544a Wednesday, February 21, 2018

Symposium: Transmembrane Signals and 2692-Symp Serial Femtosecond Crystallography of G Protein-Coupled Receptors Signaling Mechanisms Vadim Cherezov. University of Southern California, Los Angeles, CA, USA. 2689-Symp G Protein-Coupled Receptors (GPCRs) are versatile cellular gatekeepers Structure and Dynamics of Functional Chemotaxis Receptor Nanoarrays that regulate a variety of physiological processes in the human body and by NMR and Hydrogen Exchange have been targeted by a large share of pharmaceutical drugs. Structural Maryam Kashefi, Xuni Li, Elizabeth R. Haglin, Lynmarie K. Thompson. studies of this superfamily have been enabled a decade ago by multiple Chemistry, University of Massachusetts, Amherst, MA, USA. breakthroughs in technology that included receptor stabilization, crystalliza- Chemotaxis receptors bind ligands from outside the cell and transmit signals tion in a membrane environment, and microcrystallography. The recent across the membrane to direct the swimming of bacteria towards favorable emergence of X-ray free electron lasers (XFELs) has further accelerated environments. The signaling mechanism is thought to involve a 2 A˚ piston structural studies of GPCRs and other challenging macromolecules by over- displacement of an alpha helix that extends through the periplasmic and coming radiation damage and providing access to high-resolution room tem- transmembrane domains. However, it is not known how the signal then perature structures and dynamics using micrometer-sized crystals. This talk propagates an additional 200 A˚ through the cytoplasmic domain to control will summarize key technology advancements and major milestones of a kinase bound at the membrane-distal tip of the receptor. To investigate this GPCR research at XFELs, and provide a brief outlook on future develop- process, we prepare functional complexes of the aspartate receptor cyto- ments in the field. plasmic fragment with the kinase CheA and a coupling protein CheW that form extended, hexagonal arrays similar to those found in the cell. The re- ceptors in these native-like nanoarrays exhibit surprising dynamic proper- Symposium: Protein Dynamics, Folding, and ties. Mobility-filtered solid-state NMR experiments have identified a Allostery II: Dynamics and Function highly mobile region on the N terminal side of the methylation domain of the receptor, which is involved in adaptation. Mass spectrometry experi- 2693-Symp ments have revealed slow hydrogen exchange in the protein interaction Structure and Dynamics of the CheY Response Regulators from domain of the receptor, at the binding sites for CheA and CheW, and un- Rhodobacter sphaeroides usual EX1 hydrogen exchange throughout the receptor. Comparison of these Lorena Varela, Matt Smith, Lukas Stelzl, Christian Bell, Judith Armitage, properties in the kinase-on and kinase-off signaling states is being used to Christina Redfield. test and further develop models for the role of dynamics in signal propaga- Dept. of Biochemistry, University of Oxford, United Kingdom, Oxford, tion through the chemoreceptor cytoplasmic domain. This research sup- United Kingdom. ported by NIH grant GM120195. The chemotaxis signalling network of E. coli. depends on autophosphorylation of a histidine protein kinase (HPK) in response to a signal from a sensor 2690-Symp domain, with subsequent transfer of the phosphoryl group to an aspartate on Tuning the Signaling Output of Protein Kinase C response regulator (RR) proteins that bind to the flagellar motor and alter its Alexandra C. Newton. rotation. CheY is a 14kDa single domain RR that is conserved across motile University of California, San Diego, La Jolla, CA, USA. species. It is formed by 5 a-helices and 5 b-strands surrounding a conserved Protein kinase C (PKC) transduces the plethora of signals resulting phosphoryl accepting aspartate residue, and once phosphorylated diffuses to from lipid hydrolysis. Precise control of the amplitude of its signaling the flagellar motor, binding to its FliM component to cause switching of rota- output is essential for cellular homeostasis, and disruption of this control tional direction. The photosynthetic bacterium Rhodobacter sphaeroides has leads to pathophysiologies such as cancer or degenerative diseases. multiple chemosensory pathways formed by homologues of the E. coli chemo- Key to the regulation of the signaling output of most PKC isozymes is sensory proteins. It has six CheY homologues with different effects on chemo- the ability of the cytosolic enzyme to respond to the membrane-embedded taxis. Only CheY6 is able to stop the flagellar motor but either CheY3 or CheY4 lipid second messenger, diacylglycerol, in a dynamic range that prevents are also required for chemotaxis. signaling in the absence of agonists but allows efficient activation in NMR and computational methods have been used to answer questions about the response to small changes in diacylglycerol levels. This contribution de- structure, dynamics and function of two of the CheY’s, CheY3 and CheY6. scribes the regulatory inputs that control the spatiotemporal dynamics of NOEs, chemical shifts and residual dipolar couplings are used to define the PKC signaling. structures of CheY3 and CheY6 in their inactive and active states, where phos- - phorylation is mimicked using BeF3 . We have investigated fast timescale 2691-Symp 1 15 Redox Dependent Trans-Membrane Signaling backbone dynamics using the { H}- N heteronuclear NOE and have used William A. Cramer. CPMG relaxation dispersion experiments to detect low populations of alterna- Biological Sciences, Purdue University, West Lafayette, IN, USA. tive conformations. CheY6 differs from the other R. sphaeroides CheYs and Redox dependent trans-membrane (TM) signaling which activates a kinase E. coli CheY by the insertion of a ten-residue loop before the C-terminal helix. (serine-threonine ‘‘Stt7’’ in the green alga C.reinhardtii) regulates the distribu- We have deleted this loop region from CheY6 in order to determine, using tion of light energy between the two photosystems of oxygenic photosynthesis. in vivo and in vitro assays, if it plays a role in the unique function of CheY6 in R. sphaeroides. Redox reactions mediated by the cytochrome b6f complex (pdb 4OGQ) on the electrochemically positive side of the chloroplast thylakoid membrane activate the kinase on the trans (negative) side, leads to phosphorylation of light- 2694-Symp harvesting chlorophyll proteins and their redistribution between the two photo- Proteostasis Function and Disfunction: The Folding Machines that systems (‘‘state transitions’’). Cloned and expressed Stt7 was characterized in Maintain Proteome Health solution by analytical ultra-centrifugation, electron microscopy, and electro- Judith Frydman. spray mass spectrometry as a 754 residue polypeptide organized as a tetramer Stanford University, Stanford, CA, USA. with MW = 332 kDa having 55% a-helix content, which was active in vitro un- Correct protein folding and quality control are essential for normal cellular der reducing conditions. Physical interaction of b6f and Stt7 is implied by function. The accumulation of misfolded proteins is emerging as central to a perturbation of the secondary structure of Stt7 in the presence of the cyto- wide range of disease states, including many neurodegenerative disorders chrome b6f complex. However, the structure basis for the inference that the such as Huntington’s and Prion Disease. A complex network of molecular trans-membrane signaling is mediated by a trans-membrane (TM) protein chaperones facilitate protein folding and assembly and monitor all aspects of topology is complicated by: (i) the presence of 4 proline residues in the putative protein homeostasis. Chaperones assist the folding of newly translated and Stt7 TM domain, which is unprecedented in the a-helical trans-membrane stress-denatured proteins, as well as affects protein quality control. Our domain of an integral membrane protein, the problem more extreme because research investigates the mechanisms and pathways by which chaperones carry there are 16 Pro residues in the TM domain of tetrameric Stt7; (iii) the inter- out these diverse functions. Systems approaches identified a chaperone network lamellar space is limited in the stacked thylakoids; (iv) utilizing the 2.5 A˚ linked to the protein synthesis apparatus assists protein biogenesis. The emer- crystal structure of the b6f complex (pdb 4OGQ), there is insufficient space gence of this translation-linked chaperone network likely underlies the elabo- for a docking site on the lumen or p-side of the b6f complex. Support: rate co-translational folding process necessary for the evolution of larger NIHGMS-038323 and Henry Koffler Professorship (WAC) and NIHDK- multidomain proteins characteristic of eukaryotic cells. A stress-inducible 063491 (JPW). chaperone network protects cells from environmental stress and assists quality

BPJ 8705_8719 Wednesday, February 21, 2018 545a control. These chaperones also communicate with the ubiquitin-proteasome teins, b and g. Notably, in the absence of Ca2þ the g1 subunit promotes large pathway to clear misfolded proteins from the cell. Protein quality control in shifts of the BK conductance-voltage curve towards more negative voltages. the eukaryotic cytosol relies on the sequestration of misfolded cytosolic pro- However, very little is known about how a and g1 subunits interact. In partic- teins in specific quality control compartments. Our studies of chaperone func- ular, what is the association stoichiometry between a and g1 subunits. Here, we tion provide a framework to understand the link between protein misfolding propose a method to answer this question, using lanthanide resonance energy and human disease. transfer (LRET). The donor was Tbþ3 that was chelated in lanthanide binding tags (LBT) engineered either into the C-terminal of the a S1 transmembrane 2695-Symp segment, or in the N-terminal of the g1 TM segment, or in both places. The Structure and Dynamics of HIV-1 Capsid Assemblies: Insights from an In- acceptor was a Bodipy dye attached to the pore blocker iberiotoxin. We then tegrated Approach used LRET to determine the stoichiometry of the a/g1 complex using a method Tatyana Polenova. that assumes that the kinetics of LRET sensitized emission (SE) of the double- University of Delaware, Newark, DE, USA. labeled complex is the linear combination of the individual kinetics of the SE in HIV-1 capsids, assembled from 1,500 copies of the capsid (CA) protein, are single-labeled complexes. The result is that g1 associates with a subunit with a an integral part of mature virions. Conical in shape, capsids enclose the viral maximal 1:1 stoichiometry. In addition, we found that the kinetics of the SE genetic material (two copies of RNA) together with several proteins that are when only g1 is LBT-labeled is slower than that found when only a is LBT- essential for viral replication. In the assembled state, capsids are remarkably labeled, suggesting that the accessory subunit is peripherally located in the dynamic, with the CA residue motions occurring over a range of timescales assembly. We propose that this method is applicable to determine the stoichi- from nano- to milliseconds. These motions are functionally important for cap- ometry of association of proteins subunits within heteromultimeric complexes. sid’s assembly, viral maturation, and interactions with host factors. In this talk, Supported by FONDECYT grants 1150273 (to R. L.). The CINV is a an integrated MAS NMR, DNP, molecular dynamics, and Density Functional Millennium Institute. Theory approach will be presented to probe the functionally important motions in assemblies of CA and their complexes with host factor Cyclophilin A 2698-Plat (CypA), as well as assemblies of CA-SP1 maturation intermediates. The role Resolving the BK Channel Voltage Sensor Activation Transition with of dynamic allosteric regulation in capsid’s assembly, maturation, and escape Relative Atomic Coordinates Under Physiologically-Relevant Conditions from the CypA dependence will be discussed. It will be demonstrated that Antonios Pantazis1, Riccardo Olcese1,2. the integration of experimental NMR and DNP methods and theory, at classical 1Anesthesiology & Perioperative Medicine, UCLA, Los Angeles, CA, USA, and quantum mechanical levels, yields quantitative, atomic-level insights into 2Physiology, UCLA, Los Angeles, CA, USA. the dynamic processes that govern the capsid’s function. Large-conductance, voltage- and Ca2þ-gated Kþ channels (BK, Slo1) are potent regulators of cellular excitability. Our understanding of BK structure 2696-Symp 2þ Functional Dynamics of Modular Multi-Domain Proteins and Ca -dependent gating was recently greatly advanced by cryo-EM- Walter J. Chazin. derived structures; however, the mechanism of voltage-dependent BK Depts. of Biochemistry and Chemistry, and Center for Structural Biology, activation remains unclear. We applied the new optical approach, distance- Vanderbilt University, Nashville, TN, USA. resolving Voltage Clamp Fluorometry (drVCF), to determine the distances Fundamental processes driving the operation, propagation, and maintenance of between transmembrane helices in the BK voltage-sensing domains (VSDs), cells are carried out by multi-protein machines. Machines require moving parts in human channels expressed in Xenopus oocytes under the cut-open oocyte and in the case of protein machinery, the movement of the machine’s parts is voltage clamp. enabled by making the constituent proteins modular, linking multiple globular In this implementation, drVCF was enhanced with atomic ‘‘GPS’’, to determine the distance and orientation of position 203 (S4) relative to surrounding helices. domains together with flexible tethers. Flexibility between domains in these ˚ proteins is critical to the movement between the steps that provide the overall We found that, at rest, the Ca atom of position 136 (S1) is 7.5 [6.8,8.5] A (mean, 95% C.I.) away from 203Ca. This distance increases upon activation output of the machine. While clearly important for function, inter-domain flex- ˚ 136 203 203 ibility poses a significant challenge for structural analysis because the spatial to 13.7 [13.1,14.7] A. In both states, Ca is nearer Ca than Cb; that is, the W203 side-chain points away from S1. Relative to the W203 Ca-Cb organization of the domains (i.e. the architecture) is not static but rather time 136 dependent. Our laboratory applies a combination of NMR and SAXS ap- axis, Ca lies at 138 [128 ,147 ] at rest and 163 [157 ,170 ] upon activa- tion. 145Ca (S2) is 6.8 [6.2,7.6] A˚ away from 203Ca at rest, diverging to 13.1 proaches in solution to define the architectural dynamics of modular multi- ˚ domain proteins through their trajectory between functional states. In this pre- [12.5,14.1] A upon activation. S2 also sits ‘‘behind’’ W203: 146 [113,166] at rest; 147 [132,158] upon activation. Finally, 19Ca (S0) is sentation I will describe our progress in investigating human DNA replication ˚ 203 ˚ machinery, characterizing the ubiquitous eukaryotic ssDNA binding protein, 15.6 [14.1,17.0] A away from Ca at rest and 20.3 [18.5,22.3] A in the Active state. W203 points more towards S0 (Resting: 48 [38,66]; Active: 92 replication protein A (RPA), that coats template DNA as it is unwound and the DNA-dependent RNA polymerase, DNA primase, which synthesizes the [72 ,115 ]) than S1 and S2. first 10 nucleotides of primer on the template. Our results show how changes Using these spatial constraints, acquired from conducting human channels in a in the architectural dynamics can modulate RPA affinity for ssDNA and pro- cellular environment and physiologically-relevant conditions, we constructed a model of voltage-dependent BK activation. Together with the cryo-EM-derived vide a model for its function as a scaffold at the core of the DNA replication 2þ machinery. They also reveal the trajectory of changes in the architectural dy- information on BK structure, Ca sensing and gating, the two models com- namics of DNA primase from pre-initiation through elongation and hand-off bined present a first view of BK channel dual activation by ligands and mem- to polymerase a, suggesting why synthesis of the initial di-nucleotide is the brane depolarization. rate-limiting step in initiation and providing insight into changes in configura- tion and architectural dynamics as it elongates the primer. 2699-Plat Gating of BK Channels: Roles of the C-LINKER and a Potential Hydrophobic Gate Platforms: Voltage-gated K Channels II Zhiguang Jia1, Guohui Zhang2, Mahdieh Yanzdani1, Jianmin Cui2, Jianhan Chen1. 2697-Plat 1Chemistry, University of Massachusetts Amherst, Amherst, MA, USA, Determination of the Stoichiometry Between a and g1 Subunits of the BK 2Biomedical Engineering, Washington University in Saint Louis, Saint Louis, Channel using LRET MO, USA. Willy R. Carrasquel-Ursulaez1, Osvaldo Alvarez1,2, Francisco Bezanilla3, Large conductance Ca2þ-activated Kþ channels (BK channel) are synergisti- Ramon Latorre1. cally and independently activated by Ca2þ,Mg2þ and membrane depolariza- 1 Centro Interdisciplinario de Neurociencia de Valparaiso, Facultad de tion. However, the structure basis of the gating of the channel is still unclear. 2 Ciencias, Universidad de Valparaiso, Valparaiso, Chile, Departamento de A ‘‘passive spring model’’ was previously proposed, which suggests that Biologia, Facultad de Ciencias, Universidad de Chile, Santiago, Chile, pore opening is induced by mechanical pulling of the pore domain by the 3 Department of Biochemistry and Molecular Biology, The University of Ca2þ sensing domain (RCK domain) via the linker between RCK and the Chicago, Chicago, IL, USA. pore (C-linker). However, recently solved EM structures of BK channel reveal Large conductance voltage- and calcium-activated potassium channels, BK, are that the C-linker has similar structure and position between metal-bound and expressed in many mammalian tissues where they play a large variety of phys- metal-free states and is thus unlikely the spring. Here, we will report our recent iological roles. BK channels are tetramers of the main a subunit. Its function is results from atomistic simulation and experiment that together support a more modulated by the interaction with two families of tissue-specific accessory pro- active role of the C-linker in pore-gate coupling of BK channels. In addition, we

BPJ 8705_8719 546a Wednesday, February 21, 2018 will also discuss emerging evidence that supports the existence of a hydropho- channel that retains almost all its biological functions (1). In order to shed light bic gate in the BK-channel. Specifically, the relative small rotation of S6 helix on the structural and functional changes caused by the L-mutations at KcsA’s and narrowing of the cytosolic opening as observed in the metal-free EM struc- position 77, we took advantage of a novel protocol for the overexpression and ture likely represent initial steps towards gate closing. Atomistic simulations purification of KcsA (2) and we have solved the structures and conducted func- reveal that rotation of S6 helix exposes the hydrophobic face and allows further tional studies of the following L-mutants: G77A, G77C and G77T. We have collapsing of the pore. The subsequent dewetting transition near F315-P320 applied a multi-pronged approach that include electrophysiology, differential gives rise to a significant barrier for Kþ diffusion. Furthermore, F315 side scanning calorimetry and macromolecular crystallography to address these chains can rotate in and out of the pore region, providing transient access to questions. largely hydrophobic blockers such as MTSEA even in the close state of BK Funding: AHA- 11SDG5440003, NIH 1RO1GM097159-01A1 and Welch channel. Foundation BI-1757. 1.Valiyaveetil, F.I., et al. Proc Natl Acad Sci U S A, 2004. 101(49): p. 17045-9. 2700-Plat 2.Tilegenova, C., et al. Protein Expr Purif, 2016. 127: p. 53-60. Mode Shift of Shaker Isolated-Voltage Sensing Domain Juan Zhao, Rikard Blunck. 2703-Plat Physics and Physiology, University of Montreal, Montreal, QC, Canada. Calcium Binding to the Turret Region Controls Inactivation Gating of a On prolonged depolarization, the movement of the voltage sensing domain Voltage-Gated KD Channel (VSD) of voltage-gated ion channels is shifted to more hyperpolarized poten- William S. Tobelaim1, Asher S. Peretz1, Daniel Yakubovich1, Yoav Paas2, tials. This behaviour, referred to as a ‘mode shift’ or ‘relaxation’, is generally Bernard Attali1. observed in voltage-gated ion channels. The molecular mechanisms that con- 1Tel Aviv University, Tel aviv, Israel, 2Bar Ilan University, Ramat Gan, trol mode shift are still unknown. In Shaker channels, mode shift was previ- Israel. ously considered to correlate with C-type inactivation and the load imposed Inactivation is an intrinsic property of numerous voltage-gated Kþ (Kv) chan- upon the VSD by the pore domain. However, since Ci-VSP, and Hv1 chan- nels and can occur by N-type or/and C-type mechanisms. While fast N-type nels, without a linked pore domain or a C-type inactivation process, also inactivation involves the inner pore occlusion by N-terminal peptide domains display mode shift, this phenomenon was found to be an intrinsic property of a and b subunits, C-type inactivation is suggested to involve structural rear- of the VSD. In our previous study in the Shaker isolated-voltage sensing rangements in the outer pore leading to a loss of Kþ coordination sites in the domain (Shaker-iVSD), the pore domain and almost the complete C-terminus selectivity filter. In Kv7.1 channels, inactivation is invisible macroscopically were removed, the mode shift was still observed. To investigate the molecular and does not exhibit the hallmarks of N- and C-type mechanisms. However, determinants responsible for the development and modulation of VSD mode Kv7.1 inactivation is revealed by hooked tail currents, which reflect the recov- shift, we generated several Shaker pore deletion mutants, in which any confor- ery from an inactivation state. Here, we show that removal of external Ca2þ mational changes of VSD would not be affected any longer by the energetic produces a striking voltage-dependent macroscopic inactivation of Kv7.1 chan- load or by structural constraints imposed by the pore domain. Our results nels. Increasing external Ca2þ suppresses macroscopic inactivation with an indicate that Shaker pore deletion mutants produced hyperpolarization- 2þ 2þ 2þ EC50 of 1.5 mM. While Sr and Cd mimic the effects of Ca , other divalent activated ionic currents when the VSD is at rest, and exhibited pronounce cations like Mg2þ and Mn2þ are ineffective. Elevating external Kþ concentra- mode shift, in agreement with our previous findings.The magnitude of tion (50 mM) does not prevent macroscopic inactivation evoked in Ca2þ-free Shaker-iVSD mode shift can be minimized by placing a load at the end of external solutions. Experimental data and kinetic modeling indicate that S4 by S4-S5 linker or C-terminal, or eliminated by T1 domain deletion or KCNQ1 channels exhibit two distinct inactivation states. Mutagenesis studies point mutations. While our data support the hypothesis that VSD mode shift and molecular modeling suggest that external Ca2þ ions are coordinated, at is the intrinsic property of VSD, they also show that other molecular determi- least by two glutamate residues E295 and E284 located at the outer pore in nants within the VSD and pore domain are involved in the generation and the turret domain. Our results reveal a new mechanism whereby external modulation of mode shift. Ca2þ exquisitely controls inactivation gating of a Kv channel via a discrete 2701-Plat pore turret region. Measuring the Kinetics of Ion Permeation in Low Conductance Ion Channels 2704-Plat Neville P. Bethel1, Sara Capponi2, John M. Rosenberg3, Michael Grabe2. Properties of the Voltage-Gated Proton Channel Gating Currents 1 1,2 1 1Graduate Group in Biophysics, UC San Francisco, San Francisco, CA, USA, Emerson M. Carmona , David Baez-Nieto , Amaury Pupo , 1 1,3 1 1 2Department of Pharmaceutical Chemistry, UC San Francisco, San Karen Castillo , Osvaldo Alvarez , Alan Neely , Ramon Latorre , 3 Carlos Gonzalez1. Francisco, CA, USA, Department of Biological Sciences, University of 1 Pittsburg, Pittsburg, PA, USA. Centro Interdisciplinario de Neurociencia de Valparaiso, Universidad de Valparaiso, Valparaiso, Chile, 2Broad Institute of MIT and Harvard, Ion channels are essential for regulating osmolarity, maintaining transmem- 3 brane potentials, and many other functions. Molecular simulation can provide Cambridge, MA, USA, Departamento de Biologia, Facultad de Ciencias, atomistic detail of ion permeation for these channels, but the high computa- Universidad de Chile, Santiago, Chile. tional demand limits the timescales to which we can describe these functions. The voltage-gated proton channel (Hv1) is a dimer of subunits containing four For low conductance channels, this poses a problem since the simulation time transmembrane domains (S1-S4) which play the double role of harboring the needed to see several permeation events at physiological voltage is larger than voltage-sensing charges and forming the proton conduction pathway. Some it what is attainable through conventional simulation. Here, we use an enhanced biophysical properties of Hv1, as the coupling between voltage-sensing and sampling approach called weighted ensemble method to probe the kinetics of permeation pathway opening, the pH dependence of gating, the cooperativity ion permeation in KcsA, a low conductance pH gated potassium channel. between each subunit in the dimer, and the steps of charge movement during We measure single channel currents at physiological voltages, and we compare activation and deactivation can be better understood by measuring and char- these values to experiment. We also determine the primary permeation mech- acterizing the Hv1 gating currents. However, to measure gating currents, ionic anism, which has been debated in previous work. Finally, we extend our currents needs to be eliminated or greatly reduced, a feat that is particularly methods to other low conductance channels, such as TMEM16. challenging for Hv1 for which there is no effective blocker and proton concentration cannot be reduced more than 10 fold. Here, we expressed 2702-Plat the low-conducting N264R Hv1 mutant from Ciona intestinalis in Xenopus Revisiting the Role of Glycine 77 within KcsA’S Selectivity Filter: A Func- laevis oocytes and succeeded in recording gating currents membrane patches tional and Crystallographic Study with both, the monomeric and dimeric channels. In agreement with previous Cholpon Tilegenova, D. Marien Cortes, Luis G. Cuello. fluorescence measurements (Qiu et al., Neuron 77:288, 2013), the analysis of Department of Cell Physiology and Molecular Biophysics, Texas Tech the gating currents kinetics and charge movement revealed that the ON University Health Science Center, Lubbock, TX, USA. component has a double exponential decay. Additionally, changes in pH In Kþ channels a highly conserved stretch of amino acid residues, known as modulate Hv1 gating currents kinetics and Q-V curves. This suggests that their selectivity filter (TTVGYGD), is responsible for the highly selective co- the pH sensors in Hv1 are located in the voltage-sensing domain. Finally, ordination of Kþ over Naþ while allowing diffusion limited transport rates. in our experimental conditions we observed charge immobilization after acti- Previously, it was proposed based in functional analysis that replacing KcsA’s vation, which could be an intrinsic characteristic of Hv1 voltage-sensing G77 with a L-amino acid rendered, due to unknown reasons, a nonfunctional domain. Supported by CONICYT-PFCHA/Doctorado Nacional/2017- channel. Additionally, it was shown that incorporation at the same position 21170395 to E.C., Fondecyt grants ACT 1104, Fondecyt 1160261 to C.G. of a D-Alanine (in a chemically synthetized and refolded KcsA) rendered a and 1150273 to R.L.

