complex, multi-subunit machine methods. The single particle method of chemical force microscopy (CFM) al- which plays a critical role in the pathogenesis and symbiosis of many lows not only the quantitation of the virus surface charges, but can probe the gram-negative bacteria including Shigella, Salmonella, Yersinia, and E. coli. effect of purification on the charge. CFM can measure the isoelectric point By secreting proteins through a needle-like complex inserted directly into of complicated virus particles by using an AFM probe terminated with ioniz- host cells, bacteria manipulate the host response, allowing an infection to able groups. The model non-enveloped porcine parvovirus (PPV) and envel- take hold. Structural studies of the T3SS and its substrate effector proteins oped bovine viral diarrhea virus (BVDV) were used to demonstrate the use show that effector proteins must be at least partially unfolded to pass through of CFM for viral particles. The change of adhesion forces measured as a func- the channel of the T3SS and must refold once inside the host cell to carry out tion of pH is due to a change of virus surface electrostatic properties. The virus their function. Because effector proteins are mechanically labile, our early at- immobilization method used helps to maintain a more natural form of the virus tempts to characterize their unfolding using non-specific adhesion to the AFM without deformation, disassembly, or dehydration. With a thorough under- surface and tip were unsuccessful due to high surface adhesion and low standing of virus surface characteristics, virus purification and future virus throughput. To overcome these limitations, we designed a polyprotein with removal process could be significantly improved to specifically targeting viral versatile, genetically encoded peptide tags that can be functionalized with particles. This will lead to improvements in virus removal, purification and mechanically robust small molecule handles. This functionalization allows detection. for specific attachment to PEG-coated surfaces via copper-free click chemistry and a strong but reversible streptavidin–biotin linkage to PEG-coated AFM 1760-Pos Board B669 tips, drastically enhancing data quality and throughput. Specifically, we High-Throughput Single-Molecule Characterization of Antibody-Antigen achieved a 75-fold increase in high-quality data and obtained the dynamic Interaction using Centrifuge Force Microscopy Yi Luo1, Darren Yang1, Molly MacIsaac2, Wesley P. Wong1. force spectrum of a single individual polyprotein in under 2 h. When com- 1 2 bined with modified cantilevers, our polyprotein enables efficient investiga- PCMM, Boston Children’s Hospital, Boston, MA, USA, University at tion of mechanically labile proteins with 10-ms resolution and sub-pN Albany, SUNY, Albany, NY, USA. stability. With these new tools, we can characterize the unfolding and refold- Antibodies generated during the immune response display a wide range of ing of T3SS effectors and explore how their structures have evolved for effi- binding properties. Such intrinsic heterogeneity is essential for their biolog- cient secretion. ical function but can be difficult to characterize in a precise and quantitative way. Yet, the ability to rapidly measure distributions of affinities and binding 1758-Pos Board B667 epitopes for polyclonal antibodies could illuminating key processes such as Probing the Single Molecule Folding Dynamics of Mammalian Prion Pro- affinity maturation, or assist in the development of vaccines. While single- teins from Species with Different Disease Susceptibility molecule approaches have the potential to provide insight into the molecular Uttam Anand1, Craig Garen1, Michael T. Woodside1,2. heterogeneity of antibody-antigen interactions, the lack of throughput in 1Department of Physics, University of Alberta, Edmonton, AB, Canada, many traditional single-molecule methods has limited such applications. 2National Institute for Nanotechnology, National Research Council, We present a bench-top centrifuge force microscope (CFM) assay that pro- Edmonton, AB, Canada. vides an inexpensive and easy-to-operate method to study antibody-antigen The prion protein PrP misfolds to cause the family of prion diseases, interaction in a massively parallel way at the single-molecule level. including ‘mad cow’ disease, chronic wasting disease, and Creutzfeldt- Combined with DNA Nanoswitches to enable repeated interrogation of Jakob disease. PrP is highly conserved across the animal kingdom, yet dis- single-molecule pairs, we are able to achieve fast and accurate analysis of ease susceptibility varies widely, from highly-susceptible species like deer heterogeneous samples. We demonstrated such ability by repeating bond- and bank voles to highly resistant species like rabbits and horses. These spe- rupture measurements over more than 250 validated single antibody- cies differences appear to be driven by changes in just a few amino acids, but antigen pairs for 20 times or more, yielding thousands of measurements how these changes alter the misfolding remains unclear. We explored the ef- from a single experiment. Our results on the force-dependent antigen disso- fects of species-related sequence differences using force spectroscopy to ciation rate of monoclonal antibodies agreed well with the theoretical

BPJ 8643_8645 356a Monday, February 19, 2018 prediction as well as the result from ensemble gel shift assay. Furthermore, Peptide-lipid interactions are fundamental interactions in cells. Probing the en- we characterized population heterogeneity in polyclonal antibodies by ergy landscape underlying such interactions yields valuable information which analyzing per-molecule averaged data, resolving multiple subgroups with can be used to better understand mechanisms and to potentially design peptides different rupture lifetimes. Our results have demonstrated that the CFM assay for advanced future applications. Recent advances in atomic force microscopy with repeated force measurements can be a powerful tool for fast analysis of (AFM)-based force spectroscopy have allowed investigations of peptide-lipid complex biomolecular interactions in heterogeneous samples. interactions with sub-piconewton precision, a force regime more typically asso- ciated with optical and magnetic tweezers. Using this AFM-based method, we 1761-Pos Board B670 studied the interaction between SecA, the critical ATPase of the general secre- Role of Dimer Interface on the Cooperativity and Misfolding in SOD1 tory system, and a model lipid bilayer (Langmuir, 2017. 33(16): p. 4057-4065). Studied by Single Molecule Force Spectroscopy Specifically, the 10 amino-terminal residues of SecA were probed; this is a re- 1 1 1 Supratik Sen Mojumdar , Zackary Scholl , Michelle Sullivan , gion that is known to be lipophilic and is critical for translocation of proteins 1 1,2 Craig Garen , Michael T. Woodside . across the cytoplasmic membrane of E. coli. Though detailed energy land- 1Department of Physics, University of Alberta, Edmonton, AB, Canada, 2 scapes were reconstructed for differing peptide geometries, the study utilized National Research Council, National Institute for Nanotechnology, limited bandwidth cantilevers and single component lipid bilayers. Now, we Edmonton, AB, Canada. are applying state-of-the-art precision measurement techniques including Superoxide dismutase 1 (SOD1) is a dimeric cytosolic antioxidant that can un- ultra-stable tip-sample control and focused ion beam modified cantilevers to dergo prion-like misfolding in the context of the disease ALS. We studied the probe this interaction more deeply. This presentation will demonstrate progress role of the dimer interface in folding and misfolding at the level of single mol- in implementing these methods using mixtures of lipid species in an effort to ecules using optical tweezers, by comparing the behavior of dimers to that of reconstruct energy landscapes and uncover kinetic pathways in near-native isolated monomers. The unfolding and refolding of dimers was less cooperative conditions. than reported from ensemble studies, with numerous intermediate states that could be resolved. We also identified several distinct misfolded states. Several 1764-Pos Board B673 notable differences in the folding of the dimer were observed. The number of High-Speed AFM Reveals Dynamic Behavor of Antibody intermediates in dimer folding was noticeably less would be expected if the Norito Kotani, Yoko Kawamoto-Ozaki, Ryo Nakatsuka, Susumu Kondo, monomer domains acted independently, indicating that the dimer interface in- Takashi Morii, Takao Okada. creases the cooperativity. Moreover, the initial steps during unfolding were Biomolecule Metrology, Research Institute of Biomolecule Metrology, different for the dimer compared to individual monomers whereas the final un- Tsukuba, Japan. folding steps were similar, suggesting that the dimer interface modulates the High-Speed Atomic Force Microscope (HS-AFM) can observe structure shapes folding pathways by changing the relative stabilities of the portions of the and the dynamic behaviors of biomolecules in solution, as a movie, with nano- monomer structure close to the interface. Finally, we found that the dimer mis- scale order. HS-AFM can get direct image, without any chemical fixing of the folded roughly half as much as would be expected from the rate at which mono- samples or fluorescence. It means that the imaging include low effect from mers misfold. These results suggest that the dimer interface helps protect SOD1 treatments as like sampling. against misfolding, in part by reducing the prevalence of partially-folded inter- We have observed dynamic behavior of IgG in solution using HS-AFM. ‘‘Y’’ mediates that lead to misfolded states. shape of IgG was imaged clearly, and distinguished structure difference be- 1762-Pos Board B671 tween Fab and Fc regions. Atomic Force Microscopy Probing of Extra-Cellular Vesicles By HS-AFM imaging at video rate, we revealed that the Fab regions moved in Daan Vorselen1, Susan van Dommelen2, Raya Sorkin1, Richard van Wijk2, torsional direction like swinging arms. The movement depends on flexible Raymond M. Schiffelers2, Gijs J. Wuite1, Wouter H. Roos3. hinge region, connects the Fab region to the Fc region. The flexibility increase 1Natuur- en Sterrenkunde, Vrije Universiteit, Amsterdam, Netherlands, their affinity to antigens. Using these soft structures, antibodies can grope for 2Klinische Chemie en Haematologie, Universitair Medisch Centrum Utrecht, the antigens at surface and/or solutions, and facilitates the collisions to the Utrecht, Netherlands, 3Zernike Instituut, Rijksuniversiteit Groningen, antigen. Groningen, Netherlands. To understand the mechanisms, we analyzed the Fab swivel movements as There is a recent, quickly growing research interest in Extracellular vesicles Brownian motion, We traced each of the locations of two Fabs and Fc, and (EVs). Fundamental studies focus for instance on their role in cell-to-cell commu- measured the distances between every region, and estimated the flexibility me- nication and disease, and applied studies look into possibilities to use EVs as chanical property of hinge region on single IgG molecules. biomarker or drug delivery vehicle. EVs are built up from lipids and intraluminal HS-AFM measured mechanical properties about single molecule without low proteins. The exact composition is expected to influence their structural proper- effect from fluorescence or chemical fixing. Data from single molecule ties and functioning, however, while the properties of a variety of protein and movement is more useful than data from variance anchoring molecules. membrane nanoparticles have already been probed by Atomic Force Microscopy HS-AFM is the only method which can visualize the dynamic behavior of (AFM) nanoindentation [1,2,3], the material properties of EVs remain elusive. IgG in solution. Here we use AFM to characterise EVs of red blood cells (RBC) from healthy in- We suggest that the observations by HS-AFM could provide a new method to dividuals as well as from a spherocytosis patient. The EVs turn out to be packed measure the flexibility of the hinge in the antibody. It is interesting that anti- with membrane proteins, however their response to indentation fits with that of a body IgG has soft and hard parts in their structure for the functions. fluid lipid bilayer without membrane proteins. The bending modulus of RBC EVs of healthy donors agrees well with the bending modulus of the RBC membrane. 