BPJ 8705_8719 Wednesday, February 21, 2018 547a

Platform: Optical Microscopy and 2707-Plat Super Resolution Imaging of Start Transcription Factors in Yeast Superresolution Imaging: Applications Labe Black1, Jean-Bernard Fiche2, Sylvain Tollis3, Jing Cheng3, Stephen Notley1, Ben Crevier1, Michael Tyers3, Marcelo Nollmann2, 2705-Plat Catherine Royer1. Super-Long Single Fluorescent-Molecule Tracking Revealed Tension- 1Biology, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Centre de Dependent Dynamic Anchorage of Integrin for Cell Adhesion Biochimie Structurale, Universite de Montpellier, Montpellier, France, Taka A. Tsunoyama1, Kenichi GN Suzuki2, Takahiro K. Fujiwara3, 3Institute for Research in Immunology and Cancer, Universite de Montreal, Akihiro Kusumi1. Montreal, QC, Canada. 1Okinawa Institute of Science and Technology (OIST), Okinawa, Japan, The balance between growth and division in budding yeast (Saccharomyces 2Center for Highly Advanced Integration of Nano and Life Sciences (G- cerevisiae) is critical for proliferation. Commitment to division occurs at Start CHAIN), Gifu University, Gifu, Japan, 3Center for Meso-Bio Single- and is controlled by the transcription factor complexes SBF and MBF. In poor Molecule Imaging (CeMI), Institute for Integrated Cell-Material Sciences, nutrient (glycerol) environments, cells divide at a smaller size, and have a Kyoto University, Kyoto, Japan. slower growth rate compared to rich nutrients (glucose). We have recently Single fluorescent-molecule imaging-tracking is becoming an important found that the SBF and MBF copy numbers are small and increase in G1 phase, tool for studying living cells. However, photobleaching and photoblinking prior to Start, which might result in the titration of their target sites in the G1/S of the probe molecules strongly hamper SMT studies of living cells, regulon to trigger the transition. In this scenario, upregulation of SBF and MBF making it difficult to observe in vivo molecular events and evaluate their in poor carbon nutrient sources would explain why cells pass Start at a smaller lifetimes (e.g., off rates). The methods used to suppress photobleaching/ size. However, with the small SBF/MBF copy numbers when compared to their photoblinking in vitro are difficult to apply to living cells, due to their target DNA sequences, it is not known what role the 3-dimensional structural toxicities. Here, using 13 organic fluorophores, we found that by combining organization of these transcription clusters facilitate in the upregulation- low concentrations of dissolved oxygen with a reducing-plus-oxidizing sys- titration mechanism. In the present study, single-molecule localization micro- tem, photobleaching/photoblinking could be strongly suppressed, with only scopy was used to resolve sub-diffraction limited, 50-100 nm sized, nucleation minor effects on cells, enabling SMT for as long as 12,000 frames (7 min site structures of the SBF/MBF complexes within the nucleus of budding yeast at video rate, as compared to the general 10-s-order durations) with in rich and poor nutrient environments. Interestingly, poor carbon nutrients 23-nm single-molecule localization precisions. The super-long tracking of increased the nucleation site density compared to rich nutrient sources in fixed single individual integrin molecules revealed that each integrin molecule cells. In live cells, single-particle tracking revealed discrete molecular diffusion dynamically links the extracellular matrix (ECM) and the actin filament in dynamics of the SBF and MBF components, one fast, and one slowly diffusing a tension-dependent manner, with linkage (exponential) lifetimes of 80 s component, which we interpret as non-specifically and specifically bound pro- and 40 s for integrins beta 1 and 3, respectively. Such tension-dependent dy- teins. No nutrient dependence of the dynamics was observed. These observa- namic binding occurs most frequently two-thirds of the distance (1 mm) tions support our previous work, indicating increasing copy number of SBF away from the focal-adhesion center toward the distal end, consistent and MBF as a key factor controlling Start and its modulation by nutrients. with the result reported by the Waterman group (Plotnikov et al. Cell 2012). These results suggest that when an integrin molecule enters the focal 2708-Plat adhesion zone, it might incidentally bind to both ECM and the actin filament, Two-Color 3D STORM Reveals Ciliary Transition Zone Architecture and and if this linkage transmits the mechanical tension, it can lead to longer its Role in Ciliary Signaling immobilization of the integrin molecule, contributing to the cellular binding Xiaoyu Shi1, Galo Garcia2, Jeremy F. Reiter2, Bo Huang1. 1 to the ECM. Pharmaceutical Chemistry, University of California, San Francisco, CA, USA, 2Biochemistry and Biophysics, University of California, San Francisco, CA, USA. 2706-Plat The cilium is a cellular organelle that mediates the cell signaling to control the Cadherin Order and Dynamics in Calcium-Dependent and Independent cell development. A domain near the ciliary base called transition zone controls Desmosomes the protein composition of the ciliary membrane, but how it does so is unclear. Emily Bartle, Tara Urner, Tejeshwar Rao, Alexa Mattheyses. To better understand the transition zone and its role in ciliary signaling, we UAB, Birmingham, AL, USA. defined the arrangement of key proteins in the transition zone using two- Desmosomes are complex cell-cell junctions composed of adhesive trans- color 3D stochastic optical reconstruction microscopy (STORM). This map- membrane cadherin proteins and intracellular plaque proteins. Integration of ping revealed that NPHP and MKS complex components form nested rings the desmosomal complex with the keratin intermediate filament cytoskeleton comprised of nine-fold doublets. On top of this ciliary map, we found that confers strength and mechanical resistance to epithelial tissue. Desmosomes diverse signaling molecules, including a key Hedgehog signaling regulator can adopt different functional states in vivo that are critical for regulating SMO, accumulate at the transition zone in wild type cells. This observation adhesion during processes like wound healing and development. These states suggests that the transition zone is a waypoint for proteins entering and exiting are characterized by desmosome sensitivity to calcium; upon reduction of the cilium. To further understand how altered transition zone architecture af- extracellular calcium, cells with calcium-dependent desmosomes lose adhe- fects developmental signaling, we examined the localization of the SMO in hu- sion while cells with hyper-adhesive (calcium-independent) desmosomes man fibroblasts with mutations in RPGRIP1L. These mutations disrupt the maintain strong adhesion. Though defined by resistance to reduced calcium, architecture of the MKS and NPHP rings in transition zone. In the mutant cells, the mechanism that confers hyper-adhesion is not known. Interestingly, the we found SMO failed to accumulate at the transition zone and was significantly cadherin tertiary structure is dependent upon calcium binding for its rigidity. reduced in ciliary membrane. We propose that the disruption of transition zone Furthermore, electron tomography showed desmosomal cadherins form an or- architecture leads to a failure of SMO to accumulate at the transition zone, dis- dered array at the adhesive interface, and this order is hypothesized to be crit- rupting the Hedgehog developmental signaling pathway. ical for adhesion. To address the mechanism driving hyper-adhesion, we investigated the order and dynamics of the desmosomal cadherin desmoglein 2709-Plat 3 (Dsg3) in calcium dependent and hyper-adhesive states. Using fluorescence Visualizing Dynamic Microvillar Search and Stabilization during Ligand polarization microscopy, we found that Dsg3 was ordered in calcium- Detection by T Cells dependent desmosomes. When calcium was reduced, order was lost (t=5 En Cai1, Kyle Marchuk1, Peter Beemiller1, Casey Beppler1, min) faster than adhesive function was compromised (t= 15 min). When Matthew G. Rubashkin2, Valerie M. Weaver2, Audrey Gerard1, we induced hyper-adhesion by inhibiting PKCa we similarly found Dsg3 Tsung-Li Liu3, Bi-Chang Chen3, Eric Betzig3, Frederic Bartumeus4, was ordered. Surprisingly, when calcium was reduced in hyper-adhesive cells, Matthew F. Krummel1. order was lost with similar kinetics to the calcium-dependent cells, despite 1Department of Pathology, University of California San Francisco, San maintained adhesion. Measuring the order and dynamics of Dsg3 has revealed Francisco, CA, USA, 2Center for Bioengineering and Tissue Regeneration, novel differences in ultrastructure between functional states in living cells. University of California San Francisco, San Francisco, CA, USA, 3Janelia Our results demonstrate that desmosome adhesion is not directly dependent Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA, on cadherin order and cadherin trans-binding can persist after loss of tertiary 4Center for Advanced Studies of Blanes (CEAB-CSIC), Girona, Spain. structure. Because cadherins lose order regardless of adhesive state, these During immune surveillance, T cells survey the surface of antigen-presenting results suggest a central role for the desmosomal plaque in conferring cells (APCs), which may display mainly nonstimulatory peptide-loaded major hyper-adhesion. histocompatibility complexes (pMHCs) and only rare cognate antigen in a

BPJ 8705_8719 548a Wednesday, February 21, 2018 process involving close membrane apposition. Thus, T cell must solve a classic In addition to providing measurements of microviscosity in multiple compart- trade-off between speed and sensitivity. It has long been supposed that micro- ments of the cell with a single ‘molecular rotor’ probe, we also report striking villi on T cells act as sensory organs to enable search, but their strategy has been variation of microviscosity between and within individual matrices of mito- unknown. We used lattice light-sheet microscopy and quantum dot-enabled chondria, the cell’s energy repository. synaptic contact mapping microscopy to show how microvilli on the surface Furthermore, we were able to detect changes of ER microviscosity upon drug of T cells search opposing cells and surfaces before and during antigen recog- administration. The ability to measure the viscoelastic response of the ER upon nition. We uncovered fractal organization of the microvilli on T cell surfaces. stress is extremely important and highly pertinent to health and disease, as well We found that microvilli survey the majority of opposing surfaces within one as being novel fundamental science. minute through anomalous diffusion, which is equivalent to the roughly one- We believe that ROVI offers novel avenues to study cell biology and can aid to minute half-life of T cell-APC contacts in vivo. Individual microvilli dwell monitoring the progression and treatment of diseases. times were long enough to discriminate pMHC half-lives. TCR recognition re- sulted in selective stabilization of receptor-occupied protrusions as seen by 2712-Plat longer microvilli dwell times in synapse regions with pMHCs and increased A Polar Matrix Microdomain Constrains Diffusion and Regulates persistence of TCR-occupied contacts. Stabilization was independent of tyro- Intracellular Signaling 1 2 2 2 sine kinase signaling and the actin cytoskeleton, suggesting selection for avid Alex von Diezmann , Keren Lasker , Thomas H. Mann , Daniel G. Ahrens , Lucy Shapiro2, W.E. Moerner1. TCR microclusters. This work defines the efficient cellular search process 1 2 against which ligand detection takes place in T cells. Chemistry, Stanford University, Stanford, CA, USA, Developmental Biology, Stanford University School of Medicine, Stanford, CA, USA. 2710-Plat The dynamic localization of a suite of signaling proteins at the cell poles of the bac- Light-Sheet Microscopy Allows Simultaneous Imaging of Second Messen- terium Caulobacter crescentus mediates asymmetric cell division and the cell- gers in Intact Pancreatic Islets cycle-specific control of gene expression. The positioning of many of these pro- Zeno Lavagnino, Michael DiGruccio, David W. Piston. teins depends on PopZ, a 177-residue protein that self-assembles to form a 150- Cell Biology & Physiology, Washington University in St. Louis, St. Louis, 200 nm porous polymeric network at the poles. While it is known that the nucleoid MO, USA. and ribosomes are excluded from the PopZ microdomain, how proteins navigate Elucidating the regulation of glucagon release from the islet of Langerhans has this network is poorly understood. We used multicolor, three-dimensional sin- been limited by the lack of reliable methods to identify glucagon-secreting a- gle-molecule microscopy to track the motion of signaling proteins with 30-60 cells, which make up a minority of the islet cells. Transgenic mice expressing nm spatial and 20-100 ms temporal resolution relative to the super-resolved fluorescent proteins targeted specifically to a-cells can be used to overcome this PopZ microdomain. Both membrane-bound and cytoplasmic proteins implicated limitation. We have developed a mouse model that combines an improved in the developmental circuitry of Caulobacter were captured and slowed within glucagon-promoter driven Cre recombinase (Glu-iCre) with a floxed fluores- the polar microdomain, diffusing up to two orders of magnitude more slowly in- cent protein calcium sensor (GCaMP6) to express the biosensor in >95% of side the volume delimited by PopZ. Establishing that the PopZ network acts to the a-cells specifically, as demonstrated by immunostaining. The genetic partition even relatively small biomolecules, we found that proteins without polar expression of the sensor allows real-time monitoring of calcium in combination binding partners were excluded from the polar microdomain. By integrating these with other biosensors, such as for the second messenger cyclic AMP (cAMP) tracking results with biochemical measurements in a comprehensive reaction- and for a spectrally separated calcium sensor driven by the insulin promoter. diffusion model, we show that localization and transient capture by PopZ control A novel, single wavelength cAMP sensor driven by baculovirus expression is the intracellular distributions of signaling proteins. With spatially resolved point used in combination with islets from the GCaMP6 mouse to monitor calcium measurements of transcription, we found that a master cell cycle regulator ex- and cAMP simultaneously under different stimuli triggering calcium response hibited a gradient of activity in quantitative agreement with our model. Together, and glucagon secretion. Additionally, a red calcium sensor driven by the rat in- these results shed light on the structure and function of a bacterial microdomain sulin promoter allows simultaneous monitoring of calcium in a- and b-cells, and the role of membrane-less organelles in cellular organization. underlining putative paracrine interactions between the two cell types’ signaling pathways. A light-sheet fluorescence microscope (dual view inverted Platform: Cardiac Muscle Mechanics, Structure, Selective Plane Illumination Microscopy - diSPIM) permits the rapid acquisi- tion of three-dimensional data sets of the whole islet’s calcium and cAMP ac- and Regulation II tivity with isotropic spatial resolution. In this way, it is possible to obtain simultaneous information on the interplay of calcium and cAMP signaling in 2713-Plat different cell types in the whole islet of Langerhans. Length-Dependent Activation is Reduced in Myocardium from Patients with Non-ischemic Heart Failure 2711-Plat Bertrand C.W. Tanner1, Peter O. Awinda1, Cheavar A. Blair2, Rotor-Based Organelle Viscosity Imaging Maya A. Guglin3, Kenneth S. Campbell2. Marketa Kuba´nkova´1, Joseph E. Chambers2, Stefan J. Marciniak2, 1Integrative Physiology and Neuroscience, Washington State University, Marina K. Kuimova1. Pullman, WA, USA, 2Department of Physiology, University of Kentucky, 1Department of Chemistry, Imperial College London, London, United Lexington, KY, USA, 3Division of Cardiovascular Medicine, University of Kingdom, 2Department of Medicine, CIMR, University of Cambridge, Kentucky, Lexington, KY, USA. Cambridge, United Kingdom. Heart failure contributes to 1 in 9 deaths in the United States and often reflects Microscopic viscosity is a critical parameter in cellular processes such as impaired cellular-level contractile function. In healthy hearts, the myofilaments diffusion-controlled reactions and the correct folding of nascent proteins. How- become more sensitive to Ca2þ as the myocardium is stretched. This effect is ever, a lack of a suitable technology has hampered the analysis of this funda- known as length-dependent activation and is the cellular-level basis of the mental parameter within the organelles of living cells. Frank-Starling mechanism. Few studies have measured the magnitude of We have established a new technique called Rotor-based organelle viscosity length-dependent activation in myocardium from failing human hearts. Multicel- imaging (ROVI). In our technique, environment-sensitive fluorescent dyes lular preparations from the left ventricular free wall were chemically permeabi- termed ‘molecular rotors’ are directed via active targeting to specific intracel- lized and Ca2þ-activated at sarcomere lengths of 1.9 and 2.3 mm, at physiological lular compartments of live cells for the real time dynamic measurement of temperature (37C). 12 hearts were explanted from patients receiving cardiac microscopic viscosity. transplants; 6 patients had ischemic heart failure and 6 had non-ischemic Molecular rotors are small synthetic fluorophores whose photophysical proper- heart failure. Another 6 hearts were obtained from organ donors. Calcium- ties strongly depend upon the viscosity and molecular crowding of their imme- sensitivity of contraction (defined as the pCa50 value) increased by 0.07 pCa diate environment. By measuring the fluorescence lifetime of molecular rotors units as the preparations were stretched from 1.9 to 2.3 mm in samples from organ via time-correlated single photon counting fluorescence lifetime imaging donors (p<0.001) and ischemic failing hearts (p=0.003). Calcium-sensitivity did (TCSPC FLIM) it is possible to quantitatively determine the microviscosity not change with sarcomere length in preparations from non-ischemic failing of the local environment. hearts. These data show for the first time that length-dependent activation of Using ROVI, we were able to measure and compare the microviscosity in mul- contraction is preserved in myocardium from patients with ischemic heart fail- tiple cellular compartments, namely the cytosol, nucleus, ER and mitochondria. ure, but eliminated in myocardium from patients with non-ischemic heart failure. Microviscosity was measured both basally and during manipulations that alter Because length-dependent activation is an important component of the Frank- organelle function. We report significant variations of microviscosity between Starling mechanism, patients who have non-ischemic disease may exhibit a the studied organelles. diminished contractile response to increased ventricular filling. Different

BPJ 8705_8719 Wednesday, February 21, 2018 549a therapeutic strategies may be more effective in patients with ischemic and non- 2716-Plat ischemic heart failure. Live Cell Palm Techniques for Super Resolution Imaging of Murine Cardiac Myocytes 2714-Plat Yufeng Hou, Ornella Manfra, Jia Li, Xin Shen, William E. Louch. Cardiac Muscle Regulatory Units are Predicted to Interact Stronger than Institute for Experimental Medical Research, Oslo, Norway. Neighboring Cross-Bridges Since their inception, super resolution methodologies have revolutionised the Mari Kalda, Marko Vendelin. way in which biologists can visualise subcellular structures. Cardiac science Laboratory of Systems Biology, Department of Cybernetics, Tallinn has particularly benefited under these methodologies, as structures such as University of Technology, Tallinn, Estonia. the dyads of cardiomyocytes have become resolvable, thus yielding new under- In this work, we developed a model to quantitatively access the influence of standing of -their spatial organization. However, pairing of nanoscale dyadic different types of interactions within the sarcomere on properties of cardiac structure and function has been hampered by the fact that techniques such as muscle. We use the set of partial differentsial equations to describe the dy- direct stochastic optical reconstruction microscopy (dSTORM) generally namics of ensembles consisting of cross-bridge groups connected by elastic require fixed tissue. Furthermore, quantification of dSTORM images based tropomyosin. The mathematical model gives thermodynamically consistent on antibody labelling is complicated by variability in labelling efficacy, and description of isometric and shortening contractions allowing us to study bio- displacement of the fluorophore from the protein of interest. We presently cir- physical principles behind the cooperativity.Through large scan in the free en- cumvented these limitations by creating a transgenic mouse expressing a photo ergy landscape, we demonstrate the different influence of RU-RU, XB-XB, and activated red fluorescent protein tagged to ryanodine receptor 2 (PA-tag-RFP- XB-RU interactions on cooperativity coefficient of calcium binding, developed RyR2). Imaging of live, isolated cardiomyocytes was attained with a commer- maximal force, and calcium sensitivity. The model solution was fitted to repro- cial Zeiss Elyra dSTORM setup using the photo-activated light microscopy duce experimental data on force development during isometric contraction, (PALM) technique. PA Tag RFP requires dual sample illumination with activa- shortening in physiological contraction, and ATP consumption by acto- tion laser and imaging laser to produce constant photo switching. In our exper- myosin during different types of contractions. On the basis of the fits, we iments we found that by adjusting the rate of activation and photobleaching showed that RU-RU interaction leads to about 5 times larger change in the from imaging we could optimize the blinking behaviour required for live cell free energy profile of the reaction than XB-XB interaction. PALM imaging. To reduce background, HILO and TIRF illumination were em- This predicted mechanism behind cooperativity of the muscle contraction is in ployed to reduce the thickness of the optical section. Our results show RyR lo- quantitative agreement with the studies comparing the azimuthal tropomyosin calisation accuracy of 40nm in the X and Y plane for live cell images, in movement induced by binding calcium or myosin. Due to the deterministic contrast to the higher resolution of 10-20nm in fixed samples with antibody description of muscle contraction and its thermodynamic consistency for short- labelling. RyR cluster arrangements in live cells were observed to be broadly ening contractions, we envision that the developed model can be used to study in agreement with fixed samples. Ongoing work is aimed at linking RyR orga- biophysics of heart muscle contraction, its energy cost, link between calcium nization to local calcium release events (Ca2þ sparks), with focus on unravel- release and force development not just on the tissue level, but on a level of ling the functional consequences of the dynamic nature of RyR arrangement. the whole heart. 2717-Plat 2715-Plat Understanding Cardiac Tube Formation in Developing Drosophila Human Embryonic Stem-Cell Derived Cardiomyocytes: Single-Cell Embryos using Light Sheet Microscopy and Cardiac Drug Screening Mapping to Relate Twitch Kinetics to Myosin Heavy Chain Protein and Christopher MJ McFaul1, Rodrigo Fernandez-Gonzalez1, mRNA-Expression Christopher M. Yip2. Natalie Weber1, Kathrin Kowalski1, Tim Holler1, Ante Radocaj1, 1Institute of Biomaterials and Biomedical Engineering, University of Kristin Schwanke2, Alexander Lingk1, Uwe Krumm1, Meike Wendland1, Toronto, Toronto, ON, Canada, 2Chemical Engineering, University of Urs Zywietz3, Boris Chichkov3, Ulrich Martin2, Robert Zweigerdt2, Toronto, Toronto, ON, Canada. Bernhard Brenner1, Theresia Kraft1. Heart development begins with the formation of a primitive tube, both in fruit 1Molecular- and Cell Physiology, Medical School Hannover, Hannover, 2 flies and mammals. Tube formation is mediated by coordinated cell move- Germany, Department of Cardiothoracic, Transplantation and Vascular ments. In Drosophila, the heart is formed from 52 bi-lateral pairs of cardiac pre- Surgery (HTTG) and LEBAO, Medical School Hannover, Hannover, 3 cursors that migrate dorsally and medially to join their counterparts. The cells Germany, Nanotechnology, Laser Zentrum Hannover e.V., Hannover, must then undergo distinct morphological changes to control sites of adhesion Germany. and repulsion to their partner in order to form a lumen. While the genetic path- Previously we showed that in human stem-cell derived cardiomyocytes (hSC- ways that induce cardiac cell specification have been clearly defined, the CMs) a-/b-myosin heavy chain (MyHC) protein expression can be quite het- cellular and molecular mechanisms that regulate collective cell migration dur- erogeneous from cell-to-cell. Thus, to investigate how MyHC isoform and ing heart tube formation are not well understood. Leveraging the simplicity and function are related, sequential analysis of both parameters for the same indi- pharmacological tractability of the fruit fly, Drosophila melanogaster, and the vidual CM is necessary. We developed a novel, setup-independent single-cell ability to perform live imaging of its embryos, we have developed a light-sheet mapping technique for multiparametric characterization of the very same microscopy platform and quantitative image analysis to characterize cell be- hSC-CMs. Since often MyHC-mRNA is used as indirect measure for protein haviours and molecular rearrangements during heart tube formation in living expression, we also tested whether a- and b-MyHC-mRNA levels correspond Drosophila embryos . Our system allows identification and tracking of cardiac to MyHC-protein levels of the same single cells. Previously, a discrepancy of precursors and the overlying epidermal cells. Automated image analysis allows 7% a-MyHC-protein associated with 35% a-MyHC-mRNA of total MyHC- quantitative comparison of the dynamics of tube formation across embryos. To protein and mRNA, respectively, was reported for adult human ventricle. identify the pathways that regulate collective cell movements during heart hSC-CMs were cultivated for 35 days in cardiac bodies (CBs) or plated on development, we are conducting a pharmacological screen for inhibitors of car- glass coverslips. We recorded twitch contractions and X-Y-coordinates of sin- diac precursor migration. In our screen, embryos are permeabilized, exposed to gle CMs for subsequent remapping of the same CMs. Functional measurement the compound of interest at the onset of cardiac tube formation, and then al- was followed by fluorescence-in-situ-hybridization using specific probe sets lowed to develop for 1-4 hours before fixation and quantitative assessment of against a-MyHC-mRNA and b-MyHC-mRNA, and finally by immunofluores- the extent of cardiac precursor movement. Screen hits will be followed up using cent staining of MyHC-proteins. Protein staining showed for 22% of our light sheet microscopy system with living embryos. Together, our novel cardiomyocytes from CBs pure a-MyHC-protein and for 14% pure b-MyHC- tools will allow us to identify pathways critical for cardiac precursor migration, protein expression. In contrast, only 1% of plated cardiomyocytes had pure polarization, and cell-cell adhesion. a-MyHC-protein and 66% had pure b-MyHC-protein. The remaining CMs ex- pressed both MyHC-protein isoforms. Most importantly, the majority of the 2718-Plat pure a-MyHC- and b-MyHC-protein cardiomyocytes irrespective of cultiva- In Situ Replacement of cMyBP-C N’-Terminal Domains using the Novel tion strategy expressed considerable amounts of mRNA of the other isoform, Spy-C Method respectively, showing only weak correlation between mRNA and protein Katia Touma1, Sabine J. van Dijk2, Joshua Strom1, Samantha P. Harris1. (e.g. plated CMs with pure b-MyHC-protein had 17% a-MyHC-mRNA). 1University of Arizona, Tucson, AZ, USA, 2University of California, Davis, Also, relative expression of both mRNAs from cell-to-cell was highly variable, CA, USA. especially in CMs from CBs. Analysis of twitch parameters in relation to Cardiac myosin binding protein-C (cMyBP-C) is a major regulator of heart MyHC-isoform showed no difference for a-MyHC and b-MyHC, suggesting muscle contraction. To understand molecular mechanism(s) by which that MyHC-isoform does not dominate twitch kinetics. cMyBP-C affects force, most studies have focused on its first 4 N’-terminal

BPJ 8705_8719 550a Wednesday, February 21, 2018 domains because they are sufficient to affect actomyosin interactions in vitro. drial junctions. We have also observed that mitochondria form heterogeneous However, because the C’-terminal domains are required to anchor cMyBP-C clusters within localised regions of the cell. Recent studies suggest that conduc- to the thick filament, a limitation of such studies is that the precise spatial dis- tion of membrane potential through the mitochondrial networks can act as one tribution of the N’-terminal domains is lost. To overcome this limitation we of the dominant mechanisms to distribute energy across the cell. There are devised a novel protein engineering approach to remove and replace N’-termi- several other myofibril based metabolite diffusion systems present in cardio- nal domains of cMyBP-C at their exact positions in situ. The approach uses car- myocytes which can also contribute to intracellular energy transport. What is diomyocytes from gene-edited ‘‘Spy-C’’ mice that express a modified cMyBP- the interplay between these different mechanisms and the heterogeneous distri- C containing an internal TEV protease site followed by a short ‘‘SpyTag’’ bution of mitochondria? sequence. Removal of N’-terminal domains is accomplished by incubating per- We are developing detailed finite element models of cardiac ultrastructure to meabilized Spy-C myocytes with TEV protease to cut cMyBP-C and expose investigate the role that mitochondrial membrane potential, oxygen and the the SpyTag on the remaining C’-terminal domains of cMyBP-C. New recom- creatine-kinase shuttle have in homogenizing ATP supply and demand within binant N’-terminal domains are then ligated via a covalent bond formed be- a heterogeneous cellular architecture. Our results show that diffusion of O2 is tween SpyTag and its partner protein, SpyCatcher, which is encoded at the insufficient to reach interior of a cardiomyocyte in a high workload - leading ends of the new recombinant N’-terminal domains (Zakeri et al., PNAS, to a regional hypoxia at the core of the cell. Our simulations also indicate that 109:E690–7, 2012). Here we show proof-of-principle for the Spy-C method rapid diffusion of creatine and phosphocreatine may be still be required to ensure by demonstrating TEV cleavage of cMyBP-C and using immunofluorescence that metabolites like ATP and ADP are uniformly distributed in the midst of a to show that added N’-terminal domains localize properly in sarcomeres heterogeneous distribution of mitochondria that we observe in the cell. However, from Spy-C mice. Force measurements in permeabilized myocytes further our current model simulations suggest that conduction of membrane potential confirm the feasibility of using the Spy-C method to measure function of from the mitochondria at the cell periphery can provide sufficient protonomotive exchanged cMyBP-C N’-terminal domains in situ. The Spy-C method thus of- force to the mitochondria at the cell core to sustain a uniform distribution of me- fers a new approach for introducing nearly any desired modification into the tabolites and force dynamics across the cell cross section. N’-terminal domains of cMyBP-C (e.g., mutations, PTMs, FRET sensors) to study their structure and function in situ. [NIH HL080367, AHA Platform: Membrane Dynamics and Fusion II 17IRG33411051, and a UA Precision Mouse Modeling Program Award)]. 2721-Plat 2719-Plat Spatio-Temporal Dynamics and Turnover of Lipopolysaccharide in the Engineered Heart Tissues Expressing Mutant Desmoplakin Exhibit Bacterial Outer Membrane Altered Twitch Kinetics Sam Lenton1, Rosalyn M. Leaman1, Richard J. Spears2, Martin A. Fascione2, 1 2 3 4 4 Ronald Ng , Xia Li , Heather Manring , Jinkyu Park , Jiesi Luo , Dmitri O. Pushkin3, Mark C. Coles1,4, Christoph G. Baumann1. Daniel Jacoby4, Maegen A. Ackermann5, Stuart Campbell1. 1 2 1 Biology, University of York, York, United Kingdom, Chemistry, Department of Biomedical Engineering, Yale University, New Haven, CT, 3 2 3 University of York, York, United Kingdom, Mathematics, University of USA, Yale University, New Haven, CT, USA, Department of Physiology York, York, United Kingdom, 4Kennedy Institute of Rheumatology, and Cell Biology, The Ohio State University, Columbus, OH, USA, 4 University of Oxford, Oxford, United Kingdom. Cardiovascular Research Center, Section of Cardiovascular Medicine, Gram-negative bacteria have a cell envelope consisting of a peptidoglycan Department of Internal Medicine, Yale University, New Haven, CT, USA, 5 layer located between an inner cytoplasmic cell membrane and an outer- Department of Biomedical Engineering, The Ohio State University, membrane (OM). The OM is an asymmetric bilayer consisting of an inner Columbus, OH, USA. leaflet of phospholipid and an extra-cellular facing outer leaflet composed Arrhythmogenic cardiomyopathy (AC) is a disease characterized by electrical of lipopolysaccharide (LPS), in which outer membrane proteins (OMPs) abnormalities and fibro-fatty infiltration of the myocardium. It is often linked are embedded. The mechanism whereby OMPs and LPS turnover during to mutations in desmosomal genes, but molecular mechanisms whereby desmo- cell growth is not well understood. There is recent evidence that OMPs somal mutations give rise to end-stage AC remain to be fully established. In or- are not inserted uniformly across the cell surface, but at discrete spots. der to probe phenotypic consequences of an AC-linked desmosomal mutation, Once inserted, OMPs are not observed to diffuse far from these insertion we identified a candidate family with a history of sudden cardiac death and a sites, leading to patches termed OMP ‘islands’. Whether LPS is also inserted variant of unknown significance (R451G) in the gene encoding desmoplakin in discrete locations is unknown. The interactions between LPS and OMPs (DSP). After establishing statistically significant linkage between the R451G are important for OM function and integrity. However, the extent to which variant and symptomatic family members (LOD score = 3.4), a heart autopsy LPS contributes to the confinement of OMPs is not well understood. Here, specimen was obtained from a deceased DSP R451G-positive family member we have studied the spatio-temporal dynamics of LPS using metabolic incor- who had been diagnosed with AC according to the ARVC 2010 task force poration of a sugar analogue and site-specific bio-orthogonal chemical label- criteria. Immunofluorescence microscopy of this tissue showed significant ing with a fluorescent dye. Using confocal FRAP and single-molecule reduction of desmoplakin and connexin 43 at the intercalated disks. We hypoth- fluorescence microscopy techniques, we observed that LPS does not diffuse esized that mutant desmoplakin is avidly degraded by the cell, resulting in hap- freely in the OM and is inserted at discrete locations during cell growth. loinsufficiency, poor intercalated disk organization, and reduced electrical These characteristics were independent of LPS structural complexity (i.e. conduction velocity of the myocardium. In order to test these hypotheses, we deep rough, rough and O-antigen restored strains). The results are discussed created engineered heart tissues (EHTs) from an induced pluripotent stem cell in the context of OM turnover during bacterial growth and adaptation to an line generated from a symptomatic, R451G-positive family member. EHTs environmental niche. were grown under electrical stimulus and isometric conditions for 2 weeks before contraction force and electrical conduction velocity through the tissues 2722-Plat were measured. Surprisingly, we found that conduction velocities were un- The Biophysical Asymmetry of Mammalian Plasma Membranes changed between EHTs expressing mutant versus wild type desmoplakin. While Joseph H. Lorent, Eric Malmberg, Ilya Levental. there was no statistical difference in the peak contraction force, mutant tissues Integrative Biology and Pharmacology, UT Health Science Center at had a significantly slower rate of contraction: time from stimulus to peak twitch Houston, Houston, TX, USA. force was 17.6% longer in mutant EHTs. This early biomechanical deficiency in Compositional asymmetry between the two leaflets of a membrane bilayer is a DSP R451G cells may indicate poor coupling between the sarcomere lattice and fundamental feature of eukaryotic plasma membranes. Maintaining lipid asym- the intercalated disk as a fundamental disease mechanism in AC. metry is energetically demanding, requiring active transport of phospholipids from one leaflet to the other. Phospholipid asymmetry has been described 2720-Plat largely in the context of apoptosis, where translocation of the anionic lipid Creatine-Kinase Shuttle and Rapid Mitochondrial Membrane Potential phosphatidylserine from the inner to the outer leaflet of the plasma membrane Conductivity are Needed Simultaneously to Maintain Uniform Metabolite is a marker for macrophage engulfment. However, it has recently become Distributions in the Cardiac Cell Contraction Cycle evident that lipid asymmetry can also be reversible, and regulates many phys- Shouryadipta Ghosh1, Kenneth Tran2, Edmund Crampin1, Eric Hanssen1, iological processes, including immune cell signalling and cell-cell fusion. Vijay Rajagopal1. Owing to their distinct compositions, the two leaflets of the plasma membrane 1University of Melbourne, Melbourne, Australia, 2University of Auckland, are believed to have distinct physical properties. Besides the commonly noted Auckland, New Zealand. surface charge, the hypothesized differences in sphingolipid content and acyl Cardiac muscle cells are reported to contain a cell wide mitochondrial reticu- chain composition suggest that other physical properties (lipid packing, lateral lum where mitochondria are interconnected in networks though intermitochon- diffusion, permeability) would also be distinct between the two leaflets. Here,