1765-Pos Board B674 EVs from the spherocytosis patient possess another protein composition and react Direct Quantitative Detection of Micrornas by Exchange-Induced differently to mechanical stimuli. Interestingly, while RBCs become more rigid Remnant Magnetization Spectroscopy 1 1 2 1 in spherocytosis, their EVs possess a lower bending modulus than that measured Haopeng Yang , Miriam Gavriliuc , Shoujun Xu , Yuhong Wang . 1Department of Biology and Biochemistry, University of Houston, Houston, for donor EVs. Our results shed new light onto the mechanism of EV budding and 2 on the increased stiffness of RBCs of spherocytosis patients. TX, USA, Department of Chemistry, University of Houston, Houston, TX, References USA. [1] Baclayon M, Shoemaker GK, Uetrecht C, et al., Nano Letters, 2011, Vol. MicroRNAs, as a promising biomarker for the diagnosis and prognosis of a 11, 4865-4869 variety of diseases, have attracted much attention of researchers in various [2] Snijder J, Uetrecht C, Rose R, et al., Nature Chemistry, 2013, Vol. 5, 502-509 fields. To address the increasing need for its precise detection in cells as [3] D. Vorselen, F.C. MacKintosh, W.H. Roos, et al., ACS Nano. 2017, well as body fluid, a new direct quantitative detection techniques, 11:2628-2636 Exchange-Induced Remnant Magnetization Spectroscopy (ExIRMS) has arisen with a purification-free and amplification-free procedure. Here we 1763-Pos Board B672 report a technique based on thermodynamic exchange for microRNA detec- Probing a Fundamental Peptide-Lipid Interaction in E. coli using High tion. We utilize the exchange reaction between the target microRNA and the Precision Force Spectroscopy Methods probe with one mismatched base during competitive binding with the com- Kanokporn Chattrakun1, Krishna P. Sigdel1, Tina R. Matin2, plementary magnetically labelled RNA. A specific platform containing Milica Utjesanovic1, Ioan Kosztin1, Gavin M. King1,3. duplex was initially optimized and prepared: one DNA strand with a 1Physics and Astronomy, The University of Missouri-Columbia, Columbia, mismatch was titrated and immobilized on the surface; the RNA strand MO, USA, 2Weill-Cornell Medicine, New York, NY, USA, 3Biochemistry, with complementary sequence was labelled with a magnetic particle and The University of Missouri-Columbia, Columbia, MO, USA. formed a duplex with the mismatched DNA strand on the surface. When

BPJ 8643_8645 Monday, February 19, 2018 357a body fluid containing the microRNA was introduced, it prompted the improved brightness of the computationally optimized cpVermilion is verified dissociation of magnetically labelled RNA strand from the mismatched experimentally, with its brightness indeed substantially improved. These results DNA strand and led to randomization of the magnetic dipoles of the magnetic suggest that the importance of the linker region to the brightness of permuted particles, which can be measured by the atomic magnetometer. Our results red fluorescent protein and that computer aided screening can be supplemented showed that ExIRMS was sufficiently robust to achieve a high sensitivity to fluorescent protein engineering. with 105 molecules for detecting microRNAs in cell lysate as well as clinical serum. Additionally, neither purification nor amplification is required 1768-Pos Board B677 for detection, which maximally simplify the analysis process and Phenylene Ethynylene Based Sensors for the Selective Detection of TAU avoid possible loss during sample preparation for absolute quantification. Pathology 1 2 3 Overall, our technique presents a novel approach for a sensitive, quantitative Florencia A. Monge , Patrick L. Donabedian , Nicole M. Maphis , Shanya Jiang3, David G. Whitten4, Kiran Bhaskar5, Eva Y. Chi4. and reliable microRNAs profiling which can be further applied in disease 1 diagnosis. Center for Biomedical Engineering, Biomedical Engineering Graduate Program, University of New Mexico, Albuquerque, NM, USA, 2Center for Biomedical Engineering, Nanoscience and Microsystems Engineering Graduate Program, University of New Mexico, Albuquerque, NM, USA, Posters: Biosensors I 3Molecular Genetics and Microbiology, Biomedical Sciences Graduate Program, University of New Mexico, Albuquerque, NM, USA, 4Department 1766-Pos Board B675 of Chemical and Biological Engineering, Center for Biomedical Engineering, High Throughput Integrin Tension Mapping in Platelets at Submicron University of New Mexico, Albuquerque, NM, USA, 5Molecular Genetics Resolution and Microbiology, University of New Mexico, Albuquerque, NM, USA. Yongliang Wang1, Dana N. LeVine2, Margaret Gannon3, Yuanchang Zhao1, 1 4 1 The misfolding and aggregation of the microtubule binding tau protein into Anwesha Sarkar , Bailey Hoch , Xuefeng Wang . b-sheet enriched fibrillar deposits (neurofibrillary tangles NFTs) has been 1Physics, Iowa State University, Ames, IA, USA, 2Veterinary Clinical 3 linked to an array of neurodegenerative diseases. As tau aggregation is Sciences, Iowa State University, Ames, IA, USA, Veterinary Microbiology believed to lead to neurodegeneration and cognitive decline decades before and Preventative Medicine, Iowa State University, Ames, IA, USA, 4 the onset of clinical symptoms, tau aggregates are ideal biomarkers for early Kinesiology, Iowa State University, Ames, IA, USA. disease detection and therapeutic intervention. However, there is a lack of Platelets are small blood cells mediating hemostasis and thrombosis. diagnostically useful sensors to detect tau deposits. Herein we have charac- Integrin-transmitted force plays an important role in platelet adhesion, terized two sensors from a library of oligomeric phenylene ethynylene activation, spreading and contraction. Platelet force calibration is desired (OPE)-based conjugated polyelectrolytes, anionic OPE1 and cationic in both basic research and clinical applications. Previously, bulk platelet OPE2 as novel fluorescent sensors in the detection of NFTs in brain tissue force has been measured with cell traction force microscopy and sections. Previous in vitro studies demonstrated that OPE1 and OPE2 atomic force microscope. However, mapping platelet force at submicron res- display a conformation selective turn-on fluorescence when bound to path- olution has been challenging, integrin molecular tension in platelets has not ologically relevant amyloid fibrils. In this study, we characterized and quan- been calibrated either. Here we developed integrative tension sensor (ITS) tified OPE’s ex vivo sensing capability through confocal fluorescence to map integrin tension in platelets with high resolution, sensitivity microscopy and co-localization analysis using tau specific antibody and data throughput. ITS is a rupturable molecular linker with defined ten- AT180 and historically used amyloid stain Thioflavin-T. Our results show sion tolerance (Ttol) decorated with fluorophore-quencher pair. Integrin turn-onOPE1fluorescencewhenboundtoNFTsintransgenicmiceandhu- tension above Ttol would rupture the linker and free fluorophore man frontotemporal dementia brain sections with little to no non-specific from quenching, thus directly converting tension signal to fluorescent signal binding. While OPE2 also detected NFTs, it exhibited more background and enabling platelet force mapping by fluorescence imaging. ITS maps m staining possibly attributed to interactions with neuropil threads or dystro- integrin tension in hundreds of platelets at 0.4 m resolution in parallel. phic neurites. Importantly, the OPE dyes demonstrated effective sensing We found that integrin tension is in the range of 12-54 pN during platelet at a concentration (5 mM) far lower than Thioflavin-T (1.56 mM and higher). adhesion and spreading, while the tension rise beyond 54 pN during platelet Results from this study thus validated the use of OPEs for the selective contraction. We also found that platelet force distribution has strong sensing of NFTs in brain tissue, laying the groundwork for developing these polarization which is sensitive to treatment with the anti-platelet drug tiro- molecular sensors to into a tool to simultaneously and dynamically track fiban, suggesting that the ITS force map can report anti-platelet drug NFT formation for both in vitro and in vivo systems, transforming our abil- efficacy. Furthermore, ITS assay has low demand for sample volume, m ity to study the cause, diagnosis, and treatment of major neurodegenerative only requiring ten microliter ( L) finger-pricked blood in one assay. Collec- disorders. tively, ITS maps integrin-transmitted platelet force with submicron resolu- tion, rapid data acquisition, high data throughput and low sample volume. 1769-Pos Board B678 With these features, ITS will facilitate both basic research and drug Imaging Dopamine Neuromodulation in Brain Striatum and Prefrontal development of platelet inhibitors. ITS also holds great potential as a diag- Cortex nostic tool for rapid examination of platelet function or dysfunction in pa- Abraham Beyene1, Kristen Delevich2, tients, helping to better diagnose and treat bleeding and thrombotic Jackson Travis Del Bonis-O’Donnell1, Wren Thomas2, Wan Chen Lin2, disorders. Linda Wilbrecht2, Markita P. Landry1. 1Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA, 1767-Pos Board B676 USA, 2Psychology, UC Berkeley, Berkeley, CA, USA. Improving the Brightness of Circular Permutated Red Fluorescent Pro- Neurons communicate through chemical neurotransmitter signals that either teins using an in Silico Approach terminate at the postsynaptic process (‘‘wired transmission’’) or diffuse Junyi Liang, Mark Rizzo. beyond the synaptic cleft to modulate the activity of larger neuronal networks University of Maryland, Baltimore, MD, USA. (‘‘volume transmission’’). Molecules such as dopamine belong to the latter Red fluorescent proteins are a class of widely used reagents in life science class of neurotransmitters, for which real-time imaging of the signal’s spatial research for imaging studies, due to their genetic encodability, better specimen propagation would constitute a major advance in neurochemical imaging. To penetration and lower phototoxicity relative to shorter wavelength visible light. this end, we present a nanoscale near-infrared fluorescent reporter for dopa- Bright red fluorescent proteins with unconventionally oriented chromophores mine and demonstrate its efficacy for imaging dopamine volume transmission are a long standing goal because of their utility for optimizing Forster Reso- in the extracellular space of the brain striatum and prefrontal cortex. We nance Energy Transfer (FRET) biosensors. Here, we developed a bright and un- describe the design, characterization, and implementation of DNA function- conventionally oriented chromophore-containing red fluorescent protein by alized single wall carbon nanotube nanosensors and present recent progress first circular permuting a new red fluorescent protein mVermilion to modulate in imaging the dynamics of evoked and spontaneous dopamine release in the chromophore orientation and then pursuing a computational based mouse acute brain slices. Dopamine nanosensors record spatially and tempo- screening on the linker region of the permuted to evolve a planar and most rally resolved dynamic behavior of dopamine on length scales that encom- rigidified chromophore, in order to maximize its brightness. Our in silico study pass single terminals and inter-terminal distances in the striatum, and demonstrate that the length and rigidity of the linker region can directly affect enable investigation of the effect of drugs such as the antidepressant Nomi- the planarity of cpVermilion chromophore, and thus its brightness. Finally, the fensine on dopamine dynamics. We further describe how nanoparticle

BPJ 8643_8645 358a Monday, February 19, 2018 exciton engineering can be used to tune sensor performance and design Our fluorescent biosensors are built on an OpuBC-GFP fusion scaffold. OpuBC sensors best suited to in vivo experimentation. Molecular dynamics modeling is a bacterial periplasmic binding protein with two key features: a cation-p box, elucidates the physicochemical phenomena underlying the dopamine sensing favorable for binding amines well represented in drugs, and a binding-induced process and stochastic simulations reveal that optical nanosensors can capture ‘‘Venus fly-trap’’ conformational change. The suitably mutated OpuBC domain millisecond-scale changes during phasic and tonic firing of dopaminergic is connected, at the hinge regions, to a circularly permuted ‘‘superfolder’’ GFP neurons. Combining our experimental and computational work, we outline (cpGFP). Aided by structural information, we use directed evolution to create a the functional range of this technology for imaging dopamine neuromodula- family of drug-sensing biosensors meeting the criterion of DF/F0 > 1at1mM tion in biologically complex and optically dense live brain. Our experimental drug. and theoretical results show that near-infrared (nIR) neurotransmitter nano- Our proof of principle study concerns nicotine entering the ER, as measured by sensor constructs can relay information about neuronal signaling in the an ‘‘intensity-based nicotine-sensing fluorescent reporter’’ (iNicSnFR). Previ- tissue-compatible nIR optical window, with spatiotemporal scales that ous data demonstrated that exposure to nicotine causes changes in number capture both single synaptic release events and ensemble terminal behavior and stoichiometry of nicotinic receptors by chaperoning within the ER; howev- suitable for in vivo behavioral experimentation. er nicotine itself entering the ER had not yet been measured. In live cell imag- ing of an iNicSnFR targeted to the ER, we found that nicotine enters the ER 1770-Pos Board B679 within 10 s of application at concentrations experienced by a cigarette smoker. High Selectivity and Sensitivity of Oligomeric P-Phenylene Ethynylenes Moreover, we found that varenicline, a smoking cessation drug, enters the ER for Detecting Amyloid Proteins In-Vitro almost as rapidly as nicotine, helping to explain varenicline’s biochemical and Adeline M. Fanni1, Florencia A. Monge1, Arjun Thapa2, David G. Whitten2, behavioral effects. Eva Y. Chi2. We are currently developing other ‘‘iDrugSnFRs’’ for antidepressants, antipsy- 1Center for Biomedical Engineering, Biomedical Engineering Graduate chotics, and opioids. These tools to study subcellular pharmacokinetics will Program, University of New Mexico, Albuquerque, NM, USA, 2Chemical help to clarify chronic effects of several families of neural drugs. and Biological Engineering, University of New Mexico, Albuquerque, NM, Support: DA037161, GM123582, NARSAD, California TRDRP, and HHMI. USA. A common pathological hallmark of neurodegenerative diseases such as 1772-Pos Board B681 Alzheimer’s and Parkinson’s diseases is the presence of extra- or intra- G-Quadruplex Foci Revealed by Fluorescent Probe: A Universal Cancer cellularly accumulated amyloid proteins. Importantly, as these protein Biomarker? deposits are believed to initiate a cascade of events culminating in neuro- Ta-Chau Chang. degeneration and cognitive decline before the onset of clinical symptoms, IAMS, Academia Sinica, Taipei, Taiwan. protein amyloids are ideal biomarkers for early disease detection and Ta-Chau Chang, Ting-Yuan Tseng, Wei-Wen Chen, I-Te Chu, therapeutic intervention. A molecular probe that selectively detect these Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan b-sheet rich amyloid deposits could lead to early disease detection and Cancer is not a single disease but a complex progression of cellular/tissue a better understanding of these diseases. We are developing a class of mutation. Currently, no ‘‘universal’’ cancer biomarker has been docu- novel amyloid sensors based on the p-phenylene ethynylene (PPE) molec- mented. G-quadruplex structures have recently received increasing attention ular scaffold. We have shown that oligomeric PPE compounds (OPEs) as a potential target for cancer research. Bioimaging of fluorescent probes bind specifically to amyloid fibrils made of model proteins, insulin and provides a fantastic tool to visualize different cellular responses from lysozyme, which leads to fluorescence turn-on. In this study, we evaluate various cells and to monitor target/probe interaction in cells. Here using the capability of two probes, OPE1- and OPE2þ, to detect amyloid fibrils time-gated fluorescence lifetime imaging microscopy, significantly formed from Alzheimer’s disease-associated amyloid-b (Ab) peptides and more signals from 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide Parkinson’s disease associated a-synuclein proteins. Selectivity and spec- (o-BMVC) foci, characterized by the longer fluorescent decay time of ificity of OPE1- and OPE2þ to detect the amyloid conformation of those o-BMVC, were detected in five types of cancer cells than in three types proteins were quantified. We show that both OPE1- and OPE2þ had of normal cells. The large contrast in the number of o-BMVC foci is a higher selectivity toward Ab40 fibrils than the commonly used amyloid common feature in distinguishing cancer cells from normal cells. Further dye, Thioflavin-T (ThT) and that the OPE compounds detected Ab40 fi- evidence indicates that o-BMVC foci are mainly due to its binding with brils at a concentration as low as 0.5 mM. However, OPE2þ also dis- G-quadruplexes in the cells. In addition, we hypothesize that the loss of played a small fluorescence enhancement in presence of the monomeric chromatin integrity plays a role in generating more number of G-quadru- protein, which could lead to false positives. Compared to ThT, OPE1- plex foci in cells. Although the finding of the large contrast in the number more sensitively detected fibrillar aggregates of a-synuclein proteins. of G4 foci between five cancer cells and three normal cells is not sufficient Importantly, OPE1- also detected pre-fibrillar aggregates of a-synuclein to provide solid conclusions, these findings lay the foundation for the proteins that ThT did not detect. The high selectivity and specificity of development of G4 foci as a universal cancer marker that may have an OPE1- for the detection of a wider range of amyloid protein aggregates astonishing impact on cancer research. makes it a promising probe for the early detection of neurodegenerative diseases. 1773-Pos Board B682 Drinc: Co-Imaging of Dynamic Activities and Protein Localization in 1771-Pos Board B680 Super-Resolution Microscopy Using Fluorescent Drug Biosensors for ‘‘Inside-Out Pharma- Gary C.H. Mo, Jin Zhang. cology’’ Pharmacology, University of California San Diego, San Diego, CA, USA. Anand K. Muthusamy1, Amol V. Shivange2, Aaron L. Nichols2, Cell signaling is highly compartmentalized. A nanodomain can play host to Aron Kamajaya2, Janice Jeon2, Philip M. Borden3, Jonathan S. Marvin3, regulators and effectors of one or many signaling pathways, maximizing the Elizabeth K. Unger4, Huan Bao5, Edwin R. Chapman6, Lin Tian4, efficiency, speed, and specificity of protein actions. If the activity of Loren L. Looger3, Henry A. Lester2. signaling proteins is restricted to within nanodomains, however, it is below 1Division of Chemistry and Chemical Engineering, Caltech, Pasadena, CA, the diffraction limit and requires a new class of biosensors for visualization. USA, 2Division of Biology and Biological Engineering, Caltech, Pasadena, We previously resolved activity nanodomains of cAMP Protein Kinase CA, USA, 3Janelia Research Campus, Ashburn, VA, USA, 4Biochemistry (PKA) using Fluorescent fLuctuation INcrease by Contact (FLINC). We and Molecular Medicine, UC Davis, Davis, CA, USA, 5Dept. of showed that A Kinase Anchoring Proteins (AKAP) play an important role Neuroscience, HHMI and Dept. of Neuroscience, U. Wisconsin-Madison, in maintaining PKA activity nanodomain. However, it is challenging to visu- Madison, WI, USA, 6HHMI and Dept. of Neuroscience, U. Wisconsin- alize both AKAPs and PKA activity at super-resolution together to study Madison, Madison, WI, USA. their spatial relationship in live cells. Here, we present Dronpa Removed Neuropharmacology offers many tools for connecting molecular to acute FLINC (DRINC), a method to image enzyme activity and protein localization behavioral phenomena but has few tools to explain effects of chronic simultaneously at super-resolution. We utilize DRINC to determine the drugs. In the biophysically based ‘‘inside-out’’ approach to neuropharma- spatial relationship between membrane PKA activity nanodomains and com- cology, drugs bind to their nascent targets within the endoplasmic reticu- ponents of the PKA pathway such as AKAP79 and L-type calcium channel. lum (ER) (i.e. pharmacological chaperoning). Now we visualize, quantify, We further probe into the role of cytoskeleton in the distribution of active and time the first steps: drugs entering the cell and entering organelles. PKA nanodomains. DRINC is as general as FLINC. We believe its

BPJ 8643_8645 Monday, February 19, 2018 359a

development strengthens our ability to decipher signaling cohesively using ease). The development of accurate and quantitative methods to measure O2 super-resolution activity imaging. concentration in living cells, tissues and organisms has remained challenging and is subject of intense research. We developed a protein-based, fluorescent 1774-Pos Board B683 oxygen sensor that can be expressed directly in cells to monitor oxygen levels Simultaneous Imaging of Apollo-NadpD and Fucci to Correlate Beta-Cell in the cytoplasmic environment. We fused Myoglobin (Mb), a physiological NADPH/NADPD Redox State to the Cell Cycle oxygen carrier, with mCherry, a fluorescent protein, to build a fluorescence Huntley H. Chang1, Jonathan V. Rocheleau1,2. resonance energy transfer (FRET) pair where Myoglobin acts as a variable 1Institute of Biomaterials and Biomedical Engineering, University of dark acceptor. The changes in the spectral properties of Myoglobin upon Toronto, Toronto, ON, Canada, 2Toronto General Research Institute, oxygen binding translate into changes of the FRET-depleted emission inten- University Health Network, Toronto, ON, Canada. sity of mCherry, and this effect is best detected by monitoring the fluores- We recently developed a family of genetically encoded sensors based on cence lifetime of the probe. We present in this work the expression and steady-state fluorescence anisotropy to track NADPH/NADPþ redox state in characterization of the Mb-mCherry system in vitro to establish a calibration þ real time (Apollo-NADP ). NADPH critically supplies reducing equivalents curve of its response to [O2] and other relevant effectors. Further, by using to the glutathione/thioredoxin antioxidant pathway to scavenge toxic reactive Fluorescence Lifetime Imaging Microscopy (FLIM), we show that we are oxygen species (ROS) such as H2O2, and is also used in the biosynthesis of able to map the oxygenation level of the probe ex vivo in cells, providing lipids, amino acids, and nucleotides. Thus far, we have used the Apollo- a quantitative description. A genetically encoded oxygen probe based on NADPþ sensor to explore the link between NADPH/NADPþ redox state and fluorescence addresses the most common issues with currently available survival in mature beta-cells. Beta-cells are notoriously sensitive to ROS due methods to monitor O2 levels in living organisms: data collection speed, to their limited NADPH production via pyruvate cycling. Inducing beta-cell spatial resolution and localization within cells and tissues. Customization proliferation is a major therapeutic objective to treat diabetes. We therefore of the response can be achieved by exploiting the mutations of Mb to tune aimed to examine the balance between NADPH production and ROS scav- O2 binding affinity, or the response to external modulators (e.g.: lactate) pre- enging at different stages of the cell cycle. We hypothesized that biosynthetic sent in the cell. pathways required for proliferation would deplete NADPH and thus leave the þ cells more vulnerable to ROS. To correlate Apollo-NADP responses to 1777-Pos Board B686 different stages of the cell cycle, we co-expressed the fluorescence ubiquitina- Acidity at the Surfaces of Cancer Cells þ tion cell cycle indicator (FUCCI) sensor with Apollo-NADP in the INS-1E Oleg A. Andreev1, Da Wei1, Donald Engelman2, Yana Reshetnyak1. 