BPJ 8705_8719 Wednesday, February 21, 2018 551a we test this hypothesis directly by using a combination of fluorescence lifetime 2725-Plat imaging of order sensitive probes with microinjection and selective quenching. General Non-axisymmetric Shapes of Biological Membranes and their Our results show that in live cell plasma membranes, inner leaflet order is Importance in Understanding Endocytosis slightly, but significantly, lower compared to the outer leaflet. The magnitude Kranthi K. Mandadapu1, Yannick Omar2, Amaresh Sahu1, Roger Sauer2. of the difference is smaller than expected from compositional asymmetry, sug- 1Chemical and Biomolecular Engineering, University of California Berkeley, gesting that strong coupling between leaflets in asymmetric membranes pre- Berkeley, CA, USA, 2RWTH Aachen, Aachen, Germany. vents high lipid order differentials. We also find that a loss of membrane Many important biological phenomena are mediated by cell membranes and the asymmetry during phospholipid scrambling or apoptosis drastically reduces shapes accommodated by these membranes. For example, endocytosis involves the order of the plasma membrane and eliminates the order differences between bending to form a bud or a tube from a flat shaped membrane under the action leaflets. Finally, we observe that these biophysical differences are important for of proteins such as clathrin or BAR and their coupling to actin pulling forces. cell physiology, as inhibition of lipid scrambling suppresses the functional acti- Various morphological shapes of membranes are governed by thermodynamic vation of immune cells. These findings document the biophysical asymmetry of parameters such as osmotic pressure drop across the membrane or membrane the plasma membrane and suggest an important role for this asymmetry in cell tension. Most theoretical studies focusing on understanding membrane bending function. involve either small deformations about a given configuration or axi-symmetric shape transitions. In this talk, using the recent advancements in theoretical and 2723-Plat computational modeling of membranes, we show that axi-symmetric or small Formation and Stability of Membrane Necks from Molecular Simulation deformations are inadequate to understanding the morphological phase- € Rikhia Ghosh, Andrea Grafmuller, Reinhard Lipowsky. diagrams of membranes governed by protein induced spontaneous curvature Theory and Biosystems, Max Planck Institute of Colloids and Interfaces, and membrane tension. In particular, we discuss how axi-symmetric analysis Potsdam, Germany. of membranes misguide our understanding and demonstrate that non- In their fluid state, lipid membranes are able to execute a variety of shape trans- axisymmetric shape transitions are almost always energetically favorable to formations. One intriguing example is provided by vesicle budding, i.e., by the axi-symmetric ones. We apply these methods to understand the classical endo- expulsion of a small, spherical bud connected to the mother vesicle by a narrow cytosis phenomena in mammalian and yeast cells. With the newly found non- membrane neck. The formation of such necks provides an essential step of axisymmetric shapes, we discuss the physical principles governing endocytosis many biological processes (cellular uptake, secretion as well as cell division at varying levels of membrane tension. or cytokinesis). In the framework of curvature elasticity, formation and stability of such a narrow neck are determined by local stability conditions, which 2726-Plat depend both on the local mean curvature and on the spontaneous Molecular Mechanism of Microdomain Dependent Protein Trafficking curvature of the membrane. However, these conditions ignore the molecular Blanca B. Diaz-Rohrer, Kandice R. Levental, Ilya Levental. structure of the membrane, which may affect the shape of the neck. Further- Integrative Biology and Pharmacology, UT Health Science Center, Houston, more, because of their locality, the stability conditions cannot describe the TX, USA. overall response of a neck to mechanical perturbations. In order to address these Eukaryotic cells are organized into spatially and functionally distinct questions, we use molecular simulations to study the neck formation. Budding membrane-bound organelles, whose functions are defined by their composition. and neck formation are induced by small solutes such as monosaccharides that Accurate sorting of membrane components between these compartments is adsorb onto the bilayer membranes. If the two leaflets of a membrane are necessary for maintaining organelle identity. For most membrane proteins, exposed to different solute concentrations, the membrane becomes asymmetric the determinants of their steady-state subcellular localization remain unknown. and acquires a certain spontaneous curvature. We start from a spherical vesicle Lateral membrane domains (lipid rafts) provide an ideal platform for mem- that is exposed to a certain solute concentration in the exterior solution. When brane sorting processes. However, the structural determinants of protein asso- we reduce the volume of this vesicle, we observe formation of narrow mem- ciation with such domains are almost entirely unknown. We developed and brane necks provided the concentration exceeds a certain threshold value. characterized a robust experimental system for quantitative measurements of We induce mechanical perturbation using a geometry that resembles micropi- raft affinity in intact plasma membranes and used it to explore the determinants pette aspiration on the micrometer scale to observe neck opening for which we of transmembrane protein recruitment into raft domains. We identified several calculate the associated free energy landscape. structural features associated with raft affinity, and established a quantitative 2724-Plat and functional relationship between raft association and subcellular protein Diffusion of Proteins and Lipids in Protein-Rich Membranes localization. Specifically, we observed that raft association is fully sufficient Matti Javanainen1,2, Hector Martinez-Seara3, Ralf Metzler4, for PM recycling of certain proteins, and that abrogation of raft partitioning Ilpo Vattulainen1,2. for these proteins led to their degradation in the lysosomes. These findings sup- 1Department of Physics, Tampere University of Technology, Tampere, port the conclusion that ordered membrane domains mediate recycling of spe- Finland, 2Department of Physics, University of Helsinki, Helsinki, Finland, cific membrane components from the endosomal compartments to the PM. We 3Institute of Organic Chemistry and Biochemistry of the Czech Academy of have proceeded to define the molecular machinery that mediates raft lipid and Sciences, Prague, Czech Republic, 4Institute for Physics and Astronomy, protein sorting and recycling to the PM. Using a set of orthogonal transmem- University of Potsdam, Potsdam, Germany. brane proteins as probes of raft and non-raft domains, we developed a high Cell membranes are complex structures that act as hosts for membrane- throughput siRNA screen to dissect the molecular machinery and dynamics associated proteins to form dynamical complexes with lipids and other proteins. for raft-mediated sorting. We identified a number of validated hits including The process driving the formation of functional protein-lipid complexes is lateral known players of the early endocytic traffic (Rab5 and EEA1), but also novel diffusion where lipids and proteins migrate in the membrane plane in a stochastic players that appear to define a distinct class of trafficking mediators specific for but concerted fashion [1,2]. In protein-poor membranes the diffusion of lipids raft-associated proteins. This pathway is not dependent on the classical recy- and proteins is fairly well understood. Experimental studies have provided evi- cling pathways defined by Rab4 and Rab11, rather defining a novel route for dence that for (trans)membrane proteins of radius R, their diffusion coefficient D PM recycling of raft-preferring cargo. scales logarithmically as D log(1/R). The well-known Saffman-Delbruck€ (SD) theory [3] is in line with this picture. However, the challenge is to understand the 2727-Plat diffusion of proteins and lipids in membranes of living cells that are extremely Capacitive Detection of Low-Enthalpy, Higher-Order Phase Transitions in rich in proteins. Based on extensive computer simulations we here discuss Synthetic and Natural Lipid Membranes how crowding with proteins affects lateral diffusion [4-6]. We show that protein Graham J. Taylor1, Frederick A. Heberle2, John Katsaras2, crowding leads to deviations from the SD relation and to the emergence of a C. Patrick Collier3, Stephen A. Sarles4. 1 stronger Stokes-like dependence D 1/R, highlighting that the lateral dynamics The Bredesen Center for Interdisciplinary Research, UTK-ORNL, 2 in crowded membranes is significantly different from protein-poor conditions. Knoxville, TN, USA, SWC Joint Center for Neutron Sciences, Oak Ridge 3 We also demonstrate how protein crowding gives rise to dynamical correlations National Laboratory, Oak Ridge, TN, USA, Center for Nanophase Materials observed as anomalous diffusion. Finally, we discuss biological consequences of Sciences, Oak Ridge National Laboratory, Knoxville, TN, USA, 4 protein crowding and anomalous diffusion in, e.g., formation of functional multi- Mechanical, Aerospace, and Biomedical Engineering, The University of protein complexes. [1] Falck E, et al. J Am Chem Soc 130, 44 (2008). [2] Tennessee, Knoxville, Knoxville, TN, USA. Niemela PS, et al. J Am Chem Soc 132, 7574 (2010). [3] Saffman P, Delbruck,€ Lateral organization and phase separation of lipids in natural membranes play M. Proc Natl Acad Sci 72, 3111 (1975). [4] Jeon, JH et al. Phys Rev X 6, 021006 key roles in regulating cellular processes. First-order phase transitions in model (2016). [5] Javanainen M, et al. Faraday Discuss 161, 397 (2013). [6] Javanainen membranes consisting of few lipid components are well understood and readily M, et al. J Phys Chem Lett 8, 4308 (2017). detectable via calorimetry, densitometry, and fluorescence. However, far less is

BPJ 8705_8719 552a Wednesday, February 21, 2018 known about natural membranes containing numerous lipid species and high Binding of the intrinsically disordered protein to an ordered target protein plays concentrations of cholesterol, for which thermotropic transitions are undetect- a crucial role in the induced folding of the unstructured protein. The third sce- able by the above-mentioned techniques. We recently reported1 on how mem- nario where the folding superfunnel is used is the formation of aggregates from brane capacitance is highly sensitive to low-enthalpy thermotropic transitions destabilized proteins, which is an important factor in case of several conforma- taking place in complex lipid membranes. Specifically, we measured electrical tional diseases, like Alzheimer’s and Parkinson an many other ones. capacitance as a function of temperature for droplet interface bilayer model The folding superfunnel introduced here explains all three above cases success- membranes of increasing compositional complexity, namely, (a) a single lipid fully with minimal assumptions about the interaction potential. species, (b) domain-forming ternary mixtures, and (c) natural brain total lipid extract (bTLE). We observed that, for single-species lipid bilayers and some 2730-Plat ternary compositions, capacitance exhibited an abrupt, temperature dependent Dual Function of the Trigger Factor Chaperone in Nascent Protein Folding change that coincided with the transition detected by other techniques. In addi- Kaixian Liu, Kevin Maciuba, Christian M. Kaiser. tion, capacitance measurements revealed transitions in mixed-lipid membranes Johns Hopkins University, Baltimore, MD, USA. that were not detected by the other techniques. Most notably, capacitance mea- Multi-domain proteins - constituting a large group in all proteomes - often surements of bTLE bilayers indicated a transition 38 C not seen with any require help from molecular chaperones to fold productively, even before the other method. Likewise, capacitance measurements detected transitions in ribosome has finished their synthesis. The mechanisms underlying chaperone some well-studied ternary mixtures that, while known to yield coexisting lipid function remain poorly understood. We have used optical tweezers to study phases, were not detected with calorimetry or densitometry. Results signified the folding of elongation factor G (EF-G), a model multi-domain protein, as it the exquisite sensitivity of capacitance measurements to low-enthalpy mem- emerges from the ribosome. We find that the N-terminal G-domain in nascent brane transitions; capacitive sensitivity stems from changes in bilayer thickness EF-G polypeptides folds robustly. The following domain II, in contrast, fails that occur when lipids and excess solvent undergo subtle rearrangements near a to fold efficiently. Strikingly, interactions with the unfolded domain II convert phase transition. In recent work, capacitance measurements with membranes the natively folded G domain to a non-native state. This non-native state readily formed from binary saturated lipid-cholesterol (20-50%) mixtures also display unfolds, and the two unfolded domains subsequently form misfolded states, pre- signs of phase transitions which are otherwise undetected via calorimetric venting productive folding. Both the conversion of natively folded domains and methods. non-productive interactions among unfolded domains are efficiently prevented 1. Taylor et al., Langmuir 2017, 33 (38), 10016-10026. by the ribosome-binding chaperone trigger factor. Thus, our single-molecule measurements of multi-domain protein folding reveal an unexpected role for 2728-Plat the chaperone: It protects already folded domains against denaturation which re- NMR Contacts between the HIV Fusion Peptide and Lipid Support a Beta- sults from interactions with parts of the nascent polypeptide that are not folded Bowl Membrane Topology of the Peptide with Thermodynamic Preference yet. Previous studies had implicated trigger factor in guiding the folding of indi- for Peptide/Cholesterol Contact vidual domains, and interactions among domains had been neglected. Avoiding David Weliky. early folding defects is crucial, since they can propagate and result in misfolding Michigan State University, East Lansing, MI, USA. of the entire protein. Our experiments define the folding pathway for a complex The HIV fusion peptide (FP) is the 25-residue N-terminal region of the gp41 multi-domain protein and shed light on the molecular mechanism employed by protein and plays a critical role in joining virus and host cell membranes. Seg- molecular chaperones to ensure productive and efficient folding. ments of gp41 that include the FP catalyze fusion of membrane vesicles, which supports a FP contribution to viral fusion. The FP adopts intermolecular anti- 2731-Plat parallel b sheet structure in membrane that contains 30 mole% cholesterol, Unravelling the Mechanics of a Molecular Chaperone 1 1 2 3 which is typical for HIV host cells. NMR measurements support distinct pop- Katarzyna M. Tych , Markus Jahn , Hannah Girstmair , Thorsten Hugel , Johannes Buchner2, Matthias Rief1. ulations of membrane locations of the FP b sheet. The dominant population has 1 <5A˚ contacts between residues in the sheet interior and –CH groups of phos- E22 Biophysics, Physics Department, Technical University of Munich, 3 € 2 phatidylcholine (PC) and cholesterol lipids near the membrane center. Longer Garching bei Munchen, Germany, Chair of Biotechnology, Chemistry > ˚ Department, Technical University of Munich, Garching bei Munchen,€ 10 A distances are detected to lipid headgroups. By contrast, close headgroup 3 contacts are observed for residues at the sheet edges, with longer distances to Germany, Institute of Physical Chemistry, University of Freiburg, Freiburg, Germany. the –CH3 groups. There is a smaller FP population with contacts between inte- rior strand residues and the membrane surface. A FP b-bowl inserted in a single The large molecular chaperone, heat shock protein 90 (Hsp90), is a dimeric ATP- membrane leaflet is consistent with many of these contact data. The middle sec- driven molecular machine. It is one of the most abundant proteins found in eu- tions of interior strands of the bowl contact the membrane center, whereas karyotic cells, and is known to function together in complexes with many strand termini contact lipid headgroups. Most of the bowl edge strands contact different co-chaperones. These complexes are involved, amongst other things, the membrane surface. Lipids contacting the bowl adopt its curvature, which in the regulation of cell division and signalling [1]. As such, Hsp90 has emerged may reduce activation energy needed to achieve the high-curvature transition as an important target for the development of cancer therapeutics [2]. state of fusion. The NMR data also support higher population of FP- The exact mechanisms by which Hsp90 undergoes conformational changes, the cholesterol vs. FP-PC contacts. The thermodynamic preference of cholesterol roles of nucleotides, different domain orientations and how these influence the for the FP bowl may be due to higher intrinsic curvature of cholesterol vs. PC. function of the chaperone in complex with its co-chaperones are mostly unknown. Using a custom-built single molecule optical trapping experimental setup, we have so far elucidated the mechanism by which this large protein folds [3], Platform: Chaperone-assisted Protein Folding characterised the role of its flexible charged linker region [4] and performed a detailed comparison of Hsp90 orthologs [5]. Current research efforts are 2729-Plat building on these findings to characterise the dimerization of Hsp90, and to Explaining Cooperative Folding of Interacting Proteins by a Folding study the effect of nucleotide binding and co-chaperones on the conformational Superfunnel cycle and single molecule mechanics of Hsp90. Laszlo Smeller. [1] A. J. McClellan, et al. Cell 131, 121-135, (2007). Dept. Biophysics and Radiation Biology, Semmelweis University, Budapest, [2] J. S. Isaacs, et al. Cancer cell 3, 213-217, (2003). Hungary. [3] M. Jahn, et al. Proc. Nat. Acad. Sci. 113, 1232-1237, (2016). Generalization of the well-known folding funnel of proteins is presented here. [4] M. Jahn, et al. Proc. Nat. Acad. Sci. 111, 17881-17886, (2014). While the polypeptide chain is treated in the frame of the conventional funnel [5] M. Jahn and K. Tych, et al. Structure (under revision) (2017). model as an isolated object, real biological systems are considerably crowded, protein-protein interactions are fundamental phenomena during the life cycle of 2732-Plat proteins. The Exclusive Effects of Chaperonin on the Free Energy Landscape of The generalized funnel, which we call as superfunnel represents the free energy Proteins with Complex Knots surface of the whole system as function of the conformational coordinates of Joanna I. Sulkowska1, Yani Zhao2, Pawe1 Dabrowski-Tumanski3, the two interacting proteins. Szymon Niewieczerzal1. The folding superfunnel presented here explains the folding process of interact- 1Centre of New Technology, University of Warsaw, Warsawa, Poland, ing proteins in several specific biological processes. Three examples are pre- 2Institute of Physics, Polish Academy of Sciences, Warsawa, Poland, sented here. The first one is the folding of a polypeptide chain with the aid 3Faculty of Chemistry, University of Warsaw, Warsawa, Poland. of chaperones. The second important example for the protein-protein interac- A survey across the PDB shown that at least 7% of known protein structures are tions is the binding assisted folding of the intrinsically disordered proteins. entangled. Proteins can be knotted, possess lassos and links. In particular

BPJ 8705_8719 Wednesday, February 21, 2018 553a knotted structures are known to act as enzymes and DNA binding proteins, and ing advantage of TROSY, deuteration and selective labelling techniques the are also found in the mitochondria and in the membrane. However self-tying of work presented here aims to understand how J-protein – HSC70 interactions such proteins is still an unresolved question. occur as well as how information from the binding event is transmitted to Based on representative members of Ubiquitin C-terminal Hydrolases (UCHs) the nucleotide binding pocket and substrate binding domain of HSC70. Look- that contain the 52 knot in the native state, we explain how UCHs are able to ing at two different J proteins from different sub-families (DNAJC5 and unfold and refold in vitro reversibly within the structure-based model. In partic- DNAJB6) which act through distinct pathways, how HSC70 targeting is ular, we identify two, topologically different folding/unfolding pathways and achieved as well as the role played by J-protein regions outside the J-domain corroborate our results with experiment, recreating the Chevron plot. We can be understood. This work will hopefully contribute to efforts to understand show that confinement effect of chaperonin or weak crowding greatly facili- how protein homeostasis is regulated in the cell. A process which is not only of tates folding, simultaneously slowing down the unfolding process of UCHs, interest to basic cell biology but also underlies many pathologies and may un- compared with bulk conditions. The results of the work show that the crowded derpin future therapies developed to target diseases of protein folding. environment of the cell should have a positive effect on the kinetics of complex knotted proteins, especially now when proteins with deeper knots are found in 2735-Plat Saccharo- this family. These results are also the first step to the identification of possible Kinetic Mechanism of ATP-Dependent Disaggregating Motor myces cerevisiae oligomerization-prone forms of UCHs, which may cause neurodegenerative Hsp104 1 2 2 2 diseases. Clarissa L. Weaver , Meredith E. Jackrel , JiaBei Lin , Korrie L. Mack , 2 1 2 1 Based on analysis of proteins with very low sequence similarity, different Elizabeth Sweeny , Elizabeth C. Duran , James Shorter , Aaron L. Lucius . 1Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA, depths of a knot but with a similar fold, and the same type of topology 2 (VirC2, DndE and MJ0366), we found that encapsulation facilitates knotting Department of Biochemistry and Biophysics, Perelman School of Medicine, at the early stage of folding and can enhance an alternative folding route. Com- University of Pennsylvania, Philadelphia, PA, USA. parison to unknotted proteins with the same fold shows directly how encapsu- S. cerevisiae Hsp104 helps cells survive and adapt to environmental stress. lation influences the free energy landscape. Extreme stress denatures proteins, causing toxic aggregates to form. Hsp104 resolve aggregates, returning damaged proteins to their proper fold and func- 2733-Plat tion or releasing them for degradation. This function, broadly defined as protein þ A Novel Conformation of the Polypeptide-Binding Pocket Supports an remodeling, is common to AAA proteins. Because disaggregases do not Active Substrate Release from Hsp70S covalently modify their substrates, monitoring the progress of their reactions Jiao Yang1, Yinong Zong1, Jiayue Su1, Hongtao Li1, Huanyu Zhu1, has been challenging. Without a method of monitoring translocation in the Linda Columbus2, Lei Zhou1, Qinglian Liu1. absence of covalent modification, Hsp104 have been assumed to operate via þ 1Physiology and Biophysics, Virginia Commonwealth University, the same molecular mechanism as ClpA, a related AAA protein that partners Richmond, VA, USA, 2Department of Chemistry, University of Virginia, with a protease to degrade certain protein substrates. That mechanism is proc- Charlottesville, VA, USA. essive translocation, or complete threading, of the polypeptide. Our lab devel- Heat Shock Proteins 70kDa (Hsp70s) are a class of ubiquitous and highly oped a single-turnover, transient state kinetic assay to study ClpA in the conserved molecular chaperones essential for cellular protein folding and ho- presence or absence of its proteolytic partner. Recently, our lab applied this meostasis. Each Hsp70 has two functional domains: a nucleotide-binding strategy to study the molecular mechanism of ClpB, a bacterial disaggregase. domain (NBD) and a substrate-binding domain (SBD). NBD binds and hydro- In a paradigm shifting finding, we determined that ClpB is a non-processive lyzes ATP while SBD binds polypeptides in extended conformation. ATP bind- translocase, taking just one or two kinetic steps on a polypeptide substrate. ing allosterically regulates polypeptide substrate binding and release. The Here, we extend this investigation to the eukaryotic homologue, Hsp104. We chaperone activity strictly depends on this allosteric coupling. Previous crystal found that Hsp104 is also a non-processive translocase, partially threading structures have revealed an open conformation of the polypeptide-binding polypeptide substrate through the Hsp104 axial channel. pocket in the ATP-bound state, providing breakthrough insights in this allosteric 2736-Plat coupling. However, this open conformation failed to provide an explanation for a A Protean Clamp Guides Membrane Targeting of Tail-Anchored Proteins productive chaperone cycle. To illuminate the molecular mechanism of this allo- Shu-ou Shan, Un Seng Chio. steric coupling and efficient chaperone activity, here we present a novel crystal California Institute of Technology, Pasadena, CA, USA. structure of an intact human BiP, an essential Hsp70 in ER, in an ATP-bound ˚ To maintain cellular organization, many chaperones and targeting factors state at 1.85 A resolution. Strikingly, the polypeptide-binding pocket is escort nascent proteins to membrane destinations. It was generally thought completely closed, seemingly excluding any binding to polypeptide substrates. that substrate proteins preferably bind conformationally closed chaperones. Our FRET, biochemical and EPR analysis inspired by this structure suggests that However, how the nature and dynamics of the targeting complex help guide this fully-closed conformation is the major conformation for the ATP-bound substrate proteins to the target membrane is not understood for most pathways. state in solution and essential for chaperone activity, providing the first evidence We addressed this question for the conserved ATPase Get3, which targets the for an active release of bound polypeptide substrates following ATP binding. essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum Interestingly, the Hsp40 co-chaperone converts this fully-closed conformation (ER). Single-molecule fluorescence spectroscopy showed that, contrary to pre- to an open conformation to initiate productive substrate binding. In summary, vious models of a static closed Get3,TA complex, Get3 samples open confor- this study provided a mechanistic understanding of the dynamic nature of the mations on the sub-millisecond timescale upon TA binding, generating a polypeptide-binding pocket in the Hsp70 chaperone cycle. fluctuating ‘protean clamp’ that stably traps the substrate. Biochemical data showed that this dynamic opening primes Get3 for targeting of TAs to mem- 2734-Plat brane receptor sites. Our results provide a precedent for large-scale dynamics Understanding the Regulation of the HSC70 Chaperone Machine in a substrate-bound chaperone, which provides an effective mechanism to Felipe Ossa, Jason R. Schnell. retain substrate proteins with high affinity while also generating functional Department of Biochemistry, University of Oxford, Oxford, United switches to drive vectorial cellular processes. Kingdom. HSC70 constitutes one of the workhorse proteins of the cell, acting in a wide variety of roles associated with protein homeostasis. These range from protein Platform: Endocytosis, Exocytosis, and folding at the point of biogenesis to the prevention of aggregation of mature Intracellular Transport proteins, and include a plethora of pathway specific roles directly modulating protein-protein interactions. 2737-Plat The action of HSC70 like all of the canonical HSP70 family chaperones is Nanoporosity Influences Membrane Curvature and Subsequent driven by ATP hydrolysis. ATP hydrolysis is in turn regulated by HSC70’s in- Endocytosis teractions with a wide array of regulatory proteins and co-chaperones. J-pro- Alexis Belessiotis-Richards1, Molly M. Stevens1, Alfredo Alexander-Katz2. teins represent one particularly important family of regulatory proteins, 1Department of Materials, Imperial College London, London, United acting both to stimulate ATP hydrolysis and to target HSC70 to specific path- Kingdom, 2Department of Materials Science & Engineering, Massachusetts ways. This targeting of the folding machinery allows the cell to respond to a Institute of Technology, Cambridge, MA, USA. variety of different stresses and environmental changes and constitutes an The development of novel nanoneedle arrays as drug delivery vectors have important response to combat the formation of toxic protein aggregates. stemmed much interest in the use of nanostructures for influencing cell fate. The often fairly weak, transient and dynamic interactions of HSC70 and its reg- These structures, made of porous silicon, are biodegradable, non-cytotoxic ulators provide fertile grounds for productive investigation through NMR. Tak- and can deliver a wide variety of biomolecules, including nucleic acids, to cells