1 2 beta-cell line. We subsequently performed H2O2 dose responses and found Physics, University of Rhode Island, Kingston, RI, USA, Yale University, þ Apollo-NADP anisotropy decreased with increasing H2O2 concentration, New Haven, CT, USA. consistent with NADPH depletion by the glutathione/thioredoxin antioxidant Solid tumors have an acidic extracellular environment, which results from system. In contrast to our hypothesis, cells in S/G2/M showed a decreased elevated cellular glycolytic activity (Warburg effect). Cancer cells maintain response to H2O2 compared to cells in G0/G1 suggesting compensatory produc- a neutral intracellular pH (7.2) by exporting protons, lactate and carbonic tion of NADPH and greater protection from ROS. We are now probing the un- acid. One can expect that the acidity of the extracellular microenvironment, derlying mechanisms of this NADPH compensation using specific metabolic particularly near cancer cell surfaces, will be high, but there was a need to inhibitors. measure it. We recently introduced a novel approach to measure the pH at cell surfaces by using a pH-sensitive fluorescent dye, SNARF, conjugated 1775-Pos Board B684 to a pH Low Insertion Peptide (pHLIPÒ peptide), which targets plasma mem- Apollo-NADPD Expressed in the Pancreatic Beta-Cells of Living Zebrafish branes of cells in acidic tissue. We grew cancer cells in spheroids, stained Responds to Glucose and Diamide to Reveal NADPH Dynamics them with SNARF-pHLIP, washed, and then took two fluorescent images Cindy V. Bui1, Curtis W. Boswell2, Brian Ciruna2, Jonathan V. Rocheleau1. formed by a dual view optical system, one image with a 590 nm filter and 1Institute of Biomaterials & Biomedical Engineering, University of Toronto, the other with a 650 nm filter. We developed a program that aligns the images, Toronto, ON, Canada, 2Molecular Genetics, University of Toronto, Toronto, calculates ratios between the images and converts the ratios into a cell pH ON, Canada. map. The program also calculates an average pH for each cell and plots a We recently developed a family of genetically-encoded homoFRET sensors to pH histogram for a cell population. We studied several cancer cell lines. In measure NADPH/NADPþ redox state based on homo-dimerization of inactive the absence of glucose or in the presence of deoxyglucose (nonmetabolizable), glucose-6-phosphate dehydrogenase (Apollo-NADPþ). Our aim is to use the surface pHs of most cells were similar and close to the pH of the bulk so- Apollo-NADPþ to study NADPH dynamics of pancreatic beta cells in vivo lution, while in presence of glucose highly metastatic cancer cells showed during redox stress. Redox stress induces beta cell failure, ultimately resulting lower surface pHs than nonmetastatic cells, and both cell types showed sur- in Type 2 Diabetes. We previously showed that light-scattering in cell aggre- face pHs lower than the bulk solution pH. For highly metastatic cells, the sur- gates caused a shift in absolute fluorescence anisotropy values, but the relative face pH was 6.7-6.8, while the medium pH was 7.4. In tumors, where the differences between samples were maintained. We now investigate zebrafish diffusion of molecules is restricted, the pH on the surfaces of cancer cells as a low-scatter alternative platform for 3D, in vivo fluorescence anisotropy could be 6.0 or even lower. imaging. Zebrafish are an ideal platform for this work due to their optical transparency for imaging, genetic tractability, and ability to model many path- 1778-Pos Board B687 ological diseases. Here, we generated a transgenic zebrafish with stable Photonic Control of Microalgae for In-Situ and Real-Time Monitoring of Apollo-NADPþ expression targeted to the insulin-secreting beta cells of the þ Algal Cultivation zebrafish pancreas and show that Apollo-NADP responds to glucose and Minsun Song1, SoonGweon Hong1, Luke P. Lee1,2. diamide treatments. Both 2-photon microscopy and microfluidics will be 1Bioengineering, UC Berkeley, Berkeley, CA, USA, 2School of Medicine explored to track cellular NADPH dynamics and optimize imaging conditions and Faculty of Engineering, National University of Singapore, Singapore, for live transgenic zebrafish. Overall, this project aims to determine whether þ Singapore. homoFRET sensors such as Apollo-NADP can be translated in vivo to study Electrophysiological responses of light-gated ion channels through light regu- living tissue. lation have been used in many fields. Photonic control on innate light-gated ion channels of microalgae can be an ideal way to investigate microalgal 1776-Pos Board B685 physiology and microalgal applications which is of great interest due to the A Breath of Fresh Air: A Genetically Encoded O2 Probe for Direct Map- enormous potential of microalgae as a future energy, food, and medicine. ping and Quantification of Oxygenation Levels in Cells via Fluorescence Herein, we present a comprehensive, real-time, in-situ and noninvasive micro- Lifetime Imaging algae monitoring method based on electrophysiological response via photonic Alessio Andreoni1, Rozhin Penjweini1, Marie-Paule Strub1, Dan L. Sackett2, regulation of microalgae. To assess cell conditions, we design our sensor to Jay R. Knutson1. monitor photocurrent induced by light-gated ion channels (channelrhodopsins) 1NHLBI, National Institutes of Health, Bethesda, MD, USA, 2NICHD, in microalgae. We demonstrate that the temporal dynamics of the photocur- National Institutes of Health, Bethesda, MD, USA. rent over microalgae culture period are highly correlated with cell conditions Molecular oxygen is an important reporter of metabolic and physiological (i.e., metabolic activity and population). Also, the photocurrent monitoring status at the cellular and tissue level, and its concentration can be used for during high-value metabolite production period informs dynamics of the the evaluation of diseases of various nature (e.g.: cancer, coronary artery dis- metabolite accumulation in microalgae, which can be useful as a strategic

BPJ 8643_8645 360a Monday, February 19, 2018 index for algal cultivation. We believe our photonic control of microalgae Protein films derived from natural substances have garnered a considerable holds great potentials for in-situ, real-time monitoring in algal farming and amount of interest among researchers all over the world for their biodegrad- environmental surveillance. ability and excellent film forming capabilities. In this study, we have prepared the protein films from the proteins isolated from the cataractous lens emulsion 1779-Pos Board B688 for the first time. On aging, the human eye lens turns cloudy due to aggregation Monitoring Voltage Fluctuations of Intracellular Membranes Fluctuations of these proteins in the lens and after surgery; the aggregated protein mass in the of Intracellular Membranes cataractous lens is discarded. Designing protein films using this readily avail- 1 2,3 2 Masoud Sepehri Rad , Lawrence B. Cohen , Oliver Braubach , able mass that is rich in proteins makes it cost effective. The films have been 2 Bradley J. Baker . prepared using glycerol as a plasticizer, which increases the flexibility of the 1Center for Functional Connectomics, Korea Institute of Science & 2 films, and varying amounts of glutaraldehyde (10%-40% ) (w/w) of CEPI) as Technology, Seoul, Republic of Korea, Center for Functional Connectomics, cross-linker. The films were characterized by Fourier transform infra-red Korea Institute of Science & Technology, Seoul, Republic of Korea, 3 spectroscopy, nano-indentation, and thermo gravimetric analysis studies. The Department of Cellular and Molecular Physiology, Yale University School physical properties of the films were determined by total soluble matter and of Medicine, New Haven, CT, USA. moisture content. The best mechanical properties for the CEPI films were ob- In eukaryotic cells, the endoplasmic reticulum (ER) is the largest continuous tained with 20 % (w/w of CEPI) of glutaraldehyde. The films having 20 % membrane-enclosed network which surrounds a single lumen. Using a new (w/w of CEPI) of glutaraldehyde were utilized as a delivery carrier for ampi- genetically encoded voltage indicator (GEVI), we applied the patch clamp cillin sodium as a model compound. The encapsulation efficiency was found technique to cultured HEK293 cells and neurons and found that there is a to be 74%. The in-vitro pH dependent release study showed highest amount very fast electrical interaction between the plasma membrane and internal of ampicillin sodium release in phosphate buffer saline medium. These films membrane(s). This discovery suggests a novel mechanism for interaction be- can have potential use in the development of therapeutics for pharmaceutical tween the external membrane and internal membranes as well as mechanisms applications. for interactions between the various internal membranes. The ER may transfer electrical signals between the plasma membrane, the Golgi Apparatus, the nu- clear envelope, the mitochondria, and other internal organelles. The internal 1782-Pos Board B691 membrane signal is reversed in polarity but has a time course similar to that Nuclear Uptake of Thiolated Riboflavin Gold Nanoassembly: DNA Dam- of the plasma membrane signal. The optical signal of the GEVI in the plasma age and Apoptosis Induction in Cancer Cell Abhishek Sau1, Sabyasachi Sen1, Kallol Bera1, Uttam Pal1, membrane is consistent from trial to trial. However, the internal signal de- 1 1 2 creases in size with repeated trials. Biswarup Satpati , Chandrima Das , Samita Basu . 1Saha Institute of Nuclear Physics, Kolkata, India, 2Saha Institute of Nuclear 1780-Pos Board B689 Physics, HBNI, Kolkata, India. Real-Time Imaging of Lithium ‘Hot-Spots’: An Analysis of Ion Conduc- The riboflavin (Rf) is widely distributed in human tissues in free or conju- tance in Aquaporin-1 using Novel Photo-Switchable Sensor gated forms and plays an important role in formation of flavoprotein enzyme. Jinxin V. Pei1,2, Sabrina Heng3,4, Micheal De Ieso1, Georgina Sylvia3,4, The Rf that consists of an isoalloxazin moiety and a ribityl chain, possesses Mohamad Kourghi1, Andrew D. Abell3,4, Andrea J. Yool1,2. mixed features of hydrophobic and hydrophilic functionalities, which 1Adelaide Medical School, University of Adelaide, Adelaide, Australia, generate its affinity towards different biological macromolecules including 2Institute for Photonics and Advanced Sensing, Adelaide, Australia, 3School proteins and DNA. Moreover, the Rf receptor protein constitutes one type of Physical Sciences, University of Adelaide, Adelaide, Australia, 4ARC of tumor biomarker due to their over expression in malignant cells of human Centre of Excellence for Nanoscale BioPhotonics, Adelaide, Australia. breast and prostate cancer. Taking advantage of the above properties of Rf, Aquaporin-1 (AQP1) belongs to the aquaporin (AQP) protein family that herein we have engineered a smart nuclear targeting thiol-modified ribo- facilitate both the flux of water and other solutes across membranes. In flavin-gold nano assemblies, RfS@AuNPs, which accumulate selectively in each of the four channel subunits, AQP1 has pores for water conductance, nucleus without any nuclear-targeting peptides (NLS/RGD) and show photo- while the central pore of the tetrameric channel contributes the monovalent physically in vitro DNA intercalation. A theoretical model using Molecular cation conductance. Among all 13 AQPs, AQP1 is the one that plays a Dynamics has been developed to probe the mechanism of formation and sta- crucial role in cell migration. In various studies, cells with genetic knock- bility as well as dynamics of the RfS@AuNPs in aqueous solution and within down of AQP1 expression have demonstrated significant impaired cell DNA microenvironment. The RfS@AuNPs facilitate binucleated cell forma- motility, whereas reintroduction of AQP1 restored cell migration. The mech- tion that is reflected in the significant increase of DNA damage marker, anism of AQP1 action on cell migration remains unknown, it seems likely to g-H2AX and the arrest of most of the