BPJ 8705_8719 554a Wednesday, February 21, 2018 rapidly and efficiently. [1] However, the mechanism of cargo transfer between We used these measurements to constrain a mathematical model of actin poly- these nanostructures and cells is far from being understood. Indeed, the influ- merization coupled to vesicle internalization using the filament-based modeling ence of nanoneedles on cells are varied and much contention remains around software Cytosim (Nedelec and Foethke, 2007). In the model, Arp2/3 complex the biointerface between the cell membrane and such nanostructures. Recently, on the plasma membrane generates branched actin filaments, and a nascent it has been shown that cell membrane curvature induced via deformation on vesicle coated with actin-binding proteins elastically resists internalization nanopillars attracts and localizes clathrin-mediated endocytosis proteins. [2] into the cell. Actin filaments are stochastically capped and stall under load. Here, using coarse-grained molecular dynamics simulations, we show that Three-dimensional, stochastic simulations of the model revealed that Arp2/3- nanoporosity can activate similar nanoscale membrane deformation. By con- nucleated actin networks self-organize into a dendritic cone focused against trolling a set of design parameters, we can strongly control and maximize the attachment site at the base of the endocytic vesicle. The network produced such induced membrane curvature on nanoporous substrates. In addition, we 10-15 pN of force, internalizing the vesicle 100 nm in 10 s. Thus, branched show that endocytosis proteins, in particular Epsin-1, can spontaneously actin networks in mammalian cells are weak but sufficient to overcome phys- localize to these curved membrane regions confirming that such regions act iological membrane tension and carry out endocytosis. as endocytosis ‘‘hot-spots’’. The promotion of cell-active uptake of cargo due to nanoscale plasma membrane deformation could explain in part the 2740-Plat highly efficient delivery properties of nanoneedle arrays as well help us under- All in One: GTP-mediated Membrane Strangling, Fission, and Dynamin stand a piece of the complex puzzle surrounding the biointerface between cells Scaffold Disassembly Martina Pannuzzo1, Zachary McDargh2, Markus Deserno1. and nanotextured substrates. 1 2 [1] Chiappini, C. et al. Biodegradable silicon nanoneedles delivering nucleic Physics, Carnegie Mellon University, Pittsburgh, PA, USA, Chemical acids intracellularly induce localized in vivo neovascularization. Nat. Engineering, Columbia University, New York, NY, USA. Mater.14, 6-13 (2015). In the last twenty years, scientists have been debating the GTP mediated me- [2] Zhao, W. et al. Nanoscale manipulation of membrane curvature for probing chanical action of dynamin in promoting membrane fission. Two main endocytosis in live cells. Nat. Nanotechnol.12, 750-756 (2017). competing models have been proposed. According to the ‘‘two-stage model’’, dynamin dimers in the same nucleotide state self-assemble as a helical scaffold 2738-Plat and constrict the neck of budding vesicles in one stage, while GTP is spent dur- The SH3 Domain of Unconventional Myosin IB from E. histolytica ing the final cooperative dynamin disassembly. However, this model conflicts Interacts With a GEF (EhFP10) and Regulates Phagocytosis by Affecting with the observation that the Hill coefficient of dynamin vs GTP concentration Actin Bundling equals one in the assembled state, which indicates lack of cooperativity. The Gunjan Gautam. main competitor, the ‘‘ratchet/constrictase model’’, maintains that GTP hydro- School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. lysis is instead spent to drive the neck towards maximal constriction, while the Unconventional myosins form the largest class of myosin Superfamily. Unlike scaffold is automatically released once the membrane template disappears (i.e., conventional two-headed myosins, they are involved in organelle movement, after the fission event). This model, instead, fails to explain why depolymeriza- vesicle trafficking, phagocytosis, pinocytosis, membrane ruffling and the for- tion is observed to be associated with GTP hydrolysis. mation of lamellipodia. Several isoforms of class I myosins differing in struc- By employing coarse-grained Molecular Dynamics simulation, tuned to cap- ture and function have been reported in lower as well as higher eukaryotes. ture the geometry of a fission complex and the elasticity of its two main com- E.histolytica has only two myosins. The sole unconventional EhMyosin IB ponents (lipid membrane and dynamin filament), we propose a way to reconcile is a long-tailed myosin I with a c-terminal SH3. In the present work, we these two viewpoints. We present a phenomenological model which can have determined the crystal structure of the Sh3 domain of EhMyosin IB, explain how the energy of GTP can be spent to promote significant conforma- in native and PEG-bound form. Structural studies of PEG-bound EhMySh3 tional changes to both constrict the membrane and promote the subsequent domain revealed characteristics of its probable ligand. We have identified a loosening and detachment of the scaffold. We highlight the far-reaching poten- GTPase exchange factor, EhFP10 with APC basic domain to be one of the tial of a single component molecular motor that, by virtue of its peculiar binding partner of EhMySH3 and validated it through SPR studies and co- geometrical shape, can perform rather diverse actions. crystallization of the target peptide with the EhMySH3 domain. EhMyosin 2741-Plat IB co-localizes with EhFP10 during pinocytosis and phagocytosis. Interaction ESCRT Membrane Scission Revealed by Optical Tweezers with EhMySh3 was observed to inhibit the actin bundling activity by APC Johannes Scho¨neberg1, Shannon Yan1, Amir Bahrami2, Maurizio Righini1, domain at high EhMySH3: APC ratios while as the concentration of APC in- Il-Hyung Lee1, Mark Remec Pavlin1, Lars-Anders Carlson1, creases, it was able to overcome the inhibition. Our findings reveal a new Daniel Goldman1, Gerhard Hummer2, Carlos Bustamante1, James Hurley1. mode of regulation of endocytic processes in E. histolytica, a highly motile 1UC Berkeley, Berkeley, CA, USA, 2Max Planck Institute of Biophysics, gastric pathogen. Frankfurt am Main, Germany. ESCRT proteins catalyze the function of membrane budding and scission from 2739-Plat the inside of the cytosol to the outside. This process is fundamental in cell Actin-Generated Forces during Mammalian Endocytosis biology, comprising multivesicular endosome biogenesis, cytokinesis, viral 1 2 2 Matthew Akamatsu , Ritvik Vasan , Padmini Rangamani , budding (e.g. HIV, Ebola, Dengue) and other pathways. The mechanism under- 1 David G. Drubin . lying ESCRT-III-mediated membrane budding and scission remains elusive. 1Molecular and Cellular Biology, University of California Berkeley, 2 We have encapsulated within giant unilamellar vesicles (GUVs) a minimal Berkeley, CA, USA, Mechanical and Aerospace Engineering, University of ESCRT module consisting of ESCRT-III subunits and the AAAþ ATPase California San Diego, San Diego, CA, USA. Vps4. Using optical tweezers, membrane nanotubes reflecting the correct topol- Actin filaments produce force by converting polymerization energy into mechan- ogy of scission can be pulled from these GUVs. Upon photo-uncaging of ATP, ical work. During endocytosis, actin generates force todeform a smallregion of the surprisingly large forces in the tens of piconewtons were recorded and tube plasma membrane into a round vesicle for internalizing transmembrane and extra- scission could be observed. ESCRT subunit composition and concentration cellular cargo. Despite detailed knowledge of the biochemical function of individ- alter force generation and scission behavior. In combining confocal fluores- ual actin cytoskeletal proteins, the mechanism by which they work collectively to cence microscopy and optical tweezers, the scission events can be observed bend and pull the plasma membrane in cells is not understood. in both force and fluorescence and studied in detail. For the first time, the We combined live-cell quantitative fluorescence microscopy in genome-edited biophysics of ESCRT membrane budding and scission are revealed. human induced pluripotent stem (iPS) cells and mathematical modeling to investigate the mechanism by which branched actin networks generate force 2742-Plat during endocytosis. Using GFP-tagged self-assembling protein nanocages of Molecular Mechanisms Controlling Neurotransmitter Release by the defined copy number (Hsia et al., 2016), we generated a calibration curve Primed SNARE-complexin-Synaptotagmin Complex relating fluorescence intensity to molecule number in cells. Using spinning- Qiangjun Zhou1, Peng Zhou1, Thomas C. Sudhof€ 1,2, Axel T. Brunger1,2. disk confocal microscopy and semi-automated image analysis we counted 1Molecular and Cellular Physiology, Stanford University, Stanford, CA, the numbers of molecules over time of the endocytic proteins Arp2/3 complex USA, 2Howard Hughes Medical Institute, Stanford, CA, USA. and dynamin2. Compared to yeast, mammalian endocytic sites had fewer Arp2/ Synaptic transmission is essential for the process of communication between 3 complexes, implying fewer branched actin filaments, which corresponds to two neurons. It occurs upon fusion of synaptic vesicles with the plasma mem- less resistance from the plasma membrane. brane, a highly regulated and ultrafast process occurring in less than a

BPJ 8705_8719 Wednesday, February 21, 2018 555a millisecond. Synaptotagmin, complexin, and neuronal SNARE (soluble N-eth- meshwork. The meshwork poses a ‘super-viscous’ environment to the inert ylmaleimide sensitive factor attachment protein receptor) proteins mediate (non-specific) cargos while behaving as a less viscous liquid for nuclear- evoked synchronous neurotransmitter release, but the molecular mechanisms transport-factor-bound cargos. mediating the cooperation between these molecules remain unclear. We deter- mined crystal structures of the SNARE-synaptotagmin-1 complex and the Symposium: Biophysical Insights from Surface primed pre-fusion SNARE-complexin-synaptotagmin-1 complex. These struc- tures reveal a specific, Ca2þ-independent, conserved interface between Engineering synaptotagmin-1 and the SNARE complex. The structure of the primed pre- fusion SNARE-complexin-synaptotagmin-1 complex reveals an unexpected 2745-Symp tripartite interface between synaptotagmin-1 and both the SNARE complex Intercellular Mechanotransduction and complexin. Mutations that disrupt either interface in solution also severely Deborah Leckband. impair evoked synchronous release in neurons, suggesting that both interfaces Chemical Sciences, University of Illinois, Urbana, IL, USA. are essential for the primed pre-fusion state. Ca2þ binding to the This talk illustrates the use of surface engineering to investigate force trans- synaptotagmin-1 molecules unlocks the complex, allows full zippering of the duction through cadherin-based intercellular adhesions. We focused on cad- SNARE complex, and triggers membrane fusion. The tripartite SNARE- herins, which are indispensable adhesion proteins at cell-to-cell junctions complexin-synaptotagmin-1 complex at a synaptic vesicle docking site has to and identified sites of force transduction. Unlike integrin-extracellular ma- be unlocked for triggered fusion to start, explaining the cooperation between trix adhesions, it is challenging to selectively interrogate adhesion proteins complexin and synaptotagmin-1 in synchronizing evoked release on the sub- at cell-cell junctions, independent of other adhesion proteins at these con- millisecond timescale. tacts. However, by selectively tailoring of cadherin ligand presentation on hydrogel surfaces of tunable rigidity, we directly demonstrated that cadherin 2743-Plat complexes, like integrins, sense extracellular mechanics to regulate cell Run, Wander, and Drift: Multi-modal Transport in the Eukaryotic signaling and contractility. We monitored force-transduction using fluores- Cytoplasm cence based sensors and traction force microscopy. Moreover, varying the Elena F. Koslover. immobilized cadherin ligand revealed the selectivity of cadherin-based Physics, University of California, San Diego, La Jolla, CA, USA. force transduction, with consequent differences in cell adhesion and Eukaryotic cells utilize a variety of mechanisms to transport particles of signaling. We verified that findings basedonimmobilizedcadherinsub- different sizes throughout the cytoplasm. Commonly employed transport strates translate to bona fide cell-cell junctions, by exploiting the fact that modes include diffusion driven by stochastic fluctuations in the medium, the substrate composition and stiffness alter cell contractility and the endog- processive motor-driven transport along cytoskeletal tracks, and advection enous force on cell-cell junctions. Results confirmed that substrate- in a flowing cytoplasmic fluid. We use analytical theory and simulations, dependent intercellular tension also activates cadherin-force transduction. grounded in the physics of stochastic processes and fluid dynamics and Overall, our use of tailored substrates enabled the identification of coupled with analysis of in vivo data on organelle motion, to explore the ef- mechano-chemical signaling network through which cadherins, integrins, ficiency and relative contributions of these different modes of transport. Us- and the extracellular matrix coordinately regulate global cell mechanics ing several example cellular systems, we identify both the physical limits and the integrity of cell-cell junctions. and synergistic nature of multi-modal transport, where individual organelles switch between active and passive regimes. In particular, we highlight the 2746-Symp potential importance of hydrodynamic entrainment and large-scale cyto- Cells Sense and Respond to Curvature by Patterning Stress Fibers and plasmic flow associated with cell deformation to the dispersion of vesicles Undergoing Curvature Guided Migration within fungal hyphae and motile leukocytes, respectively. Peroxisome trans- Kathleen Stebe. port in hyphae is shown to lie in a regime where both passive diffusion and University of Pennsylvania, Philadelphia, PA, USA. directed ‘‘hitch-hiking’’ runs contribute substantially to the organelles’ abil- Physical cues from a cell’s environment influence cell behaviors; important, ity to efficiently find intracellular targets. We also show that spatial locali- well established examples include surface texture on the sub-cellular scale zation of neuronal mitochdria by glucose-dependent regulation of transport and substrate stiffness. Here we explore how adherent cells sense and respond is sensitive to intracellular glucose levels in a manner dependent on the bal- to the curvature of their substrates by studying fibroblasts and vascular smooth ance of mitochondrial transport modes. These results highlight the impor- muscle cells on cylinders and on substrates that we term spheres with skirts de- tance of combining together a variety of passive and active transport signed to present domains of positive and negative Gaussian curvature. On cyl- mechanisms to efficiently distribute, deliver, and organize organelles within inders, fibroblasts align two spatially distinct subpopulations of actin stress eukaryotic cells. fibers along the principal directions of the surface: the apical stress fibers avoid bending while the basal stress fibers bend maximally. On a negative Gaussian 2744-Plat curvature surface, similar subpopulations emerge. This reorganization also The Nature’s Superviscous Nano-channel: Insight from Big Data-Driven plays a role in cell migration. On planar surfaces, cells typically migrate in Biophysical Modeling the direction that coincides with apical stress fibers orientation. However, cells Ruhollah Moussavi-Baygi, Mohammad Mofrad. on a negative Gaussian curvature surface migrate in the direction nearly orthog- UC Berkeley, Berkeley, CA, USA. onal to stress fiber orientation. These results demonstrate that macroscale cur- The nuclear pore complex (NPC) is the unique gateway to the cell nucleus vature is a prominent physical cue that guides distinct pattern formation within and is primarily filled with intrinsically disordered proteins (IDPs) rich in the cell and cell migration direction. Phe-Gly (FG) repeat domains. While the microdynamics of these IDPs is still under debate, there is no information and/or consensus about their mi- 2747-Symp crorheological properties. Here, we use state-of-the-art computational Spatio-Temporal Control of Cellular Dynamics using a Cell-Friendly methods to explore, for the first time, the microrheology of IDPs confined Photoresist inside the nuclear pore. Our results indicate that the IDPs rich in FG repeat Junsang Doh. domains show an intriguing rheological behavior, which uniquely stems Pohang University of Science and Technology (POSTECH), South Korea, from the interplay of biophysical factors including hydrophobicity, charge, Pohang, Republic of Korea. the ratios of positive and negative charge content to hydrophobicity content, Many biological processes in multi-cellular organisms are regulated by various geometrical confinement, chains’ lengths, channel wall permeability, and dynamic cellular processes. However, classical tissue culture system can only the chains’ end-tethering. Performing several long-run biophysical simula- provide static environments where many dynamic events occur randomly, thus tions of tens of milliseconds, we developed detailed mechanical spectrums mechanistic study of dynamics of cellular dynamics has been limited. To over- of the polymeric meshwork inside the NPC under these physical conditions come this limitation, we developed new experimental platforms where we can over a wide range of frequencies. It appears that among these factors, the precisely position different types of cells and control their movement by illumi- chains’ end-tethering plays the dominant role in shaping the mechanical nating light to predefined areas. A thin film of a cell-friendly photoresist PDMP spectrum of FG-meshwork, particularly in the low-frequency regime. The (poly(2,2-dimethoxy nitrobenzyl methacrylate-r-methyl methacrylate-r-poly frequency-dependent viscosity of the FG-meshwork is reminiscent of (ethylene glycol) methacrylate) was used. Upon UV exposure, PDMP thin films pseudo-plasticity, and thus, the FG-repeats form a shear-thinning polymeric become soluble in near-neutral aqueous buffers such as PBS and tissue culture

BPJ 8705_8719 556a Wednesday, February 21, 2018 media with minimal cytotoxicity. To create micro-patterns on PDMP thin film, kinesin-2 motors, KIF17 and KIF3AB, which act jointly to drive intraflagellar microscope projection photolithography (MPP) technique was used. By per- transport, can explain in vivo data showing that these motors ‘‘trade off’’ while forming series of MPP on PDMP thin films and sequentially depositing cells transporting vesicular cargo along the length of cilium (Prevo at el., 2015, Nat. on UV-irradiated regions, we could successfully create single cell arrays of Cell. Biol. 17:1546-45; Milic et al., 2017 Proc. Nat. Acad. Sci. USA 114:E6830- adhering cells. Then, various dynamic cellular processes such as cell spreading, E6838). adherens junction formation, and collective cell migration were triggered by removing PDMP thin films surrounding cells in single cell arrays by UV illu- 2750-Symp mination. Using this new dynamic cell-patterning technique in conjunction Kinesin Motor Domain Dynamics during Single-Motor Stepping and with an automated microscope equipped with a motorized stage, dynamics of Multi-motor Transport each cellular process was imaged in high-throughput manners to collect large William O. Hancock. amounts of data in a single experiment. This method can be useful for screening Bioengineering, Pennsylvania State University, University Park, PA, USA. drugs targeting dynamic processes of cells. To understand mechanochemical tuning across transport kinesins, we are inves- tigating the stepping kinetics of kinesin-1 (conventional kinesin) and kinesin-2 2748-Symp (KIF3) motors. By attaching a 30-nm gold nanoparticle to one head of a dimer, The Influence of Monolayer Morphology and Dynamics on Lung Stability we are able to track head dynamics with 1 ms temporal resolution and 1-2 nm Joseph A. Zasadzinski, Amit K. Sachan, Benjamin Stottrup. spatial precision. At saturating ATP, the kinesin-1 hydrolysis cycle is roughly Chemical Engineering and Materials Science, University of Minnesota, evenly split between states with one- and two-heads bound to the microtubule, Minneapolis, MN, USA. consistent with two rate limiting steps in the hydrolysis cycle. ATP analog ex- For the past 100 years, surfactant monolayers have been studied on the planar periments support a model in which ATP hydrolysis occurs before attachment interface of Langmuir trough, even though most physiological interfaces are of the tethered head to the next binding site. It was previously shown that dif- curved at the micron to millimeter scale. Here we show that as a lung surfactant ferences in processivity between kinesin-1 and kinesin-2 result from differ- monolayer-covered bubble approaches a critical radius, the equilibrium ences in the length of their neck linker domain. We find that for both motors, morphology changes from dispersed circular solid domains in a continuous the duration spent in the one-head-bound state increases with increasing neck liquid to a continuous solid phase linear mesh separating discontinuous liquid linker length. Thus, kinesins with longer neck linkers are less processive phase domains. Monolayer dynamics and surfactant adsorption properties are because they spend a longer duration in a vulnerable one-head-bound post-hy- also strongly influenced by the interfacial curvature. The phase morphology drolysis state. Finally, to understand inter-motor coordination during multi- transition phenomena cannot be readily explained by current theories based motor transport, we have tracked the dynamics of one motor in a motor pair on isotropic domains but rather on the anisotropic properties of the semi- connected through a DNA scaffold. We find that kinesin-2 detaches more crystalline solid phase. The changes in morphology and dynamics may have frequently than kinesin-1, but also reattaches to the microtubule four-fold important physiological consequences for lung stability and function as the more rapidly. Although detachment of kinesin-2 is more load dependent than morphological transition occurs at alveolar dimensions. The anisotropy of kinesin-1, these enhanced binding dynamics allow cargo carried by kinesin-2 the condensed-phase monolayers of the primary lipid component of lung sur- to better navigate crowded microtubules. factant, dipalmitoylphosphatidylcholine, DPPC is shown in its rheological properties that differ between monolayers sheared clockwise (C) or counter- 2751-Symp clockwise (CC). The nonlinear elastic modulus and yield stress of R-DPPC Allosteric Tuning of Myosin Force Generation: New Avenues Towards monolayers are greater when sheared clockwise, i.e. against the natural CC Therapeutical Treatment winding of individual DPPC domains than when sheared counter-clockwise. Anne Houdusse. These differences disappear for racemic mixtures. Direct visualization of the Curie Institute, Paris, France. deforming domains shows a different evolution of defect structures for shear Force production by myosin motors plays major roles in muscle contraction, in the C or CC directions. We argue that the coupling between the tilt grain intra-cellular trafficking and maintenance of critical cellular structures such boundaries and applied shear can give rise to the chiral rheology we observed. as microvilli, stereocilia and invadopodia. Deficit in different myosin motors The long-time, macroscopic consequences of the underlying molecular can lead to a number of serious disease, thus myosins are important potential chirality are remarkable given the single-component, non-cross-linked nature targets for therapeutical treatment. Structures of myosins in complexes with of DPPC monolayers. small molecules reveal unsuspected allosteric sites and provide valuable in- sights for the design of specific modulators. These reveal the mechanistic con- trol of motor transitions by inhibitors and activators and provide novel Symposium: Cytoskeletal Motors understanding of the rearrangements controlling the force producing lever arm swing. Current progress and outstanding questions regarding the important 2749-Symp sequential rearrangements that lead to force production by myosins will be pre- Single-Molecule Nanomechanics of Kinesin and Kinesin-Family Proteins sented in light of recently solved X-ray structures of myosin/drug complexes. Steven M. Block. New insights into the mechanism of allosteric tuning of myosin force genera- Biology and Applied Physics, Stanford University, Stanford, CA, USA. tion is thus anticipated to lead the way in the development of new myosin- There are roughly 45 kinesin superfamily members coded by the human directed therapeutics. genome, and 25 by the fruit fly genome: these motor proteins can be classified into at least 14 different subtypes. An individual cell typically expresses up to a 2752-Symp dozen-or-more different versions at any given time. Although the nanomechan- One Motor, Many Functions: Localized Regulation of Cytoplasmic Dynein ical properties of the founding member of the superfamily, kinesin-1, are well in Neurons by Effector Proteins established, it’s been an ongoing challenge to understand the physiology of the Erika Holzbaur. other kinesin members, and in particular, how that physiology ties into their University of Pennsylvania, Philadelphia, PA, USA. varied cellular functions. It’s become clear that different kinesin motors exhibit Neurons are polarized cells with extended processes, and are particularly radically different nanomechanical properties, including wide variations in dependent on molecular motors to drive organelle transport over long distances. translocation speed, processivity, randomness, and response to load. These As the major minus-end directed microtubule motor in neurons, cytoplasmic properties dictate not only the diverse range of behaviors exhibited by individ- dynein has many functions, including axonal transport, dendritic trafficking, ual motors, but also the behavior of motors working in ensembles, for example, and axodendritic sorting. We are using optogenetics, live cell imaging and sin- when multiple motors are attached to a common cargo. For a number of years, gle molecule reconstitution assays to more fully define dynein function and my lab has been using single-molecule optical trapping to characterize the localized regulation in the neuron. To query dynein function within specific nanomechanical properties of kinesin family motors in detail, including Eg-5 compartments, we used a light-sensitive caged crosslinker to recruit motors (kinesin-5), KIF17 and KIF3AB (kinesin-2), and KIF15 (kinesin-12), as well to organelle cargos with high spatial and temporal specificity. We found that as variants of kinesin-1. A common feature that has emerged is that different kinesin-1 functions much more effectively in axons versus dendrites. In kinesin motors can all be modeled by a similar four-state, biochemical cycle contrast, dynein efficiently navigates both axons and dendrites despite signifi- involving a partial docking of the neck linker. This minimal model accounts cant differences in the polarity of the underlying microtubule cytoskeleton. We quantitatively for forward stepping, backward stepping, and eventual microtu- tested the predictions of the optogenetic assay by examining the motility of an bule release, adjusting only the transition rates among states for different mo- endogenous dendritic cargo using BDNF-bound quantum dots and found that tors. In particular, the substantial nanomechanical differences exhibited by two dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking

BPJ 8705_8719 Wednesday, February 21, 2018 557a that is critical for the transit of signaling endosomes from dendrites to the nu- 2755-Plat cleus. To further investigate the mechanisms regulating dynein-driven trans- Atomic-Level Characterization of Protein-Protein Association port, we are testing the hypothesis that motility is precisely regulated by Albert C. Pan1, Daniel Jacobson1, Konstantin Borisov1, Duluxan Sritharan1, cargo-specific effectors. We focused on Hook1, which induces superprocessive Thomas M. Weinreich1, David E. Shaw1,2. dynein motility in single molecule TIRF assays. We used live cell imaging of 1D E Shaw Research, New York, NY, USA, 2Center for Computational hippocampal neurons in microfluidic chambers to investigate the role of Hook Biology and Bioinformatics, Columbia University, New York, NY, USA. proteins in endosomal transport along the axon. Hook1 depletion significantly The association of proteins into stable complexes is central to an array of bio- reduced the flux of BDNF-positive signaling endosomes without affecting the logical processes. Predicting the structures of and understanding the mecha- overall motility of Rab5-endosomes. These observations indicate that Hook1 nisms by which proteins associate into these complexes are outstanding acts as a specific dynein effector for signaling endosomes in the axon, and sup- challenges in the field of molecular biology. Advances to our understanding port the model that differential association with cargo-specific effectors allows could also have important implications for drug discovery efforts involving for organelle-specific tuning of dynein motor function. protein-protein complexes, a large and important class of therapeutic targets where progress toward the clinic, especially for small-molecule drugs, has been challenging. To study protein-protein association, we have developed Platform: Molecular Dynamics II an approach called tempered binding that combines long timescale atomic- level molecular dynamics simulations with simulated Hamiltonian tempering. 2753-Plat We will describe the results of tempered binding simulations in which proteins Simulation of Geometrically Accurate, Multibillion Atom Cellular repeatedly associate into, and dissociate from, their crystallographically-deter- Membrane Structures mined complex structures, and discuss insights into protein-protein association 1,2 2,3 Noah Trebesch , Emad Tajkhorshid . mechanisms revealed by these simulations. 1Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2Beckman Institute for Advanced 2756-Plat Science and Technology, University of Illinois at Urbana-Champaign, A Minimal Coarse-Grained Molecular Dynamics Model of Axon Plasma Urbana, IL, USA, 3Department of Biochemistry and Center for Biophysics Membrane with its Implication on the Diffusion Behavior of Axon Mem- and Quantitative Biology, University of Illinois at Urbana-Champaign, brane Proteins Urbana, IL, USA. Yihao Zhang, George Lykotrafitis. Membranes are fundamental to the function and structure of cells and organ- Mechanical Engineering, University of Connecticut, Storrs, CT, USA. elles, and molecular dynamics (MD) simulations have the potential to reveal The axon initial segment (AIS) in the neuron has two crucial functions: main- deep insights about the basic physics and biochemistry that produce their taining the polarity of neurons and initiation of action potentials. One extraor- behavior. However, MD simulations of cellular membranes have been dinary feature of the AIS is the separation of somatodendritic and axonal extremely limited up to this point due to the geometric complexity and sheer domains which was confirmed by measuring the trajectory of beads attached size of such membranes. To overcome these limitations, we have developed to various axonal membrane proteins that there exists a diffusion barrier in xMAS (Experimentally-Derived Membranes of Arbitrary Shape) Builder, soft- the AIS restricting the motion of membrane proteins and lipids. According to ware designed to turn low-resolution 3D meshes derived from experimental the ‘‘fences and pickets’’ model, the periodic cytoskeleton and the highly techniques into atomistic membrane models that can be simulated using MD. concentrated transmembrane proteins in the AIS are capable of hindering the In the first application of this software, we have used an experimentally-derived diffusion of other proteins and lipids through steric interaction and hydrody- 3D mesh and lipid composition to develop a realistic atomistic model of a Ter- namic friction effect. In this work, we established the first coarse-grained mo- asaki ramp, a helicoidal membrane structure found at the junction between lecular dynamics model of axon plasma membrane which includes both axon sheets of the endoplasmic reticulum. The model measures approximately membrane skeleton and axonal membranes to investigate the diffusion 1.97mm by 1.59mm by 0.61mm and is composed of 36.6 million lipids behavior of membrane proteins and lipids in the AIS from a mechanics aspect. (4.5 billion atoms), making it one of the largest atomistic biological models First, we found that transmembrane proteins and proteins in the inner lipid to ever be built. Building this model with xMAS Builder involved several meth- leaflet are confined within the areas between the actin rings. We suspected odologically innovative steps, including using MD to simulate 36.6 million and proved from the computational model that the size of actin associated pro- Lennard-Jones particles while attracted to grid-based potentials to optimize tein junctions, which connected to the lipid bilayers may restrict the motion of the packing of the membrane lipids, running billion atom MD simulations to proteins in the outer leaflet. Then we showed that the spectrin-lipid attraction fix ring piercings and other complex lipid clashes using a newly developed en- level will affect the circumferential mobility of membrane proteins. Finally, ergy minimization technique, and utilizing grid-based potentials to equilibrate the highly dense membrane proteins slowed down the motion of all membrane the model while maintaining its experimentally-derived shape. Simulations of proteins even the lipids. We conclude that actin rings and spectrin filaments the cellular membrane models built using xMAS Builder will allow us to restrict the motion of membrane proteins along and around the axon, respec- computationally probe the complex interactions that give rise to structural sta- tively. However, the main mechanism forming the diffusion barrier in the bility and function at an unprecedented atomistic level of resolution, and they AIS is the accumulation of concentrated transmembrane proteins. will allow us to build better coarse models of the systems for simulation of longer timescale phenomena. 2757-Plat Dynamic Histogram Analysis to Determine Free Energies and Rates from 2754-Plat Biased Simulations Computational High-throughput Screening of Drug-Membrane Lukas S. Stelzl1, Adam Kells2, Edina Rosta2, Gerhard Hummer1. Thermodynamics 1Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Tristan Bereau. Frankfurt am Main, Germany, 2Department of Chemistry, King’s College, Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany. London, United Kingdom. The partitioning of small molecules in cell membranes—a key parameter for Transitions between metastable states govern many fundamental processes in pharmaceutical applications—typically relies on experimentally-available biology, such as protein and RNA folding. The underlying free energy surfaces bulk partitioning coefficients. Computer simulations provide a structural reso- can be obtained from simulations using enhanced sampling methods. We pre- lution of the insertion thermodynamics via the potential of mean force, but sent an algorithm to calculate free energies and rates from enhanced sampling require significant sampling at the atomistic level. Here, we introduce high- simulations on biased potential energy surfaces. Inputs are the accumulated throughput coarse-grained molecular dynamics simulations to screen thermo- times spent in each state or bin of a histogram, and the transition counts be- dynamic properties. This application of physics-based models in a large-scale tween them. For each of the states/bins optimal unbiased free energies are ob- study of small molecules establishes linear relationships between partitioning tained by maximizing the likelihood of a master equation (i.e., first-order coefficients and key features of the potential of mean force. This allows us to kinetic rate model). Unbiased rate coefficients for transitions between states/ predict the structure of the insertion from bulk experimental measurements bins can then be estimated. The resulting ‘‘dynamic histogram analysis method for more than 400,000 compounds. The potential of mean force hereby extended to detailed balance’’ (DHAMed) improves on the DHAM method. becomes an easily accessible quantity—already recognized for its high predict- DHAMed yields accurate free energies in cases where the commonly used ability of certain properties, e.g., passive permeation. Further, we demonstrate weighted-histogram analysis method (WHAM) for umbrella sampling fails how coarse graining helps reduce the size of chemical space, enabling a hier- because dynamics within the windows is slow. We illustrate DHAMed with ap- archical approach to screening small molecules. plications to a-helical peptide folding, RNA duplex formation and the confor- Menichetti, Kanekal, Kremer, Bereau, J Chem Phys, 147 (2017). mational dynamics of single-stranded RNA. DHAMed straightforwardly yields

BPJ 8705_8719 558a Wednesday, February 21, 2018 accurate free energy estimates from sets of short trajectories, which provides a Platform: Membrane Protein Dynamics way forward for computational drug design. Our rate formalism can be used to construct Markov state models from biased or replica exchange molecular dy- 2761-Plat namics (REMD) simulations and we demonstrate its practical applicability by Conformational Landscape of Sodium Glucose Transporters determining the kinetics of RNA folding from REMD. Paola Bisignano1,2, Sara Capponi1,2, John M. Rosenberg3, Michael Grabe1,2. 1Department of Pharmaceutical Chemistry, University of California, San 2758-Plat 2 Weighted Ensemble Simulation Studies of Millisecond Folder NTL9 Francisco, San Francisco, CA, USA, Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA, USA, Upendra Adhikari, Barmak Mostofian, Daniel M. Zuckerman. 3 Biomedical Engineering, Oregon Health & Science University, Portland, OR, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA. USA. Computational characterization of protein folding kinetics and mechanisms re- Sodium-dependent glucose transporters (SGLTs) exploit the sodium ion con- mains challenging because of the long timescales often involved. The weighted centration gradients to transports simple sugars across the plasma membrane. ensemble (WE) rare-events method, in principle, is an unbiased tool for exam- They work by means of an alternating access mechanism in which the trans- ining long-time behavior based on a statistically orchestrated ensemble of stan- porter first binds ligands from the extracellular side in an outward-facing dard short molecular dynamics (MD) trajectories. WE can efficiently distribute conformation and then a conformational change leads to an inward-facing computational resources across configuration space to provide ‘‘statistical conformation from which the transporter is poised to release the cargo into ratcheting’’ toward an outcome of interest. We now use WE to study fully the cytoplasm. While binding and unbinding events are relatively fast, confor- atomistic protein folding. The approach is first validated on the 10-residue mational changes are slow and pose a major challenge to simulate. Previously, hairpin chignolin by comparison to a long MD simulation. We then study the we used the weighted ensemble enhanced sampling technique coupled with a millisecond folder NTL9 under room-temperature conditions, assessing the ac- very simple Go model of the transporter to explore alternating access in the curacy and efficiency of the resulting kinetics estimates, as well as effects of the related protein Mhp1. Here, we revisit our previous computations using a starting structure(s). more chemically accurate coarse-grained description of the system, which al- lows us to explicitly explore the role of ion and substrate binding in driving the 2759-Plat conformational change. Starting from both the high-resolution apo and sugar Enhanced Sampling and Bayesian Inference to Model the Conformational bound inward-facing structures of the bacterial vSGLT from vibrio parahaemo- Dynamics of Peptoid Macrocycles lyticus, we simulated the transition to the outward-facing state using a novel Matthew Hurley, JD Northrup, Vincent Voelz, Chris Schafmeister. progress coordinate based on contact map similarity between vSGLT and the Chemistry, Temple University, Philadelphia, PA, USA. outward-facing structure of closely related transporter. Cyclic peptoid macrocycles are a compelling class of foldamers that can form stable scaffolds for the design of biomimetic molecules for diverse applications 2762-Plat such as catalysis, metal-binding, and protein-protein interactions. In collabora- Single-Molecule Analysis of Phospholipid Scrambling by TMEM16F tion with synthetic efforts, we aim to design highly preorganized peptoid-spi- Rikiya Watanabe1, Takaharu Sakuragi2, Hiroyuki Noji1, Shigekazu Nagata3. 1 roligomer macrocycles stabilized by bound metal cations. To do this, we Department of Applied Chemistry, The University of Tokyo, Bunkyo-ku, 2 3 utilize all-atom, replica-exchange molecular dynamics simulations in conjunc- Japan, Osaka University, Suita, Japan, Department of Applied Chemistry, tion with BICePs, a Bayesian Inference approach that combines theoretical Osaka University, Suita, Japan. simulation data with experimental restraints, to computationally screen and In eukaryotic cells, phospholipids are asymmetrically distributed between the select designs from a growing library of these structures synthesized by the outer and inner leaflets of plasma membranes. In various biological processes, Schafmeister group. The accurate modeling of these cyclic hexamers presents this asymmetric distribution is disrupted by phospholipid scramblases, e.g., several challenges that require enhanced sampling methods, as they form TMEM16F or Xkr-8, that translocate phospholipids bidirectionally between leaf- unique, rigid conformations that undergo very slow-timescale rearrangement lets; however, despite their importance, the biophysical features of the scrambling in the presence of ions. Our work will lead to more sophisticated methodologies reaction remain elusive due to technical difficulties to quantify the scrambling ac- for analyzing peptoids and spiroligomers, and better strategies for computa- tivities in vitro. To address this issue, we in this study developed a novel microsys- tional foldamer design. tem containing membrane bilayers with asymmetrically distributed phospholipids, and first succeeded to measure the phospholipid scrambling of TMEM16F at 2760-Plat single-molecule level, which offered key benefits over macroscopic assay methods Towards Dynamic Pharmacophore Models by Coarse Grained Molecular as it enabled characterization of the biophysical features of scrambling activities. Dynamics The single molecule observation, combined with thermodynamic analysis, re- Nicholas Michelarakis1, Zara A. Sands2, Mark S.P. Sansom1, vealed that TMEM16F transports 4.5 104 lipids s1 at 25 C, with activation Phillip J. Stansfeld1. free energy (DGz)of47kJmol1. Notably, DGz for spontaneous lipid flip-flop 1 2 University of Oxford, Oxford, United Kingdom, UCB Pharma, Braine- across membrane bilayers is about 100 kJ mol1, showing that TMEM16F signif- l’Alleud, Belgium. icantly reduces the activation free energy to achieve the efficient phospholipid Pharmacophore models play a crucial role in computer aided drug discovery e.g. scrambling. Importantly, the microsystem we have designed could also be used in virtual screening, de novo drug design, and lead optimization. Due to the to investigate phospholipid transport by other membrane proteins, e.g., flippase increased numbers of protein structures elucidated, structure-based methods and floppase, which would significantly impact in cell membrane biology. for developing pharmacophore models have started gaining in popularity and are becoming of particular importance. There has been a number of studies 2763-Plat combining such methods with the use of MD simulations to model protein flex- Measuring Membrane Surface Reactions by Diffusion: Dimerization of ibility. In the MARTINI forcefield, four heavy atoms are represented with the use Btk PH Domain and K-Ras of a single interaction centre. Four types of interactions have been parametrised Jean K. Chung1, Laura M. Nocka1, Young Kwang Lee1, John Kuriyan2, in the model, polar, charged, non-polar and apolar. These interactions coincide Jay T. Groves1. with some of the typical features found in a pharmacophore model, allowing the 1Chemistry, University of California Berkeley, Berkeley, CA, USA, CG atoms to be used as pharmacophoric probes. These probes are then used in 2Molecular and Cell Biology, University of California Berkeley, Berkeley, CG MD simulations in order to explore protein interaction propensities. This CA, USA. approach, in combination with cavity detection methods, allows for the identifi- Transient protein-protein interactions on two-dimensional membrane surfaces, cation of potential ligand binding sites and the detection of ‘hotspot’ interactions such as dimerization, play an important role in many signaling reactions. How- that enhance ligand binding. Using CDK2 as a test case, we demonstrate the abil- ever, they are often difficult to quantitatively characterize in membrane environ- ity of this method to recapitulate the positions of moieties that contribute to bind- ments. Here, the dimerization behavior of two proteins, the pleckstrin homology ing interactions commonly observed for ligands known to bind CDK2 and for (PH) domain of Bruton’s tyrosine kinase (Btk) and K-Ras GTPase, investigated which crystal structures are available. When examining the ATP binding site through the two-dimensional diffusion rates on planar supported lipid bilayers of CDK2, a comprehensive and accurate map of the interactions which play a with fluorescence correlation spectroscopy and single-particle tracking, are pre- role in ligand binding is generated. The interaction sites identified are then sented. It is shown that the Btk PH domain dimerizes in a ligand-dependent compared with holo crystal structures and are shown to correctly identify the manner, and K-Ras remains strictly monomeric over a wide range of surface den- moieties which contribute to the binding interactions. By calculating the DGInt sities, ranging from single-molecule level to dense packed. These works demon- values of the interaction maps, we are able to further focus on the areas of interest strate how protein diffusion on homogeneous membranes can be used for and identify which moieties are the driving force behind binding. quantitative measurement of weak protein-protein interactions.

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2764-Plat ments and bundles, but also likely by other membrane structures, leading to 50 - A Combined Simulation and SAXS Study of the Dynamics of Lipid 500 nm sized confinement regions in RBL cells. Nanodiscs [1]. Ritchie, K.; Iino, R.; Fujiwara, T.; Murase, K.; Kusumi, A. The fence and Tone Bengtsen1, Viktor L. Holm2, Søren R. Midtgaard2, Lise Arleth2, picket structure of the plasma membrane of live cells as revealed by single Kresten Lindorff-Larsen1. molecule techniques (Review). Mol. Membr. Biol. 2003, 20, 1318. 1Department of Biology, University of Copenhagen, Copenhagen, Denmark, 2767-Plat 2Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark. Examining Length and Charge Distribution of the Periplasmic N-tail of Nanodiscs are widely used as a model system to study membrane proteins in a Bitopic Model Proteins as Determinants of its YidC and Sec Requirement lipid environment. in E.coli The structure of the MSPDH5 nanodisc was recently described by a combina- Sri Karthika Shanmugam, Ross E. Dalbey. tion of NMR, EPR, and TEM, providing a highly-anticipated step towards a Department of Chemistry & Biochemistry, The Ohio State University, better understanding of nanodiscs. The published structure focused on the pro- Columbus, OH, USA. tein belts surrounding the lipids, and does not fully address the properties of the Membrane proteins catalyse essential functions within the cell making them lipids as well as the structural dynamics of the entire system. attractive drug target candidates. However, the general principles of membrane We combine molecular dynamics simulations with SAXS and SANS experi- protein targeting,insertion and folding are less understood. Universally conserved ments to study the structure and dynamics of MSPDH5 discs in solution. Start- membrane protein YidC is a key player in this pathway; it performs the dual func- ing from the NMR structure, we use SAXS and NMR data to refine our tion of assisting the clearance of Sec dependent proteins and inserting a subset of simulation models of the nanodiscs. In this way, we obtain an ensemble model membrane proteins independently. In this study, we tested the involvement of the that describes both the structure and dynamics of the protein and lipid mole- translocated substrate periplasmic N-tail in specifying its membrane protein cules, shows interactions between lipids and belt proteins, and reveals potential translocase requirement using a bitopic model protein Pf3-Lep which does not fluctuations in the shape of the discs. Our work demonstrates how integrating require YidC or Sec for its translocation. On systematically increasing the length NMR, SAXS, and simulations can help uncover the structural dynamics of of its N-tail with neutral spacer peptides, we found that there is a certain N-tail membrane proteins embedded within lipid nanodiscs. length requirement (35 resides) beyond which YidC translocase is required. 2765-Plat N-tails longer than 50 residues required both YidC and Sec for its translocation, Effect of the Endosomal Acidification on Small Ion Transport Through the however translocation was severely impaired beyond 60 residues N-tail length. Anthrax Toxin PA63 Channel N-tail translocation of larger periplasmic domains were studied using Maltose- Nnanya Kalu1, Antonio Alcaraz2, Goli Yamini1, Sanaz Momben Abolfath1, Binding Protein and Alkaline Phosphatase fusions to the N-tail of Pf3-Lep. Sur- Laura Lucas1, Clare Kenney1, Vicente M. Aguilella2, prisingly, these bulky N-terminal regions could be translocated by YidC-Sec even Ekaterina M. Nestorovich1. in the absence of their respective signal sequences in the N-terminal direction. 1Biology, The Catholic University of America, Washington, DC, USA, These results hint at the involvement of chaperons and other membrane- 2Laboratory of Molecular Biophysics, Department of Physics, Universitat associated components like SecA in keeping the periplasmic region in an Jaume I, Castello´n, Spain. unfolded state and facilitating the translocationof larger N-tails. Finally, we tested Tight regulation of pH is critical for the structure and function of cells and organ- the role of charges on Pf3-Lep N-tail as translocase determinants and found that elles. The pH environment changes dramatically along the endocytic pathway, crowding of positive or negative charges led to YidC-Sec dependence. This study an internalization transport process that is ‘hijacked’ by many intracellularly provides fundamental information regarding the translocation capacity of YidC active bacterial exotoxins, including the anthrax toxin. Here we investigate the and YidC-Sec mode of insertion of single-spanning membrane proteins in E.coli. role of pH ( 4 - 7.5) on single-channel properties of the anthrax toxin channel, 2768-Plat PA63. Even though the PA63 channel ability to bind and translocate the enzy- Dynamics of P-type ATPase Transport Cycle Revealed by Single-Molecule matic components of the toxin has been investigated in a number of studies, FRET pH-dependent behavior of the channel per se has so far remained largely unex- Mateusz Dyla1, Daniel S. Terry2, Magnus Kjaergaard1, plored. Using conductance and current noise analysis, blocker binding, ion selec- Thomas L-M Sørensen3, Jacob Lauwring Andersen1, Jens Peter Andersen4, tivity, and PEG partitioning measurements, we show that the channel exists in Charlotte Rohde Knudsen1, Roger B. Altman2, Poul Nissen1, two different open states (‘maximum’ and ‘main’)atpHR 5.5, while only a Scott C. Blanchard2. maximum conductance state is detected at pH < 5.5. The ‘maximum’ and 1Department of Molecular Biology and Genetics, Aarhus University, Aarhus, ‘main’ conductance states are functionally identical and do not reflect dilation Denmark, 2Department of Physiology and Biophysics, Weill Cornell of the channel at the f-clamp region. We describe two substantially distinct pat- Medicine, New York, NY, USA, 3Harwell Science and Innovation Campus, terns of PA63 conductance dependence on KCl concentration uncovered at pH Diamond Light Source, Didcot, United Kingdom, 4Department of 6.5 and 4.5. At pH 4.5, the channel conductance nearly scales with KCl solution Biomedicine, Aarhus University, Aarhus, Denmark. conductivity whereas at pH 6.5 the dependence is very weak. Lowering solution P-type ATPases are ubiquitous primary transporters that pump cations across pH does not neutralize the channel, which still remains strongly cation selective. cell membranes through the formation and breakdown of a phosphoenzyme in- We suggest that the PA63 conductance is determined by the two contributing fac- termediate. Structural investigations suggest a transport mechanism defined by tors: ionic strength-dependent electrical potential and access resistance. conformational changes in the cytoplasmic domains of the protein that are allo- 2766-Plat sterically coupled to transmembrane helices so as to expose ion binding sites to Combined High-Speed Single Particle Tracking of Membrane Proteins alternate sides of the membrane. We have employed single-molecule fluores- and Superresolution of Membrane-associated Structures cence resonance energy transfer (smFRET) to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Hanieh Mazloom-Farsibaf, Keith Lidke. 2þ 2þ Physics and Astronomy, University of New Mexico, Albuquerque, NM, USA. Ca -ATPase (LMCA1), an orthologue of eukaryotic Ca -ATPases. Using the Many experiments have shown that the diffusive motion of lipids and mem- smFRET approach, we identify key intermediates with no known crystal struc- brane proteins are slower on the cell surface than those in artificial lipid bilayers tures. Our findings further reveal reversible and irreversible steps in the transport process, wherein Ca2þ efflux by LMCA1 is rate limited by phosphoenzyme for- or blebs. One hypothesis that may partially explain this mystery is the effect of 2þ the cytoskeleton structures on the protein dynamics. A model proposed by Ku- mation and resolved by ADP and Ca release from an open E2P state. sumi [1] is the Fence-Picket Model which describes the cell membrane as a set Platform: Intrinsically Disordered Proteins (IDP) of compartment regions, each 10 to 200 nm in size, created by direct or in- direct interaction of lipids and proteins with actin filaments just below the and Aggregates II membrane. To test this hypothesis, we use a hybrid tracking and super- resolution approach on the same cell. We labeled the high-affinity FcεRI recep- 2769-Plat tor in Rat Basophilic Leukemia (RBL) cells and tracked these transmembrane The Conformation of Alpha-Synuclein on the Natural Membrane Mimics proteins at up to 1000 frames per second. The cells were fixed during tracking Inner Mitochondrial Membrane (IMM) and Neuronal Plasma Membrane and then further labeled for super-resolution imaging of actin filaments and (NPM) Revisited by Bayesian Analysis of Double Electron Electron Reso- other membrane-associated components. For best correlation of tracking and nance (DEER) Distance Distributions super-resolution, we refined a fixation protocol to prevent morphology changes Pravin Kumar1, Thomas H. Edwards2, Stefan Stoll2, Martina Huber1. during the fixation process that often go unnoticed. This sequential approach 1Huygens-Kamerlingh Onnes Lab; Department of Physics, Leiden allows use of far-red dyes for tracking and super-resolution, ameliorating chro- University, Leiden, Netherlands, 2Department of Chemistry, University of matic aberrations. Results show that protein diffusion is bounded by actin fila- Washington, Seattle, WA, USA.

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The horseshoe conformation of the membrane-bound form of alpha-synuclein cataract tissue. We also show that exposure to UV radiation, a significant risk (aS) is a still controversial. Here we reinvestigate distance distributions from factor for age-related cataract disease, produces signatures of amyloid forma- DEER of aS bound to IMM and the NPM membranes [1]. With aS spin labelled tion in healthy eye lens tissue. These results establish cataracts as an amyloid at residues 27 and 56, two-component distance distributions were found, with disease, and have implications for the recent development of small molecules distances of 4.3 nm, extended conformation of aS, and 3.6 nm. The latter dis- that appear to dissolve cataracts in animal models. tance was attributed to an expanded horseshoe conformation [1], because it was longer than the 2.6 nm distance obtained for aS27/56 in the horseshoe 2772-Plat conformation on artificial membranes [2] and micelles. The DEER distance dis- The Small Molecule anle138b Shows Interaction with a-Synuclein tributions, obtained by Tikhonov regularization, have two maxima, separated by Oligomers in Phospholipid Membranes 1 1 1 1 a trough (Figs 2C and D in [1]). Reanalysis of these data using the Bayesian Leif Antonschmidt , Riza Dervisoglu , Sergey Ryazanov , Andrei Leonov , 1 1 1 2 approach [3] shows that the distance distributions from aS on IMM and NPM Melanie Wegstroth , Karin Giller , Stefan Becker , Joon Lee , 2 3 4 5 are similar to each other, in agreement with the conclusions of [1]. However, Ratneshwar Lal , Gregor Eichele , Andre Fischer , Armin Giese , Loren Andreas1, Christian Griesinger1. the trough between the two maxima is not significant. Therefore, the distance 1 distributions should be interpreted as broad, continuous distributions, not as NMR-based Structural Biology, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany, 2Mechanical and Aerospace Engineering, two distinct distances. For aS membrane binding, this implies that rather than 3 two distinct conformations, aS has a continuum of conformations, between University of California, San Diego, San Diego, CA, USA, Genes and the extended and a more bent conformation, but does not bend as far as the pre- Behavior, Max-Planck-Institute for Biophysical Chemistry, Goettingen, Germany, 4German Center for Neurodegenerative Diseases, Goettingen, viously determined horseshoe conformation [2]. This demonstrates that analysis 5 of DEER primary data by statistical approaches contributes crucial knowledge Germany, Centre for Neuropathology und Prion Research, University of about the distance distribution and in the present case changes the interpretation. Munich, Munich, Germany. [1] Kumar et al. PLoS ONE 10(11): e0142795. Neurodegenerative diseases remain a major challenge to modern day society. A [2] Drescher et al. J. Am. Chem. Soc. 104 (2008) 7796. physiological hallmark of these diseases is the presence of deposits in the pa- [3] Edwards et al. Journal of Magnetic Resonance 270 (2016) 87. tient’s brain, which consist of various aggregates of abundant proteins such as amyloid-b (Alzheimer’s Disease) or a-synuclein (Parkinson’s Disease). 2770-Plat Growing evidence suggests, that oligomers of these proteins, formed during the Hypersensitive Termination of the Hypoxic Response by a Disordered aggregation process, constitute the major toxic species. In our and collaborating Protein Switch groups a series of 3,5-diphenyl-pyrazole derivatives have been designed and Rebecca B. Berlow, H. Jane Dyson, Peter E. Wright. synthesized to interfere with the processes associated with neurodegenerative Integrative Structural and Computational Biology, The Scripps Research diseases. Among these compounds, anle138b showed the highest efficacy in Institute, La Jolla, CA, USA. mouse models of taupathies, Parkinson’s-, Alzheimer’s- and Prion Disease. The intrinsically disordered proteins HIF-1a and CITED2 play a critical role in The success of anle138b in vivo calls for an elucidation of the underlying mech- regulating the hypoxic response. Under low tissue oxygen concentrations, the anism in vitro. By a combination of NMR-, fluorescence-, and CD-spectros- disordered C-terminal transactivation domain of HIF-1a interacts with the copy the kinetics of a-synuclein in the presence of phospholipid membranes TAZ1 domain of the general transcriptional coactivators CBP/p300 to activate were thoroughly investigated. The exact knowledge of the kinetics in return al- transcription of critical adaptive genes. One such gene is CITED2, which func- lowed the enrichment of protein oligomers in the presence of lipids. Using tions as a negative feedback regulator of HIF-1a by competing for TAZ1 binding DNP-enhanced solid-state-NMR we could find an interaction of membrane- through its own disordered C-terminal transactivation domain. This negative feed- embedded anle138b with asynuclein oligomers. Further investigation of the back loop must be tightly regulated to maintain cellular oxygen homeostasis; how- membrane-associated oligomers was done by Atomic Force Microscopy. NO- ever, little is known about the mechanism by which HIF-1a andCITED2compete ESY-spectra of the compound yielded both its location and conformation inside for their common cellular target. Structural data for the HIF-1a:TAZ1 and CIT- the lipid bilayers, which will enhance understanding of the interaction. ED2:TAZ1 complexes indicate that both HIF-1a and CITED2 bind to a partially These findings give us confidence in revealing valuable information about the overlapping region of TAZ1 through helical motifs that flank a conserved LP(Q/E) mechanism of not only the disease related proteins, but also potential therapeu- L motif which is essential for negative feedback regulation of HIF-1a by CITED2. tic targets in the aggregation process. Using a combination of NMR, fluorescence, and stopped flow kinetics, we have determined that CITED2 efficiently displaces HIF-1a from TAZ1 in an allosteric 2773-Plat process that relies on the intrinsic flexibility and binding properties of disordered Gelation and Vitrification of Tardigrade IDPs 1 1 1 proteins. CITED2 displaces HIF-1a by forming a transient ternary complex with Thomas C. Boothby , Samantha Piszkiewicz , Aakash Mehta , 2 3 3 4 TAZ1 and HIF-1a, and competes for a shared binding site via its LPEL motif. Alexandra Brozena , Hugo Tapia , Doug Koshland , Alex Holehouse , 4 5 1 CITED2 binding induces a conformational change in TAZ1 that allosterically en- Rohit Pappu , Bob Goldstein , Gary Pielak . 1Chemistry, University of North Carolina, Chapel Hill, NC, USA, 2North hances the rate of HIF-1a dissociation, allowing for rapid activation of a hypersen- 3 sitive, unidirectional molecular switch that efficiently attenuates the hypoxic Carolina State University, Raleigh, NC, USA, University of Carlifornia, Berkeley, Berkeley, CA, USA, 4Washington University in Saint Louis, Saint response. This regulatory switch highlights a novel mechanism for robust tran- 5 scriptional control that is entirely dependent on the physicochemical properties Louis, MO, USA, Biology, University of North Carolina, Chapel Hill, NC, of intrinsically disordered proteins. We anticipate that this mechanism will be USA. common amongst disordered proteins, and likely plays a key role in modulating Tardigrades (water bears) are a phylum of microscopic animals renowned for cellular responses to changes in environmental stimuli. their ability to survive a number of different environmental extremes. including desiccation, freezing, temperatures above the boiling point of water, intense ra- 2771-Plat diation, and even prolonged exposure to the vacuum of outer space. How tar- Cataracts Contain Amyloid b-Sheets: A 2D IR Study of Human Cataract digrades survive these extremes is largely unknown. To identify candidate Tissue mediators of desiccation tolerance we compared transcriptomes from hydrated Ariel M. Alperstein, Joshua S. Ostrander, Tianqi O. Zhang, Martin T. Zanni. and drying tardigrades. We found that members of a family of genes encoding Chemistry, UW-Madison, Madison, WI, USA. intrinsically disordered proteins (IDPs) are upregulated upon desiccation. Age-related cataract disease affects the sight of more than 50% of Americans These IDPs are unique to tardigrades, are required for the tardigrades to survive over the age of 80. Cataracts are caused by the aggregation of crystallin proteins drying, improve the desiccation tolerance of heterologous systems, and protect in the eye lens, leading to blurred vision, but there is not a clear understanding enzymes from desiccation in vitro. We found that these IDPs form gels, and the of the molecular structure changes that lead to the disease. In vitro, crystallin gels stabilize other proteins. Although disordered, tardigrade IDPs appear to proteins form both amorphous aggregates and amyloid fiber structures, depend- exist in a conformational ensemble resembling a dumbbell - two collapsed re- ing on the solution conditions; however, there is little evidence that the aggre- gions bridged by an extended linker. Our data indicate that this dumbbell-like gates in cataractous lens tissue contain amyloid deposits. This talk will present conformation is essential for their assembly into gels, and that assembly is two-dimensional infrared (2D IR) spectra of natural human cataract lens tissue likely mediated via interprotein beta-beta interactions between termini of collected from a deceased individual. 2D IR spectra distinguish between the different IDPs. As these gels dry, they do not form crystalline solids, but rather native b-sheet structures of crystallin lens proteins and their amyloid fiber ag- form more amorphous, vitrified (glass-like), solids. Disrupting their vitrifica- gregates through a distinct diagonal peak as well as an off-diagonal cross peak. tion correlates with a loss of protective capabilities. We propose that as tardi- Control experiments are presented on in vitro crystallin proteins, pig lens tissue, grades dry they upregulate the expression of these IDPs, resulting in increased and human non-cataract lens tissue for comparison with diagnosed cataract lens cytoplasmic concentrations of these proteins, and ultimately in the gelation of tissue spectra. The 2D IR signatures of amyloid b-sheets are clearly observed in the cytoplasm. We speculate that non-desiccation tolerant proteins are