HeLa cells at pre-G1 phase indicating involve ion fluxes. Our previous works have also reported impaired migra- cell death. Moreover, a significant upregulation of apoptotic markers con- tion of HT29 cancer cells when treated with 2 different AQP1 ion channel firms that the cell death occurs through apoptotic pathway. Analyses of the antagonists (AqB011 and Bacopaside I). To understating the role ion channel microarray gene expression of RfS@AuNPs treated HeLa cells show signif- function of AQP1 in rapid cell motility would advance our knowledge of icant alterations in vital biological processes necessary for cell survival. Our the physiological relevance of aquaporin ion channels. Here we report a study reports a unique nuclear targeting mechanism through targeting the newly designed ion sensor named ‘SHL’ that is Liþ-selective and photo- riboflavin receptors, which are upregulated in cancer cells and induce switchable. SHL has been used to monitor AQP1 ion channel activity in apoptosis in the targeted cells. living colon cancer cells to at real time by the appearance of lithium hot spots imaged using confocal microscopy. In HT29 colon cancer cells, which 1783-Pos Board B692 þ has higher AQP1-expressing, the Li hot spots are clustered in the lamelli- Myoblast Protection by Polyethylene Oxide-Polypropylene Oxide Block podial leading edges. The hot-spots are blocked by the AQP1-ion channel Copolymers against Hypo-Osmotic Stress antagonist AqB011. Lithium hot spots are not observable in cell line Mihee Kim1, Karen Haman1, Evelyne Houang2, Wenjia Zhang1, SW620 that lacks comparable membrane expression of AQP1. The correla- Demetris Yannopoulos3, Joseph Metzger2, Frank Bates1, Benjamin Hackel1. tion between the subcellular distribution and the visualized ion channel ac- 1Department of Chemical Engineering and Materials Science, University of tivity of AQP1 channels in cancer cells supports the proposed role of the Minnesota, Minneapolis, MN, USA, 2Department of Integrative Biology and AQP1 ion channel in cell migration which makes it a novel candidate for Physiology, University of Minnesota, Minneapolis, MN, USA, 3Department therapeutics. of Medicine, Cardiovascular Division, University of Minnesota, Minneapolis, MN, USA. Amphiphilic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers, known as poloxamers, have Posters: Biomaterials shown the ability to rescue cells from various types of membrane injury. However, the mechanism of cell protection by block copolymers is still un- 1781-Pos Board B690 clear. To explore the role of block copolymer architecture on membrane pro- Applications of Cross-Linked Cataractous Eye Protein Isolate Films as tection, we synthesized diblock copolymers with control over the Drug Delivery Vehicles composition, molecular weight, and endgroup hydrophilicity/hydrophobicity. Sultana Parveen1, Swagata Dasgupta1. Membrane protection efficacy was studied in cultured murine myoblasts by 1 Chemistry, Indian Institute of Technology Kharagpur, Kharagpur, India. the lactate dehydrogenase (LDH) release assay, in which cells are exposed

BPJ 8643_8645 Monday, February 19, 2018 361a to the stresses of hypo-osmotic shock and isotonic recovery, and membrane per second, while providing specific pore designs that exclude sucrose (342Da) integrity is quantitatively evaluated with or without the presence of block co- and larger solutes, glucose (180Da) and larger solutes or salt (58Da) and larger polymers. The cell protection required sufficient hydrophobicity of the block solutes. This new protein redesign and testing workflow provides us the ability copolymers, controlled by either the hydrophobic PPO block or endgroup to design specified A˚ pore size to conduct Angstrom-scale aqueous separations modification. At least 9 PPO units were needed for protection at 150 mM. by using the precisely designed membrane protein based biomimetic Addition of hydrophobic tert-butyl endgroup enhanced the protection ability membranes. at reduced concentration. With fixed hydrophobicity, increasing hydrophilic PEO block length improved protection. In addition to the cellular integrity assay, we synthesized fluorophore-labeled block copolymers and performed 1786-Pos Board B695 confocal microscopy to image subcellular localization to further understand A Fluorescent Nanoprobe for the Detection of in Situ Temperature the membrane protection mechanism. Changes during Hyperthermia Treatment of Tumors Edouard Alphandery1, Darine Abi Haidar2, Olivier Seksek2, Maxime Thoreau3, Alain Trautmann4, Nadege Bercovici4, Florence Gazeau5, 1784-Pos Board B693 Francois Guyot6, Ime`ne Chebbi1. Interactions of Lipid Multilayers in the Presence of ATP 1Nanobacterie, Bures-sur-Yvette, France, 2IMNC, IMNC - CNRS 8165, Ryan Z. Lybarger, Michele Costantino, Abhinav Ramkumar, Bruce D. Ray, Orsay cedex, France, 3Institut Cochin, paris, France, 4Institut Cochin, Paris, Horia I. Petrache. France, 5MSC, Univ Paris Diderot, Paris, France, 6CNRS UMR 7590, Paris, Department of Physics, Indiana University Purdue University Indianapolis, France. Indianapolis, IN, USA. Magnetic hyperthermia is a promising new treatment, allowing to locally One interesting class of biomaterials are multilamellar stacks of phospholipid induce a temperature increase in cancer tumors that leads to a lethal effect. bilayers. Phospholipids (lipids in short) found in biological membranes are of For this, magnetic nanoparticles are introduced in tumors and exposed to significant interest in material research due to their distinctive mechanical an alternative magnetic field which produces the wanted temperature rise. and electrical properties. In water or buffer solutions, lipids spontaneously While the final biological effect can be assessed by many techniques, the form membrane structures with thicknesses on the order of 4-5 nm. In addi- in situ temperature changes are often difficult to evaluate otherwise than tion, van der Waals attraction causes membranes to form regular stacks of with a regular thermometer. This fairly crude procedure does not allow to many layers with repeat lattice spacings (D-spacings) on the order of tens finely report changes at the tissue or cell level. In this context, we report to hundreds of nanometers. The D-spacing depends on both lipid type and here an original method based on a chemical nanoprobe designed to follow on the composition of the buffer solution in which membranes are formed. temperatures changes during hyperthermia therapy. In our work, AMB-1 Adenosine triphosphate (ATP) is a molecule involved in energy transfer in magnetotactic bacteria produce the magnetic nanoparticles (magnetosomes), biological processes and is therefore of interest in bio-inspired material since we have already shown that this type of nanoparticles had a much better research. Using three complementary experimental methods, namely small- magnetic activity than chemically synthesized particles (Alphandery et al. angle x-ray scattering (SAXS), NMR spectroscopy, and dynamic light scat- ACS Nano, 2011, 5:6279). Interestingly, by introducing rhodamine B in an tering (DLS), as well as molecular dynamics simulations (MD), we show optimized growth medium for these bacteria, we were able to extract fluores- that ATP can be used to modify the material properties of lipid stacks. In cent magnetosomes with new characteristics. Indeed, keeping their typical particular, ATP induces an ubinding transition from multilamellar to single magnetic activity useful for cancer therapy, they would also display a layers followed by reforming of multilayer structures as ATP concentration temperature-dependence fluorescence allowing to perform local measure- is increased. This effect is also seen with ADP and AMP at progressively ments at a microscopic level in biological tissues. The molecular mechanism higher concentrations. These findings can help design applications in which would be discussed, as well as results obtained with different cell types ATP and its hydrolysis products can control material properties of layered (RG2, TC1-GFP, C57NL/6 peritoneal macrophages, U87-MG) and tissues structures. (RG2-implanted rat brain).

1785-Pos Board B694 1787-Pos Board B696 Biomimetic Membrane Design Principles for Angstrom Scale Separation Biocompatible Coated Magnetosome Minerals for Application in the Tingwei Ren1, Ratul Chowdhury1, Peter Butler2, Costas Maranas1, Magnetic Hyperthermia Treatment of Tumors Manish Kumar1. Yasmina Hamdous1, Imene Chebbi1, Chalani Mandawala1, 1Chemical Engineering, Penn State University, State College, PA, USA, Raphael Le Fevre1, Francois Guyot2, Olivier Seksek3, Edouard Alphandery1. 2Biomedical Engineering, Penn State University, State College, PA, USA. 1Nanobacterie, Bures-sur-Yvette, France, 2CNRS UMR 7590, Paris, France, Functional biomimetic membranes are membrane protein-polymer based, or 3CNRS UMR 8165, Orsay cedex, France. membrane protein-lipid based hybrid membranes. Biomimetic membranes Glioblastoma are aggressive brain cancer tumors with very poor prognosis; can preserve membrane protein function and have been applied to areas they often cannot be eradicated using conventional therapies, mainly such as DNA sequencing, drug delivery and water purification. Especially because of their treatment resistance and ability to infiltrate the surrounding the discovery of Aquaporins has promoted studies into the development of brain tissue. In this context, a promising technique leading to an increased biomimetic membranes as highly selective and permeable separation mem- patient life expectancy is currently assessed: magnetic hyperthermia. For branes. This presentation will describe our work on biomimetic membrane this, magnetic nanoparticles are introduced into tumors and exposed to an design principles, including both biomaterial selection for highly integrated alternating magnetic field, inducing a lethal and localized heat increase. biomimetic membranes with compatible protein-polymer combinations and These nanoparticles are either chemically synthesized (superparamagnetic robust membrane protein redesign targeting accurate small molecule iron oxide nanoparticles (SPION)), or obtained from bacteria that naturally separations. produce such particles for their own biological activity. These "magneto- For biomaterial selection, we studied a range of amphiphilic materials used somes" attract much attention because of their specific characteristics. for membrane protein incorporation, and investigated their influence on Indeed, compared with SPION, magnetosomes are better crystallized, Aquaporin performances in biomimetic membranes. We found that average yielding to improved magnetic properties. In this work, they were extracted single protein activity remained unchanged while reconstitution density var- from MSR-1 magnetotactic bacteria, purified to remove potentially toxic ied when using different biomimetic materials. Moreover, we found that organic bacterial residues and replaced by various biocompatible coating minimizing chemical hydrophobicity mismatches between membrane mate- agents. The coated magnetosomes that we obtained were characterized by rials and proteins lead to high protein reconstitution density. This provides transmission electron microscopy (TEM), Fourier transform infrared spec- a strategy to increase the overall permeability of biomimetic membranes. troscopy (FT-IR) and CHNS analysis. In vitro studies were carried out to In addition, we also developed a method to create a membrane protein ‘‘li- evaluate biocompatibility on mouse fibroblast cells (3T3) and mouse glio- brary’’ with different pore sizes. Using Outer Membrane Protein F (OmpF) blastoma cells (GL-261) cell line. The heating properties and cytotoxicity as a ‘‘scaffold’’, we created variety OmpF mutant designs with pore sizes effect of magnetic hyperthermia on GL-261 cell line of the coated magneto- less than 4 A˚ by computational design procedures. Experimental results re- somes were also evaluated. References: 1) Y. Hamdous, et al. J Nanobio- vealed these designs maintained protein water permeability exceeding classical technol 2017. In press and 2) C. Mandawala et al. J. Mater. Chem. B AQPs by more than an order of magnitude at over 10 billion water molecules 2017. In press.