BPJ 8705_8719 Wednesday, February 21, 2018 561a encapsulated within the pores of this gel, preventing their unfolding and aggre- described as funnel-shaped, is a requirement of evolution; sequences that do gation. Upon complete desiccation, the gel vitrifies, locking the cytoplasmic not fold on physiological timescale do not provide a fitness benefit. However, contents within a protective glass. Rehydration results in the dispersion of these this requirement for a biased folding pathway does not apply to pathological IDPs glasses, releasing protected proteins back into solution where they can protein aggregates. In fact, avoidance of the pathological state will favor the carry out their normal functions. evolution of free energy landscapes that are flat or even have a negative bias. This conclusion has emerged from multiple lines of investigation that we 2774-Plat briefly review in this presentation. First, we present a simple theoretical model Hierarchical Clustering of Markov State Models Reveals Sequence Effects showing that the concentration, temperature, and denaturant dependence of in p53-CTD Dynamic Behavior fibril elongation rates can be explained by a random search over molecular 1 2 1 Hannah K. Wayment-Steele , Carlos X. Hernandez , Brooke E. Husic , alignment states. Second, we present a novel Markov simulation algorithm to 1 Vijay S. Pande . generate ensembles of aggregation trajectories using all-atom representations. 1Department of Chemistry, Stanford University, Stanford, CA, USA, 2 This algorithm captures the non-additive effects of hydrophobic mutations on Biophysics Program, Stanford University, Stanford, CA, USA. a fragment of A-beta. Again, we find that the aggregation trajectories are P53 is a highly-researched transcription factor with many implications in cancer. consistent with a random, unbiased search over binding states. Finally, we pre- Its C-terminus domain (p53-CTD) is an intrinsically disordered protein (IDP) sent an analytic theory that shows how sequence patterns affect the free energy that binds in diverse conformations to a variety of binding partners. It is of great landscape. This model predicts that A-beta and IAPP have inverted funnel importance to better characterize the full conformational landscapes of p53 and landscapes. other IDPs to understand their diverse signaling behaviors. Previous work has demonstrated that IDP conformational ensembles are well characterized by the electrostatic distribution of the sequence. In this light, we wish to determine Platform: Membrane Physical Chemistry II what elements of the landscape of p53-CTD may be attributed to its electrostatic properties and what elements are sequence-specific. We performed simulations 2777-Plat of 2000 peptides generated from amino acid distributions of reference sequences Polymeric Effects vs. Chemical Specificity – Effects of Hyaluronic Acid on that are 80% homologous to WT p53-CTD. We use Markov State Models Lung Surfactant Monolayers (MSMs) to characterize the variants in the same conformational landscape, al- Benjamin R. Slaw, Ka Yee C. Lee. lowing for directly comparing sequences in the same conformational space. Chemistry, The University of Chicago, Chicago, IL, USA. We then use hierarchical clustering to group sequences by their corresponding Lung surfactant (LS) is a mixture of lipids and proteins secreted by type II MSM transition matrices, essentially clustering sequences by dynamical pneumocytes in the alveolus. The purpose of LS is twofold: first, through the behavior. Hierarchical clustering reveals sequence-specific trends at increasing self-assembly of a lipid monolayer at the air/fluid interface, LS reduces the degrees of resolution, and highlights conformations that are most conserved alveolar surface tension to near-zero values and therefore reduces the work across the ensemble of IDP variants. We investigate which scrambled sequences associated with normal breathing. Second, LS serves to stabilize the alveoli behave most similarly to WT p53-CTD and propose essential sequence charac- through repeated inhalation/exhalation cycles by dynamically varying the sur- teristics of p53-CTD and their implications for available conformations. face tension as a function of alveolar volume. While many studies have exam- ined the behavior of model LS monolayers, a rigorous understanding of how the 2775-Plat larger physiological environment of the alveolus affects the mechanical prop- Molecular Grammar Governing Phase Behavior of Intrinsically Disor- erties of the interfacial layer is still lacking. Of particular interest in native lung dered Proteins with Prion-like Domains physiology is the existence of low molecular weight (220 kDa) hyaluronic Jeong-Mo Choi1, Jie Wang2, Alex S. Holehouse1, Simon Alberti2, acid (HA). Though well characterized in other contexts, the behavior and Anthony A. Hyman2, Rohit V. Pappu1. importance of HA in the alveolar lining fluid remains unknown. In this study, 1Washington University in St. Louis, Saint Louis, MO, USA, 2Max Planck we examine the interaction of HA with two model LS monolayers. To deter- Institute of Molecular Biology and Genetics, Dresden, Germany. mine whether the resulting behavior of the monolayers is due to the chemical There is growing interest in uncovering the rules that govern the sequence-de- nature of HA or the polymer’s behavior in solution, we also examined the LS terminants of phase separation of intrinsically disordered proteins (IDPs). This behavior when D-glucuronic acid (GA) and N-acetyl glucosamine (N; the has direct relevance for reversible formation/dissociation of membraneless or- chemical constituents of the HA monomer) were present in the underlying sub- ganelles and other biomolecular condensates. Here, we focus on a subset of phase. Our results indicate that monolayers spread on HA-containing subphases IDPs with prion-like domains (PLDs) whose phase behavior appears to be gov- exhibit different mechanical responses to isothermal compression than those on erned by a rather simple molecular grammar. To the first order, IDPs with PLDs GA or N subphases. Additionally, HA subphases yield novel phase behavior as can be reduced to a flexible polymer chain comprising multiple interacting mo- observed via fluorescence microscopy. tifs connected by spacers. The minimal motifs are individual amino acids, spe- cifically tyrosine and arginine. We present a mean-field theory that predicts 2778-Plat Emergence of Membrane Material Parameters Revealed by Solid-State critical concentrations for phase separation as a function of Tyr/Arg motif 2 numbers. The theory, which was tested in numerical lattice simulations, pro- H NMR Spectroscopy 1 2 1 vides a first-order explanation for experimentally measured critical concentra- Jacob J. Kinnun , K.J. Mallikarjunaiah , Horia I. Petrache , Michael F. Brown3. tions of a family of IDPs with PLDs. Interestingly, substitutions of Tyr with Phe 1 or Arg with Lys residues drastically weakens the driving forces for phase sep- Department of Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN, USA, 2Department of Chemistry, University of Arizona, aration, suggesting that the grammar cannot be simply reduced to the multiva- 3 lency of cation-pi interactions. Instead, phase separation requires collective Tucson, AZ, USA, Department of Chemistry and Department of Physics, interactions amongst a multiplicity of Tyr/Arg motifs that implies involvement University of Arizona, Tucson, AZ, USA. of a hierarchy of length scales and orientational effects. Finally, spacers inter- The emergence of the material properties of lipid membranes is a topic of cur- spersed between the Tyr/Arg motifs serve as secondary modulators of the rent interest in quantitative biophysics. At the smallest length scale, lipid mem- driving forces for phase separation and as primary determinants of the material branes are collections of atoms whose dynamics can be traced by molecular properties, specifically the fluidity of droplets. Here too, the choice of optimal dynamics simulations. At cellular length scales, they are continuous hydropho- spacers is non-random, with a decided preference for Gly residues, which pro- bic barriers, which together with membrane proteins regulate trafficking and motes inter-chain overlaps that appear to promote collective interactions. The cell signaling. Somewhere in this broad range, material properties emerge from the ensemble of atoms. How can we understand this emergence behavior? combined experimental and theoretical analysis enables proteome-wide predic- 2 tions of critical concentrations of disordered regions with Tyr/Arg motifs and Here we show that solid-state H NMR spectroscopy is powerful, because as begins to open the door to designing sequences with bespoke phase behavior. opposed to typical scattering experiments it provides information over a range of length scales. Specifically, solid-state 2H NMR provides atomistically 2776-Plat resolved order parameters and dynamics of flexible phospholipids within the Folding Funnels (or Lack Thereof) in Amyloid Aggregation liquid-crystalline bilayer [1]. By measuring the membrane response to osmotic Jeremy D. Schmit. stress we can disentangle how bulk material properties emerge from atomistic- Department of Physics, Kansas State University, Manhattan, KS, USA. level interactions. Examples include structural parameters such as the area per In order to fold into a functional state, proteins must explore are large config- lipid and the volumetric thickness plus the associated moduli for elastic urational space. The size of the non-native ensemble is so vast that folding on deformation. Data for samples under osmotic stress allow an insightful physiological timescale is not possible without a biased energy landscape to analysis of intermembrane interactions by distinguishing between collective guide the folding process toward the native state. This energy bias, often undulatory and quasi-elastic contributions at large length scales versus

BPJ 8705_8719 562a Wednesday, February 21, 2018 short-range non-collective (molecular) effects. By calculating the area elastic that formation of this inverted-cone-shaped lipid, which stabilizes positively modulus over a substantial range of osmotic pressures, collective bilayer prop- curved structures, is involved in the process of vesicle formation. erties are found to emerge over very short length scales, on the order of the bilayer thickness or even less. Notably the emergence of lipid material proper- 2781-Plat ties on this length scale determines what model is most appropriate to study Formation of Membrane Tubular Protrusions upon Localized Application lipid-protein interactions [2]. A continuum flexible surface model (FSM) for of Calcium Ions to the Surface of Giant Lipid Vesicles 1 1 2 lipid-protein interactions on the mesoscopic length scale can optimally capture Tatsiana Lobovkina , Baharan Ali Doosti , Weria Pezeshkian , Dennis S. Bruhn2, John H. Ipsen2, Himanshu Khandeli2, Gavin D.M. Jeffries1. the essential material features that govern biomembrane functions. [1] J.Kinnun 1 2 et al. (2015) Biochim.Biophys.Acta 1848, 256. [2] M.F.Brown (2017) Annu. Chalmers University of Technology, Gothenburg, Sweden, University of Rev.Biophys. 46, 379. Southern Denmark, Odense M, Denmark. We present a study on the formation of lipid nanotubular protrusions as a result 2779-Plat of localized application of calcium ions to the membrane of giant lipid vesicles. Determining the Bending Moduli of Asymmetric Bilayers by Simulation Our findings demonstrate that a point source of calcium ions acting upon a Sophia Wheeler, Marley Samways, Jonathan Essex. negatively charged membrane generates spontaneous curvature sufficient to Department of Chemistry, University of Southampton, Southampton, United induce membrane bending and the formation of nanotubular protrusions. The Kingdom. observed membrane behaviour can be explained as the response to the trans- Biological membranes are almost always asymmetric in terms of lipid compo- bilayer mismatch of surface charge density upon calcium ion binding from sition. Such asymmetry is thought to play an important part in biological func- the exposed side of the bilayer. By using molecular dynamics simulations, tions via alteration of the membrane’s elastic properties, including its bending we validate this experimental observation, finding that calcium ion binding modulus. Recently experimental techniques have been developed which enable to a lipid membrane is sufficient to generate spontaneous curvature, bending the preparation of asymmetric GUVs and therefore allow the measurement and the membrane away from the ion source. The observed calcium-ion guided tu- direct comparison of bending moduli for pure bilayers, mixed symmetric bila- bulation in lipid membranes can be a significant step towards gaining insights yers and asymmetric bilayers composed of similar lipid species. These exper- into cell membrane re-shaping in response to chemical stimulation, and can iments show that asymmetry does indeed have a marked effect on bilayer further our understanding of the fundamental mechanisms by which lipids stiffness. In order to reproduce these data and develop a molecular level under- redistribute within cell membranes. standing of them, we have taken a computational approach using both atomistic 2782-Plat and coarse grained forcefields for a variety of system sizes and lipid species. Punching Membranes: How Lipid Bilayers Withstand and Propagate We have performed analysis of our trajectories using a range of theoretical Mechanical Load models based on a variety of different analysis techniques including those Florian Franz1, Camilo Aponte-Santamarı´a2, Sergi Garcia-Manyes3, based directly on membrane area compressibility; those based on spectral anal- Frauke Gr€ater1,4. ysis involving 2D discrete Fourier transforms; and those based on the distribu- 1IWR, Heidelberg University, Heidelberg, Germany, 2Max Planck Tandem tion of angles of splay. Our results show that the different models employed Group on Computational Biophysics, Universidad de los Andes, Bogota´, provide consistent results across a range of system sizes both with each other Colombia, 3Department of Physics, Kings College London, London, United and the experimental data, as well as suggesting promising new directions Kingdom, 4Molecular Biomechanics, Heidelberg Institute for Theoretical for experimental investigation. Studies, Heidelberg, Germany. Mechanical perturbations are ubiquitous in living cells and many biological 2780-Plat functions are strongly dependent on the mechanical response of lipid mem- The Soft Side of Extracellular Vesicles branes. We asked how lipid bilayers withstand and also propagate high me- 1 2 1 1 Raya Sorkin , Rick Huisjes , Filip Boskovic , Daan Vorselen , chanical load using large-scale atomistic and coarse-grained Molecular 2 3 4 € 5 Silvia Pignatelli , Yifat Ofir-Birin , Joames K.F. Leal ,Jurgen Schiller , Dynamics simulations. 6 4 3 Wouter H. Roos , Giel Bosman , Neta Regev-Rudzki , We perforated stacked lipid bilayers using a spherical indenter, closely 2 1 Raymond M. Schiffelers , Gijs J.L. Wuite . mimicking novel force spectroscopy techniques that capture the fracture of in- 1Department of Physics and Astronomy and LaserLab, Vrije Universiteit 2 dividual membranes, while avoiding substrate effects by probing stacks of Amsterdam, Amsterdam, Netherlands, Department of Clinical Chemistry model bilayers. We observe a step-wise fracture of individual bilayers in close and Haematology, University Medical Center Utrecht, Utrecht, Netherlands, agreement with experiments. Dwell times of the physiologically more relevant 3Department of Biomolecular Sciences, Weizmann Institute of science, 4 free standing bilayers are lower than those of stacked bilayers. Interestingly, the Rehovot, Israel, Department of Biochemistry, Radboud University Medical rupture mechanism as well as the dwell time distribution depend on the nature Centre, Radboud Institute for Molecular Life Sciences, Nijmegen, of the indenter: Only a hydrophilic intender yields the expected log-normal dis- Netherlands, 5Institute of Medical Physics and Biophysics, Medical Faculty, 6 tribution by inducing a pore-growth mechanism. University of Leipzig, Leipzig, Germany, Departement of Molecular Upon subjecting the bilayer to a longitudinal rather than a perpendicular pertur- Biophysics, Zernike Instituut, Rijksuniversiteit Groningen, Groningen, bation, we observe the propagation of a mechanical pulse through the lipid Netherlands. bilayer with the experimentally known speed of sound of 1 km/s, again in Extracellular vesicles (EVs) are emerging as important mediators of cell-cell both atomistic and coarse-grained systems. The computed dispersion follows communication, as well as potential disease biomarkers and drug delivery ve- proposed continuum visco-elastic models and is remarkably low, allowing hicles. However, the mechanical properties of these vesicles and their role in stress propagation over tens of nanometers before damping Our results provide vesicle function are largely unknown. We present a detailed AFM force- a better understanding of membrane nanomechanics and indicate that mem- spectroscopy study of mechanical properties of natural extra-cellular vesicles, branes play a role as efficient wave guides for signalling through the cell. with several key findings. First, we demonstrate that vesicles originating from very different cells, such as red blood cells (RBCs) from healthy donors, Plas- 2783-Plat modium falciparum(Pf)-infected RBCs and a fibrosarcoma-derived cell line, Regulation of Lipid Droplet Formation by Membrane Tension cultured for 20 hr at 37C, are surprisingly similar in their mechanical proper- Abdou Rachid Thiam. ties. However, the vesicles produced by RBCs differ with incubation time, with Ecole Normale Superieure de Paris, Laboratoire de Physique Statistique, vesicles isolated after 2 hrs incubation being significantly softer than after 20 Paris, France. hrs of incubation. Furthermore, the mechanical properties vary with the incuba- Cells store excess energy in the form of neutral lipids that are synthesized tion temperature: at 4C, very soft vesicles are secreted, with a bending within the intermonolayer space of the endoplasmic reticulum bilayer. At a modulus of 550.8 kBT, whereas the highest bending modulus value of certain concentration, the lipids demix from the bilayer to form droplets, which 1351kBT is obtained at 22 C and a value of 6.851kBT is obtained after in- surprisingly mainly bud off toward the cytosol. These droplets are called lipid cubation at 37C. Detailed lipid and protein analysis show a large variation in droplets (LDs) and represent dynamic organelles that play key roles in cellular the lipid and protein content between different incubation temperatures, but energy metabolism and homeostasis. How exactly LDs form or bud off from also per temperature from donor to donor. In contrast, the mechanical proper- the bilayer is still a not well-understood question for which membrane ties are very robust at given conditions. We therefore propose that changes in biophysics can bring good insights. We reconstituted LD formation topology mechanics of natural vesicles can’t be explained by a change in specific lipids by using two complementary methods: a droplet interface bilayer method, or proteins, but instead the ratio of total amount of proteins to the total amount which allows to easily prospect the role of membrane physical chemistry, of lipids might determine the mechanical behavior. Moreover, we find lyso- and giant vesicles embedded with artificial LDs, which enable to prospects phosphatidylcholine (LPC) in vesicles (but not in RBC ghosts), suggesting the effect of membrane physical properties. These approaches enabled us to

BPJ 8705_8719 Wednesday, February 21, 2018 563a decipher that LD budding is controlled by membrane physical chemistry with in epigenetic regulation. This model addresses the physics of chromatin respect to the physics of three-phase wetting systems, i.e. dictated by the equi- compaction and aids in the interpreting experimental observed structure and librium of surface tensions. Indeed, the equilibrium between the tension of the epigenetic sequence. In building our physical model, we capture a range of bilayer membrane and the tension of the two monolayers enclosing a forming biological contributions using a small number of model specific parameters, LD determine the budding shape of the latter. Based on the physics of wetting, each of which has a physical explanation. We focus on the compaction of we identified that an asymmetry in monolayer tensions is an important param- chromatin caused by methylation of the ninth lysine of histone protein 3 eter that controls LD budding side. Our results thus offer new insights on the (H3K9), which leads to preferential binding of heterochromatin protein 1 how proteins might regulate LD formation by dynamically controlling surface (HP1), which can oligomerize to bind genomically distant locations. We tensions. explore the thermodynamics and configuration statistics of this system using a polymer based Monte Carlo simulation that leverages a course graining pro- 2784-Plat cedure that allows us to simulate an entire human chromosome. For a given Nucleation and Dynamics of Rupture and Chromatin Herniation in HP1 concentration and H3K9 methylation profile - which can be measured Deformed Nuclei by chip-seq - the model predicts which genomic regions compact to form het- Dan Deviri. erochromatin. We show that the combined effect of many H3K9 methylations Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, to either side in the epigenetic sequence determines the condensation state at Israel. any particular location in the chromatin. The basic observations from our During migration of cells in vivo, in both pathological processes such as can- model provide a novel approach to interpreting chip-seq data that provides cer metastasis or physiological events such as immune cell migration through insight into the functional relationship between the epigenetic code and the tissue or embryogenesis, the cells must move through narrow interstitial resulting chromatin state. spaces which can be smaller than the nucleus. This can induce deformation of the nucleus which, according to recent experiments, may results in bleb for- 2787-Plat mation and rupture of the nuclear envelope that can lead to chromatin herni- Probing Chromatin Organization by Sorting of Short Sequence Fluores- ation and damage and to cell death, if not prevented or healed within an cence Correlation Spectroscopy appropriate time. The nuclear envelope, which can be modeled as a double Melody Di Bona1,2, Simone Pelicci1,2, Giuseppe Vicidomini3, lipid bilayer attached to a viscoelastic gel (lamina) whose elasticity and vis- Eugenia Cammarota4,5, Davide Mazza4,5, Alberto Diaspro1,2, Luca Lanzano`1. cosity primarily depend on the lamin composition, may utilize mechanically 1Nanoscopy, Italian Institute of Technology, Genova, Italy, 2Physics, induced, self-healing mechanisms that allow the hole to be closed after the University of Genoa, Genova, Italy, 3Molecular Microscopy and deformation-induced strains are reduced by leakage of the internal fluid. Spectroscopy, Italian Institute of Technology, Genova, Italy, 4Experimental Here, we present a unified model of the nucleation and evolution of holes Imaging Center, Ospedale San Raffaele, Milano, Italy, 5Fondazione CEN, in the nucleus and blebs by deformations of the nucleus and estimate the her- European Center for Nanomedicine, Milano, Italy. niation of chromatin through the hole and its relation to the lamin expression The nucleus is the cellular organelle that harbors the genetic information into levels in the nuclear envelope. DNA, which forms with histone proteins the fundamental unit of chromatin. Chromatin is organized by physical and molecular forces, like phase separation Platform: Chromatin and the Nucleoid and molecular crowding, into functional domains whose formation and mobility correlate to chromatin compaction influencing the genome outcome 2785-Plat through gene expression regulation [1]. For these reasons, it is important to Architecture of the Heterochromatin Unit Revealed by Cryo-EM study chromatin compactness from both a spatial and a dynamic point of Yoshimasa Takizawa1, Shinichi Machida2, Masakazu Ishimaru2, view. Fluorescence Correlation Spectroscopy (FCS) can probe chromatin Satoshi Sekine2, Yukihiko Sugita1, Jun-ichi Nakayama3, accessibility and molecular crowding in live cells by measuring fast diffusion Hitoshi Kurumizaka2, Matthias Wolf1. of molecules in the range between microseconds and milliseconds [2]. Single 1Okinawa Institute of Science and Technology, Okinawa, Japan, 2Waseda point FCS (spFCS) has a high temporal resolution but lacks spatial information. University, Tokyo, Japan, 3National Institute for Basic Biology, Aichi, Japan. Conversely, spatially-resolved methods, like scanning FCS, have limited tem- Chromatin formation is essential for the organization of genomic DNA in eu- poral resolution. Here we introduce an innovative method that samples several karyotes. A basic unit of chromatin is the nucleosome, in which 147 base- positions by scanning the beams in a slow orbit: analyzing the data in short time pairs of DNA are wrapped around the histone octamer composed of two copies sequences, we preserve the spFCS temporal resolution while probing different each of histones H2A, H2B, H3 and H4. The formation of higher order structure nuclear compartments. Using two excitation lines, for the probe and for a of chromatin is regulated by histone modifications and histone variants, and is DNA marker, we use the intensity trace to sort the short sequences into average involved in transcription, replication, recombination, and repair. Heterochro- ACFs associated to different compartments. We show GFP measurements in matin, a condensed higher order form of chromatin, modulates suppression cells subjected to ATP-depletion or osmotic treatment, and discuss the implica- of genomic DNA activities. To organize the heterochromatin, binding of het- tion of molecular crowding on diffusion slowdown. In addition, we study erochromatin protein 1 (HP1) to a trimethylated lysine 9 of histone H3 the mobility of p53, a transcription factor related to cancer, in different chro- (H3K9me3), which is the epigenetic maker of heterochromatin, is crucial fac- matin compartments: in perinuclear heterochromatin, containing p53 binding tor. However, the formation of heterochromatin by HP1 has not been fully un- sites, it is reduced. Finally, we discuss coupling this method with STimulated derstood. Here we present the 3D structure of the H3K9me3-containing Emission Depletion (STED)-FCS, for performing FCS at sub-diffraction spatial dinucleosome complex with human HP1 determined by cryo-electron micro- scales [3]. scopy (cryo-EM) using a volta phase plate. Our cryo-EM structure of the [1] Strom et al, Nature, 2017; Nozaki et al, Mol Cell 2017. HP1-dinucleosome complex shows that HP1 bridges two H3K9me3 nucleo- [2] Bancaud et al, Embo J 2009. somes, and that the linker DNA connecting two nucleosomes does not directly [3] Lanzano` et al, Nat Commun 2017. associate with HP1. This work allowed us to propose novel features of the het- erochromatin unit and to discuss the mechanism of heterochromatin formation 2788-Plat in eukaryotes. Interphase Chromatin Dynamics in Response to Double Stranded DNA Breaks 2786-Plat Jonah Eaton, Alexandra Zidovska. A Polymer Physics Model for Epigenetic Control of Chromatin Physics, New York University, New York, NY, USA. Compaction The dynamic organization of chromatin inside the eukaryotic cell nucleus is Quinn MacPherson, Sarah Sandholtz, Andrew Spakowitz. essential to the regulation of the genome such as maintaining its integrity, Stanford, Stanford, CA, USA. gene expression and replication. However, the physical picture of chromatin The epigenetic code regulates gene expression by modulating the spatial structure and dynamics remains unclear and requires identifying the dynamical configuration of chromatin. Therefore, understanding how epigenetic modifi- and structural signatures of specific biological processes. In this work, we study cations determine chromosomal organization is critical to interpreting both the the chromatin dynamics in interphase, time between two cell divisions, in epigenetic and genetic codes. During interphase, regions of DNA segregate response to DNA damage. As shown previously, chromatin dynamics across into densely packed heterochromatin and more loosely packed and, hence, the whole nucleus change dramatically after DNA damage; chromatin’s transcriptionally available euchromatin. Our goal is to build a physical model coherent motion is eliminated, while its local mobility increases and its for chromosomal DNA that combines the fundamental polymer physics of compaction decreases [1]. Here, we explore the local chromatin dynamics DNA with the experimentally determined behavior of the proteins involved and compaction in response to local DNA damage in human cells. To do so,

BPJ 8705_8719 564a Wednesday, February 21, 2018 we induce double stranded DNA breaks (DSBs), which we visualize using polyA tracts and the effect becme more pronounced for longer tracts. This is 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics consistent with the fact that polyA tracks disfavor nucleosome formation in using single particle tracking. Simultaneously, we use histone H2B-GFP as a vivo, and that nucleosome free regions near transcription start site often contain reporter on the chromatin compaction, to compare DSB dynamics inside the polyA tracks. Because the polyA-dependent directional bias was seen only after heterochromatin and euchromatin. To elucidate the dynamic signature of the remodeling, sequence-dependent remodeling activities may indeed be impor- local DNA damage response, we vary the number of DSBs as well as explore tant for proper nucleosome positioning in vivo. In order to gain additional the ATP-dependence of these processes. Our preliminary results show that with insight about sequence-dependence, we inserted a semi-randomized sequence an increasing DSB count the local subdiffusive dynamics of the DSBs remains and performed Slide-seq. We found that DNA shape can have a large effect unchanged. We also explore the relationship between the protein aggregation, on sliding direction and amplitude. diffusion coefficient and chromatin compaction at a DSB. [1] Zidovska A, Weitz DA, Mitchison TJ, Proc. Natl. Acad. Sci. USA, 110(39), 2791-Plat 1555560, 2013. Local DNA Sequence Controls the Asymmetry of DNA Unwrapping from Nucleosome Core Particles 2789-Plat Alexander Mauney, Lois Pollack. High-Speed Atomic Force Microscopy of SMC Proteins Cornell University, Ithaca, NY, USA. Je-Kyung Ryu1,2, Allard Katan1,2, Masashi Minamino3, Celine Bouchoux3, The interactions between DNA and proteins within nucleosome core particles Shveta Bisht4, Jorine Eeftens1,2, Christian Hearing4, Frank Uhlmann3, are major factors in the dynamics of DNA availability in vivo. Since enzyme Cees Dekker1,2. access to DNA is a major component of transcriptional regulation, the effects 1Bionanoscience, TU Delft, Delft, Netherlands, 2Kavli Institute, Delft, of the DNA sequence on wrapping and unwinding is critical to gaining an Netherlands, 3The Francis Crick Institute, London, United Kingdom, overall understanding of transcription. We report on the use of small angle 4EMBL, Heidelberg, Germany. X-ray scattering with contrast variation (CV-SAXS) to analyze the unwrap- Condensin and cohesin belong to Structural Maintenance Complex (SMC) pro- ping of DNA from nucleosome core particles, when different DNA sequences tein family that are involved in chromosome organization and chromosome are used. Increasing concentrations of salt destabilize the electrostatic interac- segregation. The key feature of these SMC protein complexes is the ring- tions between the histone proteins and the nucleic acid. As these interactions shaped structure consisting of 3 major components - two structural maintenance are destabilized many unwrapped states are accessible and the DNA explores complex (SMC) subunits and one kleisin that together form a ring, as well as 1 many possible unwrapping pathways. The CV-SAXS data report on the en- or 2 vital co-factors. Through a mechanism where the SMC protein embraces sembles of structures present at any condition. They are fit using realistic the DNA, cohesin holds sister chromatids together after DNA replication while sequence-dependent unwrapped structures with appropriate thermal varia- condensin condenses DNA using ATP as an energy source, possibly related to tions, selected by an ensemble optimization method1. The unwrapping paths the linear motor function that was recently discovered [1]. Resolving dynamic and equilibrium constants reveal interesting difference between DNA se- conformational changes of the SMC complexes is key to resolve the mecha- quences. These differences are correlated with features in the nucleic acid nism underlying these important functionalities [2]. High-speed atomic force sequence. microscopy (HS-AFM) is uniquely fit to address this question since it allows 1. Mauney A., Tokuda J.M., Gloss L.M., Gonzalez O., Pollack L. Local DNA video-imaging of entire protein structure with high spatial resolution (1 sequence controls the cooperativity and asymmetry of DNA unwrapping from nm) as well as high temporal resolution (50 ms). Here, we will show HS- nucleosome core particles. Manuscript in Preparation. AFM video imaging of condensin [3] and cohesin proteins in physiological buffers. Conformational changes of SMC dimers of condensin and cohesin 2792-Plat are directly imaged for various conditions (þ/- ATP, þ/- DNA). The real- Precise Genome-Wide Mapping of Single Nucleosomes and Linkers time imaging reveals how structures of these protein complexes evolve over In Vivo time, from which the structure-function relationship of condensin and cohesin Razvan V. Chereji1, Srinivas Ramachandran2, Terri D. Bryson2, can be obtained. These results provide insights into the molecular mechanism Steven Henikoff2. of chromosome organization and segregation. 1NICHD, National Institutes of Health, Bethesda, MD, USA, 2Fred References [1] T. Terakawa et al. (2017), ‘‘The condensin complex is a mech- Hutchinson Cancer Research Center, Seattle, WA, USA. anochemical motor that translocates along DNA’’ Science. [2] J. Eeftens, We have developed a chemical cleavage method that allows precise mapping of (2017) ‘‘Real-time detection of condensin-driven DNA compaction reveals a both single nucleosomes and linkers genome-wide in budding yeast. We multistep binding mechanism’’ BioRxiv, https://doi.org/10.1101/149138s confirm that S. cerevisiae promoters are sites of strong nucleosome depletion, [3] J. Eeftens, (2016), ‘‘Condensin Smc2-Smc4 dimers are flexible and dy- but attribute the putative nucleosome depletion seen at termination sites to namic’’ Cell Rep. MNase bias. Our nucleosome mapping data has the highest resolution among the currently available techniques, and this accuracy allows us to distinguish 2790-Plat alternative rotational positions that nucleosomes occupy in different cells. Slide-seq: Probing Sequence-Dependence of Chromatin Remodeling Furthermore, we show that linker DNA has quantized lengths for individual Activities in High Throughput genes. By comparing our nucleosome dyad positioning maps to existing Sangwoo Park, Jessica Winger, Gregory Bowman, Taekjip Ha. genomic and transcriptomic data, we evaluate the contributions of sequence, Johns Hopkins Univ, Baltimore, MD, USA. transcription, histone H1 and the H2A.Z variant in defining the chromatin land- Chromatin remodeling enzymes utilize ATP hydrolysis to remodel the chro- scape. We show that DNA sequence has a very limited effect on establishing matin by ejecting, exchanging or sliding histones and nucleosomes and their the nucleosome organization as observed in vivo. Moreover, we find that the activities are essential for for chromatin assembly, organization, and transcrip- degree of gene compaction, measured by the spacing between neighboring nu- tion regulation. Previous studies showed that yeast remodeler Chd1 mobilizes a cleosomes, correlates with the transcription level, amount of histone H1 bound nucleosome formed on the widely used 601 sequence with a directional bias to the gene, and the amount of H2A.Z variant that is incorporated in the nucle- and the bias can be reversed by inserting a stretch of polyA. Because osomes closest to the gene promoter (‘‘þ1 nucleosomes’’). Furthermore, we sequence-dependent sliding activities may play a role in positioning nucleo- present a biophysical model based on simple physical principles, which shows somes in precise locations in vivo, we developed ‘‘Slide-seq’’, a high- that steric exclusion between neighboring nucleosomes suffices to explain the throughput in vitro nucleosome sliding and mapping assay through combining salient features of nucleosome positioning and the complex nucleosome histone contact mapping with single base resolution and next generation phasing pattern that is observed genome-wide. sequencing. We created a synthetic DNA library by inserting polyA tracks of Relevant References: 3 to 25 base pairs and scanning them across the 601 sequence. Before sliding, Chereji RV et al. - Phys. Rev. E 83, 050903 (2011). nucleosomes assembled on the canonical position except for those with a long Chereji RV, Morozov AV - J. Stat. Phys. 144, 379 (2011). polyA track at the dyad. After sliding by Chd1, we observed a small set of Chereji RV, Morozov AV - PNAS 111, 5236 (2014). dominant positions in both sides of the dyad, suggesting that sliding can occur Chereji RV, Morozov AV - Brief. Funct. Genomics 14, 50 (2015). in both directions. The sliding amplitude and directional bias was dependent on Chereji RV et al. - Nuc. Acids Res 44, 1036-1051 (2016). polyA length and location. Chd1 generally slides away the nucleosome from Chereji RV et al. - Mol. Cell 65, 565-577 (2017).

BPJ 8705_8719 Wednesday, February 21, 2018 565a

Posters vitreously frozen specimens. Using the Aquilos DualBeamÔ microscope, re- gions of interest are thinned down to the appropriate thickness of 200-300 nm while maintaining cryogenic conditions. The thinned cellular sample is then Posters: Protein Structure and Conformation: transferred to an automated, high-throughput cryo-TEM for high-resolution imaging by electron cryo-tomography opening windows into cells at molec- Experimental Methods ular resolution. 2793-Pos Board B1 Local Conformational Dynamics of Bacteriorhodopsin as Revealed by 2796-Pos Board B4 In-Situ Isotopic Labeled Ultrafast Two-Dimensional Infrared Spectros- Quantifying Binding-Induced Conformational Changes of Proteins using copy Hydrodynamic Protein Size Measurements Jianping Wang. Joanna Deek, Friederike Moeller, Thomas Weber, Daisylea de, Souza Paiva, Institute of Chemistry, Chinese Academy of Sciences, Beijing, China. Ulrich Rant, Wolfgang Kaiser. Ultrafast two-dimensional infrared (2D IR) spectroscopy has been greatly Dynamic Biosensors, GmbH, Martinsried/Planegg, Germany. advanced in recent years in a variety of aspects and is becoming an exciting We present a novel method for the measurement of the Stokes radii of pro- vibrational tool for understanding molecular structures and dynamics in teins, and demonstrate how this approach can be used to detect analyte condensed phases. Local conformational dynamics of bacteriorhodopsin (bR) binding-induced conformational changes in proteins. The proteins of interest has been monitored in the electronic ground state using time-resolved 2D IR are immobilized onto DNA-functionalized gold microelectrodes on the spectroscopy. 13C=15N isotopic labeling on the protein amide unit at a series biosensor surface (using generic conjugation methods). By applying alter- of joint residues has been introduced as vibrational probes through the expres- nating electric fields to the microelectrodes, the fluorescently labeled and sion of bR in a synthetic medium with selectively 13C and 15N labeled residues electro-switchable DNA nanolevers are actuated through the solution, which in Halobacterium salinarum R1M1 without any structural perturbations. Site- effectively oscillates the target protein at high frequencies close to the specific red shift in the amide-I mode vibrational frequency region of the linear biosensor surface. The speed of this oscillatory movement is observed in IR spectroscopy of bR has been observed. This allows one to follow the confor- real-time by fluorescence energy transfer. The hydrodynamic drag incurred mational dynamics of bR with femtosecond time resolution and chemical-bond upon conjugation of the protein to the nanolever determines the speed of structural resolution. Vibrational lifetime measurement of the amide-I excited oscillation, and correlates to the target protein’s Stokes diameter. The method state and computational analysis on local structures have also been carried out. is particularly sensitive for sizes in the range of 1 - 20 nm, encompassing Local structural fluctuations and hydration dynamics at the isotopically labeled peptide and protein targets in the kDa to MDa molecular weight range. sites has been obtained and characterized from the diagonal- and off-diagonal Thus, this fluorescence-based detection method of protein target oscillation 2D IR signals. speed provides a quantitative and easy-to-interpret means for the analysis of protein size and shape. Changes in the Stokes radius can be detected with an accuracy of 0.1 nm. As the signal due to the target protein’s confor- 2794-Pos Board B2 mational change can be distinguished from a pure analyte binding event, an- Transient Interactions in Multidomain Proteins Identified by FRET alyte binding and target structural changes can be screened for 1 2 1 3 Inna S. Yanez Orozco , Junyan Ma , Feng Ding , Mark E. Bowen , simultaneously. Therefore, in addition to absolute Stokes radii, titration 1 Hugo Sanabria . and flow experiments can yield K values and kinetics (on- and off-rates) pa- 1Physics and Astronomy, Clemson University, Clemson, SC, USA, D 2 3 rameters for analyte to target binding. Here, we demonstrate the range of Chemistry, Clemson University, Clemson, SC, USA, Physiology and applicability of this technique by presenting examples for the analysis of Biophysics, Stony Brook University, Stony Brook, NY, USA. conformational changes and allosteric regulation in small proteins - such Biomolecular dynamics cover a wide range of timescales from local motions as kinases and STING - as well as for large proteins, including transglutami- occurring on the femtosecond to nanosecond time scales, to long range mo- nase and the insulin receptor protein. tions resulting from changes in macromolecular conformation, which occur at much slower timescales (sub-milliseconds to seconds). In multidomain proteins, the same forces that govern the folding of individual domains 2797-Pos Board B5 also regulate interdomain configurations. However, the surface of folded do- Conversion of a Peptide TAG to Sub-nanomolar Affinity for Single- mains is generally polar so the affinity of intramolecular interactions can be Molecule Analysis of Protein Machinery much lower; thus, capable of sampling various spatial arrangements with Wei-hau Chang. similar free energy. Here, we use FRET to characterize the structural dy- Chemistry, Academia Sinica, Taipei, Taiwan. namics of the N-terminal PDZ1-PDZ2 tandem of PSD-95. PSD-95 is a ca- Analyzing proteins as individual molecules by microscopy-based imaging nonical multidomain protein containing five protein-interacting domains. techniques depends on labeling methods. Here, we modified a peptide tag Previous studies of the PDZ tandem with NMR, crystallography and TIRFM to increase its affinity to sub-nanomolar regime in the context of protein com- based FRET produced divergent structural models. By integrating FRET plex and introduced it to RNA polymerase II (pol II) for single-molecule experiments, with ps resolution, and multiscale molecular dynamics simula- FRET measurement of the proximity between a pol II subunit and TFIIF tions, we identify at least two low-energy conformations: a closed conforma- in the pol II-TFIIF complex. Our localization of a domain in TFIIF through tion stabilized by surface-exposed hydrophobic residues and an open nano-positioning agrees with that based on recent cryo-EM structure by an ˚ conformation stabilized by internal salt bridges. Further, we discuss how error less than 10A. Our work has converted the peptide tag into a versatile the low energy barrier between conformations and the crystal contacts could tool for extending single-molecule method to protein complexes and illus- be the source of contentious results between NMR, X-ray crystallography and trates a general approach of designing, incorporating and characterizing a FRET studies. bio-orthogonal moiety in a protein complex.

2795-Pos Board B3 2798-Pos Board B6 Taking the Next Step in Structural Biology: Enabling Cellular Structural Structure and Orientation of a Small Protein on a Gold Nanoparticle Biology in situ with Cryo-FIB Sample Preparation Surface Gregor Heiss1, Alex Rigort2. Nicholas C. Fitzkee, Y. Randika Perera. 1Thermo Fisher Scientific, Vienna, Austria, 2Thermo Fisher Scientific, Department of Chemistry, Mississippi State University, Mississippi State, Germany, Germany. MS, USA. Thorough understanding of biological specimen requires the analysis of their Protein-nanoparticle interactions are an important consideration in the design 3D ultrastructure. In past years Cryo Tomography based techniques have of functionalized nanoparticles. Proteins will spontaneously adsorb to sur- evolved as particularly suitable approaches to obtain 3D Structural informa- faces, disrupting the activity of molecules already attached to the nanoparticle, tion in situ. This technique enable studying subcellular architecture as well as and potentially introducing new complications to the desired surface chemis- cell-to-cell interactions on a molecular scale. Here, we will present the latest try. Such interactions are particularly important in nanoparticle-based thera- developments for Cryo Tomography providing the highest resolution base on peutics, where protein misfolding on the nanoparticle surface could the FEI Aquilos cryo focused-ion-beam milling (cryo-FIB) platform. Cryo potentially initiate an unwanted immune response. While understanding Tomography allows for structural resolution imaging of cellular structures surface-bound protein structure is highly desirable, traditional approaches close to native state through staining free vitrification. However, as many for protein structure determination fail for nanoparticle bound proteins: Nano- cells are too thick to study intact with cryo-TEM, cryo-FIB is used to thin particles are too large for traditional solution NMR approaches, and

BPJ 8720_8723 566a Wednesday, February 21, 2018 nanoparticle-protein conjugates are not easily crystallized. Previous work single-molecule multi-parameter fluorescence detection (MFD) is a useful from our group suggests that the GB3 protein remains globular when adsorbed tool for sp-FRET, and when combined with nanosecond alternating donor/ to gold nanoparticles (AuNPs), but it is unclear whether the tertiary structure acceptor excitation, it provides a useful and robust readout of both donor and is retained. Here, we apply several novel NMR-based approaches to probe the acceptor dye performance. Using FRET restrained structural modeling (FPS), structure and orientation of GB3 bound to AuNPs. We have developed a which performs accessible volume calculations, we selected suitable residues method for monitoring hydrogen-deuterium exchange (HDX) on the AuNP for the used FRET pair (Atto488/Alexa647) on SecA. The different positions surface, and we find that HDX rates of surface-bound GB3 are highly corre- were mutated to cysteines for the creation of at least 30 different FRET- lated with GB3 in solution. Overall, rates are approximately 20 times slower pairs, and optimal conditions for labeling with maleimide dyes were deter- for the adsorbed protein, suggesting that GB3 is stabilized and largely retains mined. Until now 7 FRET-pairs, which includes all the different domains, its native structure on the surface. Methyl labeling of lysine residues suggests were selected for creating a dynamic 3D model of SecA. At the moment, we that the orientation of GB3 is fixed on the AuNP, with the helical face are investigating the effect of binding partners (e.g. pre-proteins such as exposed to solution. Using differential isotopic labeling, we have determined PhoA) and co-factors (e.g. nucleotides, ADP and ATP) which will lead to that adsorbed GB3 molecules do not readily exchange with GB3 in solution, very detailed SecA dynamics. We already showed that the addition of ADP re- and any exchange that happens occurs on a timescale much longer than 18 hr. sults in a more stable SecA conformations. Later on we will me move to an These experiments provide strong structural evidence that GB3 adopts a sta- in vitro translocation assay to study the mechanism of substrate docking and ble, native-like fold and orientation on the AuNP surface, and they open the –translocation on SecYEG, based on functional existing assays which are door for future investigations of protein structure on surfaces. compatible with single-molecule experiments. 2799-Pos Board B7 2802-Pos Board B10 Investigation of Optimal Cooling Methods in Macromolecular Cryocrys- Probing the Conformational Changes of a Model Protein by In-Cell Foot- tallography printing Coupled with Mass Spectrometry Kaitlin Harrison, Brian Wu, Douglas H. Juers. Lisa M. Jones, Dante Johnson. Physics, Whitman College, Walla Walla, WA, USA. Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Macromolecular structure determination via diffraction is commonly carried out MD, USA. at cryogenic temperature. Crystals are typically cooled via plunging into liquid In recent years, protein footprinting coupled with mass spectrometry has been cryogens or placing directly into a cold gas stream. Here we compare these two used to identify protein-protein interaction sites and regions of conformational approaches using two different crystal forms of thermolysin. We find that fast change through modification of solvent accessible sites in proteins. Hydroxyl plunge cooling of 300 mm crystals into liquid nitrogen yields higher mosaic- radical-based footprinting (HRBF) approaches utilize hydroxyl radicals to ities than gas stream cooling using the vial mounting approach (Farley et al., oxidatively modify the side chains of solvent accessible amino acids. One 2014). In some cases low mosaicities can also be achieved by plunging slowly HRBF method, fast photochemical oxidation of proteins (FPOP), utilizes an ex- through the cold gas layer above the liquid nitrogen. The observed effects are cimer laser for photolysis of hydrogen peroxide to generate hydroxyl radicals. more pronounced for a cryosolution of DMF (10% contraction) than for a cryo- To date, HRBF methods have been used in vitro on relatively pure protein solution of D-xylose (3% contraction) . The results are consistent with a model in systems. We have further extended the FPOP method for in cell analysis of pro- which non-homogeneous cooling-induced strain is amplified by faster cooling of teins. This will allow for study of proteins in their native cellular environment greater contracting materials (Kriminski et al., 2003). and be especially useful for the study of membrane proteins which can be diffi- Farley, C., Burks, G., Siegert, T. & Juers, D. H. (2014). Acta Cryst. D 70, cult to purify for in vitro studies. We have designed and built a single cell flow 2111-2124. system to enable uniform access of cells to the laser. Results demonstrate that in Kriminski, S., Kazmierczak, M. & Thorne, R. E. (2003). Acta Cryst. D 59, 697- cell FPOP (IC-FPOP) can oxidatively modify over 1300 proteins in various 708. cellular compartments. We have recently tested the ability of IC-FPOP to detect conformational changes in-cell using GCaMP2 as a model protein. GCaMP2, a 2800-Pos Board B8 chimeric protein of GFP and calmodulin, undergoes a conformational change Investigating the Protease Active Site Environment with Vibrational upon calcium binding. IC-FPOP successfully identifies regions of conforma- Reporters and X-Ray Crystallography tional change similar to in vitro FPOP. This represents the first time in-cell foot- Christopher N. Eaton, Meiqi Lou, Gwendolyn Fowler, Scott H. Brewer, printing coupled with mass spectrometry has be used to probe protein structure Edward E. Fenlon, Christine M. Phillips-Piro. in-cell. Chemistry, Franklin and Marshall College, Lancaster, PA, USA. The active sites of proteases were probed utilizing inhibitors modified with 2803-Pos Board B11 vibrational reporters and protein X-ray crystallography. Specifically, the in- Characterization of Reverse Micelle Surfactant Mixture for Biophysical hibitors 1-cyano-benzene-4-sulfonylfluoride (CBSF) and 1-azido-benzene- and Biomedical Applications 4-sulfonylfluoride (ABSF) were synthesized containing the nitrile and azide Cara Mawson, Joshua Berg, Hannah Work, Charles Hughes, vibrational reporters, respectively and reacted with the serine proteases sub- Nathaniel V. Nucci. tilisin and trypsin to probe the active sites of the enzymes. Protein crystal Physics & Astronomy, Rowan University, Glassboro, NJ, USA. structures of a number of these novel protein-inhibitor complexes have Reverse micelles are spontaneously organizing nanoscale complexes composed been determined using X-ray crystallography to illustrate successful incor- of surfactant and small volume of water in a bulk non-polar solvent. Typical poration of these inhibitors exclusively into the active site of these enzymes. reverse micelle mixtures form approximately spherical structures capable of This work shows that the tertiary structure of the active site closely matches encapsulating aqueous solutes, including biological macromolecules, in that of previously published inhibitor-bound protease structures. Details nanoscale spaces. Recently, a novel reverse micelle mixture composed about the X-ray crystal structures of the various protein-inhibitor complexes of lauryldimethylamine-N-oxide (LDAO) and 1-decanoyl-rac-glycerol will be discussed. Complementary infrared spectroscopic results will also be (10MAG) was described. This mixture shows broad applicability for encapsu- presented to illustrate the ability of this combined approach to effectively lation of proteins while maintaining their structural fidelity. We have utilized probe the active site of proteases with inhibitors containing vibrational dynamic light scattering and fluorescence spectroscopy to determine the sizes, reporters. shapes, and partitioning behavior of reverse micelles composed of this mixture. Early tests of this mixture for use in delivery of macromolecules to the cellular 2801-Pos Board B9 interior are also presented. These efforts are aimed at characterization of the E. coli The Sec Reaction Pathway for Cellular Protein Sorting under a mixture as a potential vehicle for biological drug delivery. Single Molecule Loupe Niels Vandenberk. 2804-Pos Board B12 Chemistry, KULeuven, Heverlee, Belgium. Enhanced Protein Structural Characterization using Microfluidic Protein targeting and secretion is an essential biological process in all life Modulation Spectroscopy forms. In E. coli, the Sec translocase machinery consists of a conserved Jeffrey A. Zonderman. protein-conducting channel (SecYEG), which associates with cytoplasmic part- RedShift BioAnalytics, Burlington, MA, USA. ners such as SecA. Although busily studied, important mechanistic details on RedShift Bioanalytics has developed a powerful new tool for protein structural bacterial protein targeting and secretion are still lacking. Since protein confor- analysis based on Microfluidic Modulation Spectroscopy, MMS. This technol- mation and dynamics seems to be key to the function of translocases, we there- ogy shows significant increases in sensitivity, dynamic range, accuracy and fore carried out quantitative studies on the dynamic conformation of SecA utility for determination of protein secondary structure, quantitation, similarity, in vitro using single-pair Fo¨rster resonance energy transfer (sp-FRET). Here, stability and aggregation. The analyzer utilizes a tunable mid-infrared quantum

BPJ 8720_8723 Wednesday, February 21, 2018 567a cascade laser to generate an optical signal 1000X brighter than conventional to be realized to comprehend the function of KRE2 in the P. lutzii. Therefore, sources used in FTIR spectroscopy. This also allows the use of simpler detec- these results represent advancement in the development of new therapies for the tors without the need for liquid nitrogen. Additionally, the sample (protein) so- fungal infections treatments with worldwide relevance. lution and a matching buffer reference stream are automatically introduced into a microfluidic flow cell, and the two fluids are rapidly modulated (e.g. 5 Hz) 2807-Pos Board B15 across the laser beam path to produce nearly drift-free background subtracted Structural Characterization and Crystallization of Human TMPRSS2 measurements. Protease Gideane Mendes de Oliveira, Aisel Valle Garay, Amanda Arau´jo Souza, 2805-Pos Board B13 Jonatas Cunha, Barbosa Lima, Napolea˜o Fonseca Valadares, Sonia Maria, The Functional Characterization of the Heterologous Acid Phosphatase de Freitas, Joa˜o Alexandre, Ribeiro Gonc¸alves Barbosa. from Trichoderma harzianum Celular Biology, Universidade de Brası´lia, Brası´lia, Brazil. Amanda A. Souza1, Viviane Castelo Reis2, TMPRSS2 is a transmembrane serine protease predominantly associated with Marcelo Soller Henrique Ramada3, Gideane Mendes Oliveira1, the luminal face membrane of the prostatic epithelium. The TMPRSS2 gene Azadeh Mehdad1, Fernando Araripe Torres2, Cirano Jose Ulhoa4, is androgen-driven and overexpressed in patients with prostate cancer. This Raphaela De Castro Georg4, Sonia Maria De Freitas1. protease induces tumorigenesis through the proteolytic conversion of proHGF, 1Laborato´rio de Biofı´sica, Universidade de Brası´lia, Brası´lia, Brazil, activation of c-Met signaling and engagement of the PI3K-Akt-mTOR 2Laborato´rio de Biotecnologia de Leveduras, Universidade de Brası´lia, pathway. The enzyme participates in viral infection by performing cleavage Brası´lia, Brazil, 3Laboratorio de Espectrometria de Massas, Universidade and activation of glycoproteins. Due to its crucial role in prostate cancer and Cato´lica de Brası´lia, Brası´lia, Brazil, 4Laborato´rio de Enzimologia, viral replication, structural studies of TMPRSS2 are a strategic part of the Universidade Federal de Goia´s, Goiaˆnia, Brazil. design of potent and selective therapeutic drugs. Thus, the objectives of this Trichoderma harzianum is a saprophytic fungus known for its potential as a bio- work were to express, purify and perform structural studies of the protease logical control agent and its crucial role in the release of carbon, nitrogen and domain of TMPRSS2. The gene corresponding to the zymogen of the catalytic phosphorus to the environment. Phosphorus is released through the action of extracellular domain was expressed in E. coli BL21 (DE3). The solubilized pro- phosphatases, converting organic phosphate into a soluble inorganic form by hy- tease was purified and renatured by affinity chromatography. The secondary drolysis. This is important for the recycling of organic phosphate and bioreme- structure profile of TMPRSS2 was determined by circular dichroism (CD). These studies showed changes in the secondary structure to abroad pH varia- diation process. In the present study, an acid phosphatase (ACPase) from T. harzianum was expressed in Komagataella phaffii and functionally character- tion, and thermostability between 25 and 95 C. In addition, the homology ized. The ACPase was purified by hydrophobic interaction and size exclusion modeling of the catalytic domain of TMPRSS2 and docking with the benzami- chromatography. This enzyme presented 22% of glycosylation and a molecular dine inhibitor was performed. The model is in agreement with the content of the mass of 85 kDa. The optimum pH and temperature were 4.0 and 70C. ACPase secondary structure found in the CD experiments. The predicted TMPRSS2- benzamidine complex showed the residue Asp189 present at the S1 site binds has a Km of 0.033 mM and Vmax 11.17 mM min-1 to pNP-P substrate. Interest- ingly, this enzyme was shown to bind specifically to phytic acid. The activity form hydrogen bonds to the amidine group of the inhibitor, as in other trypsin- like enzymes. The TMPRSS2 was crystallized from sitting drops and the reser- was highly inhibited by KH2PO4 and sodium tungstate. The tertiary structure of the ACPase was susceptible to broad pH variation. Circular Dichroism studies voir solution composed of 50 % PEG 200, 100 mM Potassium Phosphate and showed that enzyme remained stable between 25 to 95C at pH 4.0. The Dy- Sodium pH 6.2 and 200 mM Sodium Chloride. Afterwards, the protein tridi- namic Light Scattering assays showed that ACPase present profile organized mensional structure is going to be defined and inhibition assays are going to into monodisperse tetramers in solution. The homology model of the ACPase be realized aiming to find potent and selective drugs for the TMPRSS2. is in agreement with the secondary structure and presents a conserved catalytic 2808-Pos Board B16 site corresponding to His131, His408 and Glu409 residues. In sum, the heterol- Chicken Nanog Protein Self-Associates via a Novel Folding-upon-Binding ogous enzyme is similar to the native enzyme and can be applied as biotechno- Mechanism logical tool for removing phosphate from soils in the bioremediation processes. Jeong-Yong Suh. Seoul National University, Seoul, Republic of Korea. 2806-Pos Board B14 NANOG plays a pivotal role in pluripotency acquisition and lineage specifica- Paraccocidioides lutzii Characterization of the Molecular Target KRE2 of tion in higher vertebrates, and its expression is restricted to primordial germ cells Aiming at Development of New Antifungal Therapies (PGCs) during early embryonic development. Mammalian NANOG self- 1 1 Patrı´cia Alves Silva , Thyago Jose Arruda Pacheco , associates via conserved tryptophan-repeat motifs in the C-terminal domain 2 3 Ana Karina Rodrigues Abadio , Erika Seki Kioshima , (CTD) to maintain pluripotency. Avian NANOG, however, lacks the conserved 4 4 1 Nahum Hernandez Valente , Hector Mora Montes ,Soˆnia Maria de Freitas , motifs, and the molecular mechanism underlying the biological function is not Maria Sueli Soares Felipe1, Joa˜o Alexandre Ribeiro Gonc¸alves Barbosa1. 1 2 clearly understood. Here, using the spectroscopic and biochemical methods Biologia Celular, Universidade de Brası´lia, Brası´lia, Brazil, Biologia and cell-based assays, we report that chicken NANOG (cNANOG) oligomerises Celular, Universidade do Estado do Mato Grosso, Nova Xavantina, Brazil, through its CTD via a novel folding-upon-binding mechanism. The CTD of 3Department of Clinical Analysis and Biomedicine, Universidade Estadual de 4 cNANOG is disordered as a monomer, and associates into an a-helical multimer Maringa´, Parana´, Brazil, Divisio´n de Ciencias Naturales y Exactas, driven by intermolecular hydrophobic interactions. Mutation of key aromatic Universidad de Guanajuato, Guanajuato, Mexico. residues in the CTD abrogates the self-association, leading to a loss of the pro- The increasing number of cases of systemic infections has been the cause of liferation of chicken PGCs. Our results demonstrate that the CTD of cNANOG great concern worldwide. The therapeutic options currently available are belongs to a novel intrinsically disordered protein that switches into a helical limited, and another problem is the pathogens resistance to the classical anti- oligomer via self-association, enabling the maintenance of PGCs. fungal agents resulting in the requirement for the development of new anti- fungal agents. Preliminary results obtained by our group using comparative genomics, homology modeling and docking identified possible target genes Posters: Protein Structure and Conformation III and compounds of low molecular weight (small molecules) that potentially have the ability to inhibit these targets. A gene highly conserved in human path- 2809-Pos Board B17 ogenic fungi Kre2 or Mnt1 encodes alpha-1,2 mannosyltransferase. It is an Unique Conformational Dynamics and Dimer Tuning of mGluR7 important protein for cell viability and virulence of the Paracoccidioides lutzii Chris Habrian. within the host. The molecule inhibitory of KRE2 was called molecule 3. The University of California, Berkeley, Berkeley, CA, USA. heterologous enzyme was expressed in Komagataella phaffii. The kinetic pa- Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled re- rameters of the KRE2 was determinated using labeled substrate with [14C] ceptors (GPCRs) that operate at excitatory and inhibitory synapses. Of eight sub- and showed apparent Km 3.7 pM demonstrating the high affinity for the sub- types, group III member mGluR7 stands out with an extremely low glutamate strate in comparison to data in the literature. Indeed, the enzymatic inhibition affinity. Ensemble and single molecule FRET between the ligand binding was available using the molecule 3 against KRE2 and model enzyme (MNT1 domains (LBDs) of an mGluR7/7 homodimer show that, unlike other mGluRs, of Candida albicans). The results demonstrated that the inhibitor reduced the even saturating glutamate leads to very low occupancy of the activated confor- enzymatic activity in 60% to KRE2 and 20% to MNT1. Recently, the purifica- mation. The low affinity and efficacy are associated with an unusually stable tion was concluded with a high degree of purity by chromatography ion ex- resting conformation and unstable activated conformation. We find that when change (DEAE) and molecular exclusion Superdex S75, respectively. mGluR7 and mGluR2 heterodimerizes both agonist affinity and efficacy of Subsequently, crystallography and conformational characterization are going mGluR7/2, as well as the stability of its activated conformation, are far greater