BPJ 8643_8645 362a Monday, February 19, 2018

1788-Pos Board B697 The design of nanoparticles that can induce specific structural transitions in All Aqueous Synthesis of Silica Encapsulated Quantum Dots with Func- nucleic acids is important for nanotechnology applications including gene tional Shells delivery and nanoelectronics. It is known that in biological systems, the Huanhuan Feng1, Xing Ma1, Tingting Zheng2, Jan Bart ten Hove3, binding of cationic proteins induces structural changes in DNA or RNA, Aldrik H. Velders3, Joris Sprakel3. which can affect gene expression or cause the compaction of DNA into chro- 1Harbin Institute of Technology(Shenzhen), Shenzhen, China, 2SPH Medical matin. The anionic backbone of the nucleic acids DNA and RNA allow for Center, Shenzhen, China, 3Wageningen University, Wageningen, non-specific electrostatic interactions with cationic proteins, nanoparticles, or Netherlands. dendrimers. The interaction of nucleic acids and nanoparticles may be tuned All-aqueous synthesis of silica-encapsulated quantum dots with functional shells through changes in nanoparticle size, charge, polarity, or shape. However, Huanhuan Feng[abc], Jan Bart ten Hove[d], Tingting Zheng[ae], Aldrik H. the factors that affect structural transitions are not fully understood. We per- Velders[d] and Joris Sprakel*[a] formed atomistic molecular dynamics simulations of the binding of nucleic We present a simple yet versatile method for encapsulating water-dispersed acids to monolayer-protected gold nanoparticles to elucidate structural quantum dots (QDs) in a silica shell. As our approach, does not require ligand changes that take place for nanoparticles and DNA upon binding. Results exchange, the colloidal stability of the quantum dots is maintained throughout from these simulations were analyzed to determine modes of DNA and the process, which results in monodisperse core-shell particles of individual RNA bending with nanoparticles. Our simulations show that highly charged quantum dots with a well-defined silica shell whose thickness can be accurately nanoparticles cause DNA to bend with little damage to the helix structure, controlled. While other methods often result in a reduction of luminescence, our similar to DNA in the nucleosome. Nanoparticle shape as well as charge approach can retain all or even increases the quantum efficiency of the semicon- is shown to affect the wrapping of nucleic acids with the nanoparticle. ductor dots. We also show how amine-functional groups can be expressed at the Low salt concentrations and high nanoparticle charge cause greater surface of the silica shell, while retaining QD photoluminescence properties, al- disruptions to DNA structure. We find that the roll parameter is the most lowing further surface functionalization. Finally, we demonstrate the versatility important base-pair parameter for DNA bending. Requirements for bending of this strategy by including dopants into the silica shells to tailor the spectral differed significantly between DNA and dsRNA. The degree of DNA response of the core-shell particles by energy transfer. bending is controlled by the charge of the NPs, but ligand flexibility played [a] Physical Chemistry and Soft Matter, Wageningen University & Research, a more significant role in dsRNA bending. We have shown that functional- Stippeneng 4, 6708 WE Wageningen, the Netherlands ized gold NPs can be designed to wrap and compact both RNA an DNAs [b] Dutch Polymer Institute (DPI), P.O. Box 902, 5200 AX Eindhoven, the with fine control of binding strength through NP charge and ligand Netherlands chemistry. [c] School of Materials Science and Engineering, Harbin Institute of Technol- ogy (Shenzhen), China 1791-Pos Board B700 [d] Laboratory of BioNanoTechnology, Wageningen University & Research, Antibacterial Properties of Curcumin Loaded Graphene Oxide Flakes Bornse Weilanden 9,6708 WG Wageningen, The Netherlands Valentina Palmieri1, Francesca Bugli1, Margherita Cacaci1, [e] Shenzhen Key Laboratory for Drug Addiction and Medication Saftey, Riccardo Di Santo1, Alberto Vitali1, Riccardo Torelli1, Maura Di Vito2, Department of Ultrasound, Peking University Shenzhen Hospital & Biomed- Claudio Conti3, Maurizio Sanguinetti1, Marco De Spirito1, ical Research Institute, Shenzhen-PKU-HKUST Medical Center, Shenzhen Massimiliano Papi1. 518036, China. 1Universita` Cattolica del Sacro Cuore, Roma, Italy, 2Universita` di Bologna Alma Mater Studorium, Bologna, Italy, 3Institute for Complex Systems, 1789-Pos Board B698 Roma, Italy. De Novo Designed Proteins for Colloidal Stabilization and Improvement of Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for Cellular Uptake serious hospital infections worldwide and represents a global public health Tingting Zheng1,2, Felipe Perona Martı´nez3, Ingeborg Maria Storm2, problem. Curcumin, the major constituent of turmeric, is effective against Wolf Rombouts2, Joris Sprakel2, Renko de Vries2, Romana Schirhagl3. MRSA but at high and cytotoxic concentrations or in combination with an- 1 2 Shenzhen-PKU-HKUST Medical Center, Shenzhen, China, Physical tibiotics. The major issue in curcumin-based therapies is the poor solubility 3 Chemistry and Soft Matter, Wageningen, Netherlands, Faculty of Medical of this hydrophobic compound and its cytotoxicity at high doses. In this Sciences, Groningen, Netherlands. work, we describe the efficacy of GOCU, a composite made of curcumin Fluoresence nanodiamonds (FNDs) are gaining increasing attentions in cutting- and Graphene Oxide (GO) in the MRSA infections treatment. GO is a edge of biophysical fields (e.g. molecular imaging, biosensing and cell tracking, nanomaterial with a large surface area and high drug loading capacity. etc.), owing to its special properties, including biocompatibility and special op- GO has also antibacterial properties due mainly to a mechanical cutting tical and magnetical properties. In general, the intensity of its fluorescence signal of the bacterial membranes. We investigated GOCU properties by means relies on number of diamond defects, which is strongly related to size of FNDs. of Dynamic Light Scattering, Electron and Atomic Force Microscopy and For instance, FNDs around 100 nm is one of the most commonly used candidates Zeta Potential. We analysed growth curves and Minimal Inhibitory and for magneto-optic sensors. However, especially for FNDs R 100 nm, their weak Bactericidal concentrations (MIC and MBC), and cytotoxic effects on cells colloidal stability and poor cellular uptake are common problems that limiting its by MTT assay. We report the capacity of GO to support and stabilize biological applications. Lots of beautiful works report that stable FNDs colloids curcumin molecules in water environment. Curcumin creates a corona are achieved via surface chemical modifications. However, rare is reported about around Graphene Oxide sheets and modifies surface charge towards more achieving this in other manners apart from chemical bond formation. Here we positive values. We demonstrate the efficacy of GOCU against MRSA at present de novo designed protein polymers C4 and C4K12, which strongly bind concentration down to 2 mg/ml, while GO alone is effective only at concen- on surface of FNDs in a physical adsorption way. C4 is randomly wrapping trations higher than 100 mg/ml. However, GOCU displays low toxicity on around FNDs (MC4:MFNDs=420:1), while C4K12 is brush-like attaching on fibroblasts cells in respect to bare GO. The low MIC of GOCU against FNDs (MC4K12:MFNDs=430:1). Dynamic light scattering results verify that MRSA and absence of cytotoxicity open new applications of GOCU nano- both proteins significantly increase FNDs stabilities in salt, while naked FNDs material in the global health challenge of the antibiotic resistant infections aggregates immediately upon normal saline. In addition, cellular uptake results treatment. via confocal microscopy shows that protein decorated FNDs have less aggrega- tion in buffer and have better cellular phagocytosis. Fluorescence and electron 1792-Pos Board B701 spin resonance (ESR) measurements indicate that proteins coating on surface Graphene Oxide Laser Printing for Controlled STEM Cells Differentia- of FNDs neither affect their magneto-optical properties, nor their biocompatibil- tion and Antibacterial Effects ities. Hence, protein coating is a promising way to stabilize FNDs colloids. Valentina Palmieri1, Marta Barba2, Lorena Di Pietro2, Silvia Gentilini3, Francesca Bugli2, Rosanna Larciprete3, Wanda Lattanzi2, 1790-Pos Board B699 Maurizio Sanguinetti2, Marco De Spirito1, Claudio Conti3, Design of Histone-Mimic Nanoparticles for DNA and RNA Compaction Massimiliano Papi1. using Molecular Modeling 1Physics, Universita` Cattolica del Sacro Cuore, Rome, Italy, 2Universita` Matthew Manning, Jessica A. Nash, Yaroslava G. Yingling. Cattolica del Sacro Cuore, Rome, Italy, 3CNR, Rome, Italy. Materials Science and Engineering, North Carolina State University, Raleigh, Graphene and Graphene oxide (GO) are capable of inducing stem cells differ- NC, USA. entiation into bone tissue with variable efficacy depending on reductive state of

BPJ 8643_8645 Monday, February 19, 2018 363a the material. Thus, controlling the oxidative state of the GO it is possible, in lular granules or as net-like extracellular structures.PHAs are biodegrade to principle, to modulate the osteogenic process and the bone mineral density harmless products and non-toxic materials. Additionally, they are highly ver- distribution. In this study, we laser-printed GO surfaces in order to obtain satile and have a broad range of physicochemical properties, depending on both a local photo/thermal GO reduction and the formation of nano-wrinkles monomer composition. Typically scl-PHA has properties close to conven- along precise geometric pattern. Calvarial-derived Mesenchimal Stem cells tional plastics such as polypropylene or polyethylene, while mcl-PHA has (CMSC) were isolated in primary culture from skull calvarial fragments and elastomeric properties. One of the appealing features of PHA is its biodegrad- their behaviour was characterized on GO patterns. Initially, after cells adhesion ability, which gained the biopolymer interest as a potential alternative to on the surface, stem cells migrated and accumulated on the reduced/wrinkled traditional petrochemical plastics. One of greatest advantages of PHAs surface. When the local density of the stem cells on the reduced stripes was over other biodegradable polymers is their ability to degrade under both high, cells started to proliferate and occupy the oxidized/flat area. The designed aerobic and anaerobic conditions. Biodegradation is defined as a set of surfaces morphology guided stem cell orientation and the reduction accelerated chemical reactions through which macromoleclues are broken down into differentiation. Furthermore, the reduced nano-wrinkles greatly inhibited the smaller molecules by different microorganisms. PHAs are excellent biocom- growth of S. aureus. We propose a promising laser-printing technology for patible materials due to the lack of toxicity against human tissues and blood. tissue engineering and regenerative medicine, based on graphene to enhance In vivo tests have shown that these polyesters are biocompatible to osteo- cell growth and differentiation in a controlled way. We show that by adjusting blastic and epithelial cells. PHA is a product of cell metabolism, and together the laser emission is possible to finely tune the GO reduction and shape sub- with the fact that 3-hydroxy butyric acid (the product of degradation) is nor- strate landscape to spatially control bone regeneration and microbial infection. mally present in the blood at concentrations between 0.3 and 1.3 mmol l-1 it This strategy can offer a revolution in present and future trends of medicine and creates potential medical applications in wound management, implants for surgery. tissue engineering and drug delivery.The main goal of research project is to produce a range of novel functionalized polymers to be used in infected 1793-Pos Board B702 wound treatment, for production of implant coatings and tissue regeneration Electrospun Poly(Amino Acid) Based Nano GEL Fiber Matrices and Their scaffolds. Biocompatibility and Biodegradability This work is supported by grants from the Polish Ministry of Science and Kristof Molnar1, Constantinos Voniatis1, Daniella Feher2, Andrea Ferencz2, Higher Education 7150/E-338/M/2017 and National Center for Research and Gyorgy Weber2, Miklos Zrinyi1, Angela Jedlovszky-Hajdu1. Development 0090/L-7/2015. 1Department of Biophysics and Radiation Biology, Semmelweis University, Budapest, Hungary, 2Department of Surgical Research and Techniques, 1795-Pos Board B704 Semmelweis University, Budapest, Hungary. On the Sucrose-Induced Self-Assembly Kinetics of HM Pectin Interest in the development of biocompatible and biodegradable polymer Daniela Giacomazza1, Donatella Bulone1, Pier L. San Biagio1, matrices has increased with their usability in wide range. Artificial polymer Rosamaria Marino2, Romano Lapasin3. based fibrous scaffolds resemble to the native extracellular matrix. Electrospin- 1CNR-IBF, Palermo, Italy, 2Silvateam Food Ingredients, Rende (CS), Italy, ning technique enables the possibility to prepare a wide variety of matrices built 3Dipartimento di Ingegneria e Architettura, University of Trieste, Palermo, of fibers in the nano or micrometer diameter range. These matrices can be Italy. tailored for tissue engineering, however the use of water soluble polymers is Many researches have been dedicated to the comprehension of the mecha- inappropriate because of their fast dissolution in biological fluids. By utilizing nisms underlying the dynamical arrest of gelling systems due to their reactive electrospinning, chemically cross-linked gel fibers can be created involvement in several technological fields, from food to biotechnology. In which without enzymatic or chemical degradation will not dissolve in physio- particular, the attention is focused on the structural organization character- logical conditions. izing the final gelled state. The question about a unifying point of view for Poly(amino acid)-based polymers that have desirable chemical, mechanical colloidal gelation, colloidal glass transition and chemical gelation is still un- and biological properties have recently emerged as promising new class of answered. In the present work, we have studied the rheological properties of biomaterials. The aim of our research was to make poly(amino acid) based solutions containing few percent of pectin and increasing concentration of su- scaffolds available for tissue engineering. In the present work have utilized crose, from 0 to 65% w/w, with the aim of rheologically investigating the the anhydrous form of poly(aspartic acid), the poly(succinimide) as base role of the co-solute on the gelation properties of the polysaccharide. Our polymer. To prevent the polymer fibers from early dissolution poly(succini- data suggest that sucrose plays a crucial role in governing the structural char- mide) molecules were grafted by thiol side chains. During the electro- acteristics of high methoxyl pectin macroscopic gelation in a way that is spinning process at cross-linking reaction took place between these side extremely sensitive to small co-solute concentration changes. The time evo- chains. For the characterisation AFM, SEM, IR was used. Investigation of lution of the mechanical properties is in both qualitative and quantitative swelling behaviour due to the change of pH was carried out on the macro- terms strictly affected by the sucrose presence and the transition from sol scopic matrix of nanofibers. It was followed by in vivo experiments on 6-6 to gel state occurs when the excluded volume effects and the attractive inter- albino rats for 72 h and 1 week period. We found that the fiber matrix hydrol- actions are able to create a three-dimensional network by freezing the poly- ise in 72 hours and turns into poly(aspartic acid) based hydrogel matrix and mer dynamics, as it occurs in attractive glasses or weakly attractive colloidal degrades in 1 week. Histology showed no inflammation and irritation. These dispersions. preliminary experiments proved biocompatibility and biodegradability of our material. 1796-Pos Board B705 Financial support: OTKA K 115259, NKFIH FK 124147, Supported BY the Adhesive Nanomaterials Derived from the Barnacle Amphibalanus Am- U´ NKP-17-3-III-SE-18 New National Excellence Program of the Ministry of phitrite Polymerize by Molecular Recognition of Sequences Human Capacities. Elizabeth A. Yates1,2, Ashley M. Schenck1, Catherine M. Yip1, Kenan P. Fears3, Christopher R. So3, Kathryn J. Wahl3. 1794-Pos Board B703 1Chemistry, United States Naval Academy, Annapolis, MD, USA, Novel Biocompatible Polymers for Biomedical Applications 2Chemistry Department, Code 6176, GLSIP Greater Laboratory Scientific Tomasz Witko1, Maciej Guzik2, Kamila Sofinska 2, Karolina Stepien3, Interaction Program, Naval Research Laboratory, Washington, DC, USA, Karolina Podobinska4. 3Chemistry Division, Code 6176, Naval Research Laboratory, Washington, 1UJ Faculty of Physics, Astronomy and Applied Computer Science, DC, USA. Jagiellonian University, Cracow, Poland, 2Jerzy Haber Institute of Catalysis Arthropods, e.g., spiders, moths, and flies, commonly use fibrous materials to and Surface Chemistry, Polish Academy of Sciences, Cracow, Poland, interact with their environment. Interestingly, this has also been found in 3Department of Pharmaceutical Microbiology, Medical University of aquatic and marine members of arthropod that use silks as underwater adhe- Warsaw, Warsaw, Poland, 4Department of Experimental and Clinical sives. Recently added to this group are barnacles, which are arthropods that Physiology, Medical University of Warsaw, Warsaw, Poland. produce a micron-thick layer of proteinaceous glue structured like amyloid Biopolymers represent one of the leading sectors for bio-based products. nanofibers that provide permanent wet adhesion. To understand sequences Their expected growth is foreseen to be significant within the next 4 years. critical to cement assembly, we derive seven synthetic peptides from Polyhydroxyalkanoates (PHAs) represent a class of optically active biode- consensus barnacle cement sequences of Amphibalanus amphitrite glue pro- gradable polyesters, accumulated by numerous bacteria as discrete intracel- teins. These barnacle cement derived peptides (BCDPs) contain conserved

BPJ 8643_8645 364a Monday, February 19, 2018 segments of Gly/Ser/Ala/Thr residues, which are the building blocks of silk of MT transport behaviors using biocompatible 3D structures that are gener- associated materials. We hypothesize that each BCDP sequence yields unique ated by chemically modifying surfaces with self-assembled monolayers aggregation kinetics and amyloid-like morphologies, providing a means for (SAMs) and silicifying mammalian cells. The SAMs provide a facile the organism to control polymerization rate and physical properties of wet approach to regulate cell morphology and achieve a range of 3D surfaces adhesive biomaterials. To understand cement protein assemblies, we interro- capable of supporting MT transport. For example, carboxyl-terminated gate BCDP sequence interactions using multiplexed Thioflavin T polymeri- SAMs produced an elongated cell morphology that appear to affect the tra- zation assays. Time-resolved fluorescence measurements demonstrate that jectory, but not the velocities, of MT transport. This approach allows for the polymerization is sequence specific with varying activity and onset times: production of topographically unique and preserved surfaces, enabling the BCDP 1 and 2 form short aggregate fibrils, while BCDP 6 forms discrete study of MT gliding and ability to guide motion on complex 3D substrates. and long-ranged nanomaterials. Other BCDPs are dormant and have no activ- Sandia National Laboratories is a multi-mission laboratory managed and ity. Active BCDPs form distinct amyloid-like fibrils with beta-sheet structure, operated by National Technology and Engineering Solutions of Sandia, characterized by atomic force microscopy and Raman spectroscopy. Further- LLC., a wholly owned subsidiary of Honeywell International, Inc., for the more, BCDP6 fibrils are observed to polymerize dormant peptides and U.S. Department of Energy’s National Nuclear Security Administration un- demonstrate specific sequence recognition. Small, synthetic BCDPs are der contract DE-NA-0003525. shown here to produce nanomaterials similar in morphology and molecular structure to complex barnacle glue. Synthetic peptides offer an encouraging 1799-Pos Board B708 means to scale up and further understand native barnacle cement within ma- Mapping Spatial Distributions of Pericellular Stiffness in a Naturally rine biofouling. Derived Extracellular Matrix Mark Keating, Elliot Botvinick. 1797-Pos Board B706 University of California, Irvine, Irvine, CA, USA. Engineered Caf1 Protein Polymers form Tuneable Bioactive Hydrogel Many cell types in vivo are surrounded by an extracellular matrix (ECM), Scaffolds the molecular and mechanical scaffolding of natural tissues. Quantifying the Helen Waller1, Gema Dura2, Daniel T. Peters1, Adrian Yemm1, mechanical interactions between the cell and its ECM both spatially and Jeremy H. Lakey1. temporally, at a scale relevant to the interaction, is imperative to study 1 Institute for Cell and Molecular Biosciences, Newcastle University, how cells are regulated in physiological and pathological processes. Here 2 Newcastle upon Tyne, United Kingdom, Department of Chemistry, we use optical tweezers based active microrheology (AMR) to measure Newcastle University, Newcastle upon Tyne, United Kingdom. the distribution of pericellular stiffness surrounding isolated dermal fibro- Here, we describe a new animal-free biomaterial with potential uses in blasts embedded within collagen gels and observe important new insights 3D-tissue culture and regenerative medicine due to its low cost, high stabil- into how cells modulate their mechanical microenvironment in a contrac- ity and definable bioactivity. Capsular antigen fraction1 (Caf1) is a protein tility and matrix metalloproteinase-dependent manner. The pericellular from the plague bacterium Yersinia pestis that is secreted via the chaperone- stiffness surrounding isolated DFs was measured in four conditions: normal usher pathway and protects the pathogen from phagocytosis by forming a media (control), ROCK inhibitor (Y27632) for 1 hour, broad spectrum non-stick protective layer around the cell. The 15.5kDa monomer has an MMP inhibitor (BB94) for 24 hours, or a combination of Y27632 and Ig-like fold and resembles the extracellular matrix protein fibronectin. The BB94 for 1 and 24 hours respectively. With respect to these results, coop- subunits polymerise via donor-strand complementation, forming a highly eration may be necessary between both MMP activity and cellular contrac- stable non-covalent polymer. In this work, we demonstrate the production tility in order to create a normal pericellular mechanical topography within of recombinant Caf1 polymers via batch fermentation using Escherichia the complex material of a type I collagen system. There is a growing body coli. These are secreted by the bacterium into a flocculent layer above of correlations between bulk ECM stiffness and cell phenotype in tissue the cell pellet, and can be easily extracted and purified in large quantities. models, which tend to be performed with a set of ECMs, each with unique We show the polymer retains its robust thermostability in a variety of but homogenous bulk stiffness, with shear moduli spanning 30 to 1000 Pa. chemical conditions by circular dichroism and SDS-PAGE, and observe If we estimate shear moduli values from observed a values (via GSER, the large size of polymers by electron microscopy and SEC-MALS. This which inherently introduces error), then remarkably this same range was analysis revealed polymers containing up to 250 subunits, with observed by AMR around single cells in our study. This begs the question: lengths >1.5 mm and weights in the MDa range. Additionally, we have which stiffness value is important? We speculate that no single value of selectively reversed the natural non-stick behaviour of the WT polymer stiffness guides cells, rather it is the evolution and distribution of stiffness by introducing an integrin binding sequence, RGDS, into loop5 that can that must be accounted for within mechanics focused cell-ECM hypothesis promote fibroblast adhesion to the polymer surface. Additional bioactive testing. peptides (up to 19 amino acids in length), including bone morphogenic pro- tein2, collagen and laminin motifs, were then introduced at different posi- 1800-Pos Board B709 tions within Caf1. Finally, PEG based chemical cross linkers were used Scaffold Stiffness at Microscale Directs Stem Cell Lineage Specification to form stable 3D hydrogels with designed porosities and tuneable stiffness, Yang Song1,2, Kang Xu1, Sixiang Wang1, Jeremiah W. Woodcock2, ideal for use in cell culture and drug delivery applications. The combination Xiaoling Liao3, Martin Y.M. Chiang2, Li Yang1. of these motifs into tuneable Caf1 hydrogels will help expand the function- 1School of Bioengineering, Chongqing University, Chongqing, China, ality of this exciting new biomaterial for use in a variety of biomedical 2Material Measurement Laboratory, National Institute of Standards and applications. Technology, Gaithersburg, MD, USA, 3Institute of Biomedical Engineering, Chongqing University of Science and Technology, Chongqing, China. 1798-Pos Board B707 This study is to demonstrate scaffold stiffness at microscale strongly directs Microtubule Transport on 3D Biocompatible Nanostructures the stem cell lineage specification in 3D environment. This demonstration Haneen Martinez1, Matthew N. Rush2, Jimin Guo3, Jeff Brinker1, has been carried out through investigating the osteogenic potential of dental Geroge D. Bachand1. follicle stem cells (DFSCs) grown within fibrous scaffolds. Electrospinning 1 2 Sandia National Labs, Albuquerque, NM, USA, Los Alamos National labs, process blending PCL/SF/CNC (polycaprolactone / silk fibroin/ cellulose 3 Los Alamos, NM, USA, University of New Mexico, Albuquerque, NM, nanocrystals) was adopted to fabricate scaffolds composing of nanofibers USA. mesh with multi-scale macroscopic and microscopic stiffness. The micro- Nanoscale transport using the kinesin-microtubule (MT) system has been structure of fibrous scaffold was detected through scanning electron micro- successfully used in a wide range of nanotechnological applications scope (SEM); while the macroscopic and microscopic stiffness of scaffolds including self-assembly, nanofluidic transport, and biosensing. For the were tested through a conventional tensile test and the colloidal probe force most part, transport has been achieved using physical or chemical patterns spectroscopy using atomic force microscopy (AFM), respectively. Biocom- to guide MT motion in the gliding motility geometry, in which surface- patibility, osteogenic differentiation, and mechanosensitive gene expression adhered kinesin motors bind and translate MT filaments across a planar sur- of DFSCs to scaffolds were assessed via various bioassays. CNC-based scaf- face. These approaches require lithographically patterning features onto a fold forms web-like microstructure composed of rigid-flexible nanofibers surface, which limit the MT trajectories, where MTs can escape the barriers with bimodal diameter distributions. This heterogeneity increases with and lead to stalling or complete loss of MTs. Here, we explore the regulation CNC content. Also, the macroscopic stiffness of scaffold increases with

BPJ 8643_8645 Monday, February 19, 2018 365a the content, while the microscopic stiffness decreases to a level comparable against radial position r to the equation u = Ar-n,whereA and n are fitting to the cell stiffness. In vitro tests indicate biological efficacy increases with constants. For a classically linear elastic material, n = 2, whereas the data the content as well. While the macroscopic stiffness of scaffold is important for collagen fits to n z 1, indicating displacements propagate over a longer as a load-bearing construct during the regeneration process, this study range in fibrous materials than classically linear elastic ones. In ongoing strongly reveals that a maximum mechanical interaction between cells and research, we are testing the mechanism for long range displacements. Prelim- scaffold can occur to efficaciously direct stem cell osteogenesis when cell inary results indicate the presence of two mechanisms, alignment of fibers stiffness is comparable to the scaffold stiffness at microscale. under tension and weakening of the network under compression. It remains Acknowledgement: Innovation and Attracting Talents Program for College and to be determined whether contractile cells use these mechanical mechanisms University (‘111’ Project) (B06023); the National Institute of Standards and to reorganize fibrous tissues or to communicate mechanically with their Technology [ADE12017-0000]; The National Natural Science Foundation of distant neighbors. China (11532004, 31271014). 1803-Pos Board B712 1801-Pos Board B710 NMR Studies of Secondary Structure and Compaction of Minielastin Biomechanical Characterization of Fibroblast-Populated Collagen Tissue Ma. Faye Charmagne Carvajal1, Kelly Greenland2, Jonathan Preston2, Models Ronald Koder2, Richard Wittebort1. Zheng Yie Yap1, Chen Wai Kok1, Ting Wei Law1, Melville Vaughan2, 1Chemistry, University of Louisville, Louisville, KY, USA, 2Physics, The Gang Xu1. City College of New York, New York, NY, USA. 1Department of Engineering and Physics, University of Central Oklahoma, Tropoelastin, the soluble precursor of elastin, is highly flexible. Thus, struc- Edmond, OK, USA, 2Department of Biology, University of Central ture elucidation has been and remains a challenge. Minielastin constructs Oklahoma, Edmond, OK, USA. mimic the structure of tropoelastin with alternating hydrophobic and Fibroblasts play an important role in tissue growth and wound healing cross-link modules. Simpler minielastin constructs allow elucidation of by generating contractile force or tension within collagen network. As a the secondary structure with residue-specific resolution using NMR spec- result, fibroblast-populated collagen lattices have become a common tissue troscopy. Complete chemical shift assignments of the backbone and side model for studying the biology and biomechanics of wound healing. Howev- chain were obtained via 2D and 3D NMR experiments. Secondary shifts re- er, it is challenging to estimate the amplitudes of the cellular tension in the vealed disorder in the hydrophobic domain and weakly helical structure of engineered tissue in normal and wound healing conditions. In this study, the cross-link domain. Amide proton NOE suggested compaction in the we engineered the tissue that is made up mainly of human dermal fibroblasts cross-link module. Furthermore, using PFGNMR we have shown that this and type I collagen. We used the Optical Coherence Tomography to compaction increases at increasing temperature. These findings will help image and quantify the development and reorganization of the collagen in understanding the coacervation process of tropoelastin. Coacervation is network driven by fibroblast cells. We also probed the mechanical tension important in the synthesis of insoluble elastin and is thought to be favored in the tissue by introducing a circular dissection on the tissue and quantifying by hydrophobic effect. the ensuing wound expansion. Finite element models were created to simulate the wound expansion and estimate the magnitude of the associated tension. Quantifying how mechanical forces regulate the tissue generation will help 1804-Pos Board B713 us understand the biophysical mechanisms behind wound healing. NMR Study of Elastin’s Elasticity Mechanism Nour Jamhawi, Richard Wittebort. 1802-Pos Board B711 Department of Chemistry, University of Louisville, Louisville, KY, USA. Mechanical Response of Fibrous Materials to Local Contractile Loads Elastin is the most abundant elastomer in nature. The recoil mechanism is Brian Burkel, Maria Proestaki, Peter Grimmer, Jacob Notbohm. believed to be entropically driven for which there re two molecular level Engineering Physics, University of Wisconsin-Madison, Madison, WI, USA. theories. Recoil in one theory is a rubber-like mechanism in which the When cells contract or migrate within a three-dimensional fibrous matrix, decrease in entropy with stretch is due to a decrease in conformational en- they pull on the fibers surrounding them, generating displacements within tropy from backbone ordering. The alternative is a decrease in solvent en- the fibrous network. The resulting displacement field decays over distance tropy with stretch from solvent ordering, i.e., the hydrophobic effect. from the cell. As the constitutive relationship between stress and strain for Using a novel double quantum (2Q) pulse sequence in solid state NMR; fibrous materials remains to be determined, the connections between force, water ordering at elastin fiber surface was studied quantitatively. We displacement, and the fibrous structure are unclear. This is especially true find that water is weakly oriented at the Elastin-water surface when the for nonuniform forces such as those applied by a contractile cell. Here we fiber is relaxed and stretching the fiber or increasing the temperature explore the mechanics of fibrous materials using microscale, contractile significantly increases the 2Q signal, i.e., increases the ordered water frac- displacement fields that mimic those due to a cell. We apply displacements tion. Furthermore, the 2Q signal decreases with the addition of high mo- using poly(N-isopropylacrylamide), a hydrogel that undergoes a phase tran- lecular weight polyethylene glycol (20kDa or 6kDa PEG) that is known sition when heated, resulting in a >50% decrease in volume. By embedding to precipitate proteins and decrease their hydration in solution. We believe particles made of this gel into networks of fibrous collagen I and controlling PEG causes less water to be available on the fibers surface and thus less the temperature, we can generate well-controlled, repeatable contractile ordered water. Also, this is likely the reason why the fibers become less forces within the network. We quantify the resulting displacements by imag- flexible. Our preliminary results in both water and high molecular weight ing the collagen fibers with a confocal microscope and applying digital image PEG experiments directly correlate the recoil mechanism with the hydro- correlation. To analyze the displacement field, we fit radial displacements u phobic effect.

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