BPJ 8720_8723 568a Wednesday, February 21, 2018 than in mGluR7/7 and agonist efficacy is even higher than in mGluR2/2, making tin mediates fusion of homotypic three-way junctions in the endoplasmic mGluR7/2 a super-receptor. mGluR7 and mGluR2 have overlapping expression reticulum (ER). Dynamin 1 (Dyn1) is responsible for fission of endocytic patterns introducing the possibility that the mGluR7/2 heterodimer may provide clathrin-coated vesicles from the plasma membrane. Mutations in any of these presynaptic nerve terminals with uniquely high efficacy glutamate sensitivity. proteins can lead to neuropathies including Dominant Optic Atrophy, Hereditary Spastic Paraplegia, and Charcot-Marie-Tooth, among others. Currently, struc- 2810-Pos Board B18 tural and biochemical data is limiting for Opa1 and the Mfns, particularly in a Towards the Structure of DNase1L3 lipid environment. We have developed a protocol for expressing and purifying Jon J. McCord1, Faraz Harsini1, Sukanyalakshmi Chebrolu1, Peter Keyel2, biologically relevant and biochemically active shortened isoforms (Opa1GG Roger Bryan Sutton1. and Mfn1GG) in sufficient quantity to begin cryo-EM studies. We have shown, 1Cell Physiology and Molecular Biophysics, Texas Tech University Health 2 using analytical ultracentrifugation (AUC) and size exclusion chromatography Sciences Center, Lubbock, TX, USA, Biological Sciences, Texas Tech (SEC) that an N-terminal heptad repeat of Opa1 is required and sufficient for University, Lubbock, TX, USA. dimerization of the proteolytically processed short form, Opa1S. Opa1GG, lack- Systemic Lupus Erythematosus is a debilitating autoimmune disease that af- ing this N-terminal region, migrates differently under multiple nucleotide flicts about 1.5 million Americans today. Although SLE is a multifactorial dis- conditions by SEC. In addition, to verify work performed by x-ray crystallog- ease, SLE can arise with disruptions in two genes: Dnase1 and Dnase1L3. raphy, we have begun probing the GTPase cycle of Atlastin using single particle While the structure is known for DNase1, the structure for Dnase1L3 is un- cryo-EM methods. We have recently calculated a 4.3 angstrom resolution struc- known. To study the structure and function of the Dnase1L3 enzyme, we de- ture of the constricted state of Dyn polymers on lipid by cryo-EM. signed a heterologous expression system and purification protocol for mouse DNase1L3 in E. coli. This system will allow us to characterize the enzymatic 2813-Pos Board B21 activity of the wild-type enzyme. Towards the purpose of solving its 3D struc- Clinical and Biophysical Characterization of a Mutation in the N-Helix ture with X-Ray crystallography we proceeded to the crystallization step of Region of Cardiac Troponin C: Evidence for an Allosteric Mechanism of Dnase1L3. A variety of culturing conditions, purification methods, and charac- Contractile Dysfunction terization techniques were performed on purified DNase1L3. After testing a Jamie R. Johnston1, Mayra de A. Marques2, David Gonzalez-Martinez1, few different methods, we successfully expressed DNase1-L3 in E. coli using Guilherme A.P. de Oliveira2, Einat Birk3, Nili Zucker3, Rosetta-gami cells as a fusion protein to Maltose Binding Protein. The fusion Maicon Landim-Vieira1, Adolfo H. Moraes4, P. Bryant Chase5, enzyme was isolated via amylose affinity resin. After elution from the affinity Jerson L. Silva2, Yael Wilnai6, Jose R. Pinto1. column, the enzyme was cleaved with TEV protease and further purified on 1Biomedical Sciences, Florida State University, Tallahassee, FL, USA, S-resin as a defined peak. Finally, Dnase1L3 was purified using size exclusion 2Instituto de Bioquı´mica Medica Leopoldo de Meis, Instituto Nacional de chromatography. The activity of purified DNase1-L3 was assessed through Biologia Estrutural e Bioimag, Universidade Federal do Rio de Janeiro, Rio digestion of plasmid DNA. The identify of Dnase1L3 was confirmed by West- de Janeiro, Brazil, 3Cardiology, Schneider Children’s Medical Center, Tel ern blot. Now that we have obtained an efficient purification method, verified Aviv University, Petah Tikva, Israel, 4Bioquı´mica e Imunologia, the activity, and confirmed identity with specific antibodies, we are interro- Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 5Biological gating the structural aspects of the enzyme through X-Ray crystallography. Science, Florida State University, Tallahassee, FL, USA, 6Genetics Unit, The structural composition of this enzyme will be invaluable as pertaining to Schneider Children’s Medical Center, Tel Aviv University, Petah Tikva, possible use in treating Systemic Lupus Erythematosus. Israel. 2þ 2811-Pos Board B19 Troponin is a heterotrimeric, Ca -binding protein responsible for regulating Proteolytically Resistant Cellular Prion Protein Construct Retains Metal muscle contraction in the heart. Missense mutations in genes encoding the sub- Driven CIS-Interaction while Generating Toxicity in Cells units of the cardiac troponin complex are associated with inherited dilated car- Graham P. Roseman1, Alex J. McDonald2, Bei Wu2, David A. Harris2, diomyopathy (DCM). However, the underlying molecular mechanisms of Glenn L. Millhauser1. disease pathogenesis are not fully understood. Here, we report a clinical case 1Universtiy of California Santa Cruz, Universtiy of California Santa Cruz, of a 1-year-old female who presented with severe DCM and hypotonia. Whole Santa Cruz, CA, USA, 2Biochemistry, Boston University School of exome sequencing revealed a previously unreported de novo heterozygous > Medicine, Boston, MA, USA. variant in exon 1 of the TNNC1 gene (c.12C G), resulting in an Ile4Met mu- Transmissible Spongiform Encephalopathies, or prion disorders, are caused by tation located in the N-Helix region of cardiac troponin C (cTnC). We utilized the misfolding and aggregation of the cellular prion protein (PrPC). The precise various biophysical and biochemical techniques to examine the molecular basis C of this pathogenic variant and provide evidence for allosteric communication physiological role of the properly folded 209 amino acid GPI anchored PrP is 2þ unknown. PrPC deletions have been used to study the role of certain domains within the Ca -binding subunit of the troponin complex. In vitro extraction of native cTnC and reconstitution with either recombinant WT or I4M cTnC within the protein. Interestingly, one of these mutations, DCR (D105-125 PrP), 2þ which deletes 21 amino acids in the highly conserved central region of the protein, in cardiac muscle preparations revealed decreased Ca sensitivity of isometric causes neonatal fatality in mice and massive neurodegeneration. Furthermore, the force development and slower kinetics of tension redevelopment (ktr) for I4M compared to WT cTnC. Steady-state fluorescence measurements on isolated toxicity that is caused by DCR is similar to that of prion disorders except without 2þ the aggregation of PrPC. Biophysical studies showed that DCR causes two main cTnC using Bis-ANS indicated enhanced Ca binding at the C-terminal domain of I4M compared to WT. Furthermore, cTnC-I4M displayed a smaller changes: weakening of the metal driven cis-interaction between the N- and C- ter- 2þ minal domains and deletion of the region of the protein where regulatory prote- magnitude of Ca -induced hydrophobic exposure compared to WT. Finally, olysis occurs. What is not known is if DCR neurotoxicity is a result of a IANBD fluorescence (singly labeled at cysteine 84) titration studies revealed tighter binding of cardiac TnI to I4M compared to WT. Altogether, these results weakened cis interaction or elimination of proteolytic sites. In this study, we 2þ test DCR’s toxicity source by replacing the central region with an uncleavable suggest that perturbed contractile kinetics and altered Ca -binding, perhaps by flexible glycine-serine (GS) rich linker that retains the cis interaction of wild an allosteric mechanism, likely contribute to the contractile dysfunction type PrPC while blocking the cleavability of this region. NMR studies show the observed in this proband. Experiments using solution NMR spectroscopy are cis interaction is preserved, cell and in vitro based cleavage assay’s show that ca- currently underway to determine how a pathogenic mutation in the N-helix af- nonical cleavage is blocked, and electrophysiological measurements show that fects the overall structure and dynamics of cTnC. NIH-HL128683. these uncleavable linkers generate spontaneous currents, which correlate to toxicity that DCR exhibits. Therefore, these results show that the cleavability 2814-Pos Board B22 of PrPC is a crucial regulatory function and that blocking it generates toxicity. Computational and Experimental Studies of Divergent Clinical Effects in Proximate Thin Filament Mutations 2812-Pos Board B20 Anthony Baldo1, Salwa Abdullah2, Andrea Deranek3, Melissa Lynn4, Structural and Biochemical Assay of Dynamin-Like GTPases Michael Williams1, Jil C. Tardiff4, Steven D. Schwartz1. Andrew Kehr1, Leopold Kong1, Huaibin Wang1, Shunming Fang1, 1Dept. of Chemistry and Biochemistry, The University of Arizona, Tucson, Matt Martin2, Jenny Hinshaw1. AZ, USA, 2Dept. of Cellular and Molecular Medicine, The University of 1NIDDK, National Institutes of Health, Bethesda, MD, USA, 2University of Arizona, Tucson, AZ, USA, 3Dept. of Biomedical Engineering, The Maryland, College Park, MD, USA. University of Arizona, Tucson, AZ, USA, 4Dept. of Medicine, The University Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and of Arizona, Tucson, AZ, USA. vesiculation of cellular lipid membranes throughout the cell. The dynamin-like The cardiac thin filament controls the contraction and relaxation of cardiac proteins Optic Atrophy 1 (Opa1) and Mitofusin (Mfn) 1 and 2 are responsible for muscle. Mutations in this molecular machine have varying effects on the reg- the fusion of the mitochondrial inner and outer membranes, respectively. Atlas- ulatory function of the cardiac sarcomere, and have been linked to both

BPJ 8720_8723 Wednesday, February 21, 2018 569a hypertrophic (HCM) and dilated cardiomyopathy (DCM). In the current study discovered that in addition to binding acetyl-lysine, the BRG1-BD associates we describe both computational and experimental investigations of 2 cardiac with DNA, a novel function for BDs. In addition, we have demonstrated that Troponin T (cTnT) mutations that occur in similar regions of the thin filament an adjacent AT-hook contributes to a multivalent mechanism of association but result in divergent clinical phenotypes. Both experimental (Time resolved with DNA, increasing affinity and specificity (Morrison et al., Nature Commu- FRET (TR-FRET)) and computational results show that the D160E mutation nications, 2017). Notably, the newly identified DNA binding interface harbors repositions the flexible linker, moving it closer to the C-terminus of tropomy- several cancer-associated mutations. Here we present our continued studies on osin. This in turn alters weak electrostatic binding that decreases linker flexi- this newly recognized composite DNA binding domain. We use systematic bility, ultimately leading to HCM. By contrast, Arg173Trp (R173W) also evolution of ligands by exponential enrichment (SELEX-seq) to generate a bio- lies within the linker domain on cTnT but results in a dilated cardiomyopathy physical model of sequence specificity for the domain that we are exploring (DCM). Computation shows both average structure and time dependent dy- structurally using NMR spectroscopy and X-ray crystallography. To determine namics are differentially modified in this mutational state. Specifically, the in the kinetic and thermodynamic basis of association we use biolayer interferom- silico average structure of the thin filament shows an increased distance be- etry (BLI). In addition, we are investigating how DNA binding is altered in the tween the linker region and the C-terminus of tropomyosin due to the context of the nucleosome, and the effect of known cancer mutations on the R173W mutation. Differential scanning calorimetry on reconstituted thin fila- DNA binding activity. Together, our results are revealing the molecular details ments both with and without the R173W mutation suggest an impact on thermal of how this novel DNA binding domain functions. stability and thus the thermodynamics of protein interaction. Our simulations and TR-FRET results illuminate the resultant perturbations to the WT structure 2817-Pos Board B25 and dynamics caused by the R173W mutation, and we contrast these effects to Prion Protein’s Zn2DDriven CIS Interaction Weakened by N-Terminal those found in the D160E mutation. Our findings provide a new framework for Deletions understanding the varying pathogenic mechanisms that drive clinical pheno- Kate Markham, Glenn Millhauser. types in HCM and DCM in proximate thin filament mutations. Chemistry and Biochemistry, UCSC, Santa Cruz, CA, USA. The prion protein has been studied because of its role in Transmissible Spongi- 2815-Pos Board B23 form Encephalopathies (TSEs), prion diseases, which is the accumulation of Magic Angle Spinning Solid State NMR Studies of Oxidized Apolipopro- misfolded prion protein in the brain. PrPc is a 210 amino acid protein comprised tein A-I Aggregates of an unstructured N-terminal domain, and a predominately helical C-terminal þ þ þ Jennifer C. Boatz1, Gary Chan2, Andrzej Witkowski2, region that is GPI anchored. PrPc binds both Zn2 and Cu2 , where Zn2 is bound þ Patrick C.A. van der Wel1, Giorgio Cavigiolio2. by the octarepeat (PHGGGWGQ) of the N-terminal domain and Cu2 is bound 1Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, in a more complicated, multi-component fashion utilizing the entire N-terminal USA, 2Children’s Hospital Oakland Research Institute, Oakland, CA, USA. tail. Until recently, the N-terminal domain and C-terminal domain were thought Cardiovascular disease is the foremost cause of death in developed countries to be function separately. Spevacek et al. recently observed a N-Terminal/C-Ter- and is often the outcome of a process of atherosclerosis, in which artery walls minal (cis) interaction upon addition of Zn2þ, and that upon addition of Zn2þ, the harden upon accumulation of fatty deposits and foam cells. While lipid oxida- more positive N-terminal tail docks onto the more negatively charged C-termi- tion is commonly associated with early pro-atherogenic events, the role of nus (helices 2 and 3) creating a ‘‘global fold’’. In attempts to decipherer the aggregating proteins in the etiology of atherosclerosis remains unclear. Apoli- cellular function of the protein it was found that PrP-/- in mice showed no signif- poprotein A-I (apoA-I), the main protein component of high-density lipopro- icant change, however, deletion of a 21 hydrophobic amino acid stretch in the N- teins, is a known anti atherogenic factor. Notably, high levels of lipid-free terminus, DCR (D105-125 PrP), was neonatal lethal. Also, this 21 amino acid apoA-I and large amounts of amyloid deposits mainly composed of apoA-I stretch is only neonatal lethal if PrP has its polybasic extreme N-terminus. To are found in atherosclerotic plaques. We previously demonstrated that upon study the non-octarepeat N-termini’s influence of the cis upon Zn2þ binding, oxidation of methionine residues (Met(O)-ApoA-I), apoA-I becomes aggrega- we created 15N labeled mouse PrP and PrP N terminal deletions for NMR studies. tion prone. Here, the structure and dynamics of aggregated Met(O)-ApoA-I was We titrated Zn2þ into the protein and examined the changes in the HSQC spectra. probed using magic angle spinning (MAS) solid state NMR. The observed The results from this study will help to determine whether Zn2þ plays a in the NMR chemical shifts report on the local secondary structure of individual res- physiological function of PrP. idues and are exquisitely sensitive to structural changes, allowing for a direct comparison between soluble and aggregates states of the protein (1). Our results 2818-Pos Board B26 show that Met(O)-ApoA-I forms aggregates featuring a b-rich amyloid core Single-Molecule Atomic Force Microscopy of Blood Coagulation Factor flanked with a-helices that are retained from the native structure. Unlike the XIII and its Subunits monomeric intact protein that has a highly unstructured C-terminal domain, Anna D. Protopopova1, Andrea Ramirez2, Rustem I. Litvinov1,3, there are relatively few highly dynamic and unstructured residues in Met(O)- John W. Weisel1. ApoA-I aggregates. ApoA-I aggregates formed from seeding the intact protein 1Cell and Developmental Biology, University of Pennsylvania School of with Met(O)-ApoA-I aggregates were found to be structurally similar to the Medicine, Philadelphia, PA, USA, 2University of Texas at El Paso, El Paso, original seeds, with some differences noted in the secondary structure. This TX, USA, 3Institute of Fundamental Medicine and Biology, Kazan Federal shows that methionine oxidization in apoA-I can promote seeded aggregation University, Kazan, Russian Federation. of the intact protein, which is a previously unknown mechanism whereby Coagulation factor XIII (FXIII) is a precursor of the active transglutaminase apoA-I dysfunction may contribute to atherosclerosis. (FXIIIa) that catalyzes covalent cross-linking within polymeric fibrin to in- 1. Boatz, J.C., et al. (2017) Cataract-associated P23T gD-crystallin retains a crease mechanical and proteolytic stability of blood clots and thrombi. Inactive native-like fold in amorphous-looking aggregates formed at physiological FXIII is a heterotetramer (A2B2) with two catalytic A-subunits and two inhib- pH. Nature Communications, 8, 15137. itory B-subunits, which are dissociated upon thrombin-induced conversion of FXIII to the enzymatically active dimeric (A2) form FXIIIa. FXIII-A2 has 2816-Pos Board B24 been characterized crystallographically, while the atomic structures of the Characterization of the Novel DNA Binding Activity of the BRG1 FXIII-A2B2 complex and B-subunits remain unknown. Our goal was to visu- At-Hook-Bromodomain and Effect of Cancer Mutations alize and characterize the molecular structure of FXIII-A2B2, FXIIIa-A2, and Julio C. Sanchez, Liyang Zhang, Amber Liu, Miles A. Pufall, individual B-subunits using high-resolution atomic force microscopy (AFM). Catherine A. Musselman. Commercially available FXIII and FXIIIa (Enzyme Research Laboratories, University of Iowa, Iowa City, IA, USA. USA) were adsorbed on the surface of freshly cleaved graphite rendered hydro- The packaging of eukaryotic DNA with histones into chromatin acts to both philic with an amphiphilic hydrocarbon-glycine modifier followed by high- compact it into the nucleus as well as provide a mechanism for regulation of resolution single-molecule AFM imaging in air. FXIII was visualized as a the genome. Spatial and temporal remodeling of chromatin structure is critical globule with two filamentous extensions, while in FXIIIa the protruded elon- for all DNA-templated processes and dysregulation of these pathways is asso- gated portions were separated from the globule, suggesting that the globular ciated with a number of diseases. The human switching/sucrose non-fermenting structure represented a compact A2-dimer and the extensions comprised indi- (SWI/SNF) complex, is an ATP-dependent remodeling complex that plays an vidual B-subunits. The contour length of the B-subunits increased from important role in gene regulation, several subunits of which are mutated in hu- 2156 nm in FXIII to 3656 nm in FXIIIa after their dissociation from the glob- man cancers. The Brahma related gene 1 (BRG1) subunit of hSWI/SNF, which ular part of the protein, implying that 40% of each B-subunit was invisible provides the ATPase activity, contains a bromodomain (BD) at its C-terminus. due to tight association with the globular A2-dimer. These observations BDs are well-characterized readers of acetylated lysines on histones, and the strongly suggest that two B subunits are partially wrapped around the A-sub- BRG1-BD has been shown to bind H3K14ac. However, we have recently units, probably through their N-terminal portions. The dimensions (height) of

BPJ 8720_8723 570a Wednesday, February 21, 2018 the globular part decreased upon activation from 3.550.6 nm in FXIII to tion of the Arf1:AP-1 clathrin adaptor complex and subsequent clathrin cage 2.650.6 nm in FXIIIa as a result of B-subunits separation and conformational assembly. changes in FXIII-A2. These results provide molecular structural characteristics of FXIII and FXIIIa molecules as well as individual A and B subunits and their 2822-Pos Board B30 interactions within FXIII. Biochemical Characterisation of Lectin from Indian Hyacinth Plant Bulbs with Potential Inhibitory Action Against Human Cancer Cells Sanjit Kumar1, Sanjay Naik2, Ravindra Singh Rawat2. 2819-Pos Board B27 1 2 Cryo-EM Studies of ATPases and Substrate Engagement CBST, VIT-University, Vellore, India, School of Bio Science & Colby Sandate. Technology, VIT-University, Vellore, India. The Scripps Research Institute, San Diego, CA, USA. This work describes purification and characterisation of a monocot The AAA ATPase superfamily is a class of enzymes which couples the bind- mannose-specific lectin from Hyacinth bulbs. The purified lectin has a mo- ing and hydrolysis of ATP to perform work within the cell. Numerous near- lecular mass of 30kDainreducingaswellasinnon-reducingSDS- atomic structures have recently been determined of ATPase hexamers bound PAGE. In hydrodynamic studies by Dynamic Light Scattering (DLS) to substrate peptides. These interactions are mediated by pore-loop tyrosines, showed that purified lectin was monomeric in nature with a molecular 5 which are conserved across AAA ATPases. We explore how ATP binding or- size of 2.38 0.03 nm. Agglutination activity of purified lectin was ganizes individual subunits into an active hexamer, and the network or confirmed by rabbit erythrocytes and its agglutination activity was in- subunit-subunit interactions that enable inter-protomer communication hibited by D-mannose and glycoprotein (ovalbumin). Glycoprotein nature required for substrate translocation. These analyses provide the foundation of purified lectin was confirmed by Periodic Acid Schiff’s (PAS) stain. Pu- rified lectin showed moderate pH and thermal stability by retaining hemag- for future studies into the mechanism of how ATPases perform work on substrates. glutination activity from pH 6 to 8 and temperature up to 60 C. It also suppressed the growth of human colon cancer cells (Caco-2) and cervical 2820-Pos Board B28 cancer cells (HeLa) with IC50 values of 127 mg/mL and 158 mg/mL Shedding Light on Night Owl Behavior: How the Disordered C-Terminal respectively, after 24-hour treatment. Morphological studies of treated cells Tail of CRY1 Modulates Circadian Timekeeping (Caco-2 and HeLa) with hyacinth lectin by AO/EB dual staining indicated Gian Carlo Parico1, Ivette Perez2, Carrie Partch2. that purified lectin was capable of inducing apoptosis. 1 2 University of California, Santa Cruz, Santa Cruz, CA, USA, UC Santa 2823-Pos Board B31 Cruz, Santa Cruz, CA, USA. Structural Studies of Magnesium Transporter CNNM Circadian rhythms are driven by a negative feedback loop in which the het- Yu Chen, Meng Yang, Rayan Fakih, Guennadi Kozlov, Kalle Gehring. erodimer CLOCK:BMAL1 promotes transcription of clock-controlled Biochemistry, McGill University, Montreal, QC, Canada. genes, including its own repressors Period (PER) and Cryptochrome Magnesium is the most abundant intracellular divalent cation and is involved (CRY). The deregulation of circadian rhythms can lead to delayed sleep in over 600 enzymatic reactions including energy metabolism and protein phase disorder (DSPD), where the afflicted ‘‘night owls’’ are only able synthesis. Cyclin M (CNNM) has been proposed to function as a Mg trans- to fall asleep hours after midnight and have difficulty waking up early in porter. Structurally, CNNM contains an N-terminal extracellular domain, a the morning. CRY proteins contain a photolyase homology region (PHR) transmembrane domain, and a large cytosolic region containing a CBS and an intrinsically disordered C-terminal tail; previous studies identified domain and a putative cyclic nucleotide (cNMP)-binding domain. CNNMs an essential role for the CRY1 PHR in generating circadian rhythms, while interact with oncogenic PRLs (phosphatases of regenerating liver), which deletion of the tail was shown to alter the length of the circadian period. are frequently overexpressed in malignant human cancers. When PRL binds Our lab recently identified how the CRY1 PHR binds to both CLOCK CNNM, intracellular Mg level is increased, thereby allowing cancer cells to and BMAL1 to induce repression and close the negative feedback loop. proliferate. Here, we report the crystal structure of cytosolic domains of To date, little is known about how the CRY1 PHR and tail might interact CNNM2 and CNNM3. We found that CNNMs dimerize through its C-termi- to control this process. One prevalent mutation in DSPD patients leads to nal cNMP-binding domain, and that the CBS domain dimerization is depen- deletion of CRY1 Exon 11, a region that encodes for 25 amino acids in the dent on Mg and ATP binding. Our study provides insights into the tail, resulting in enhanced co-immunoprecipitation by CLOCK:BMAL1 mechanism in which CNNM senses intracellular ATP-Mg level and mediate and a longer circadian period. We hypothesized that the CRY1 tail might Mg transport. bind directly to the PHR to regulate interactions with CLOCK or BMAL1. Here we show that the C-terminal tail and CLOCK compete for binding to 2824-Pos Board B32 the CRY1 PHR, and that the peptide encoded by Exon 11 is sufficient for Insight into Ligand Binding and Conformational Dynamics of Kynurenine this interaction. Therefore, our data demonstrate how the tail can directly 3-Monooxygenase regulate CRY1 interactions with CLOCK:BMAL1 to provide initial insight Rajni Verma, Katie Mitchell-Koch. into the molecular basis of DSPD. Department of Chemistry, Wichita State University, Wichita, KS, USA. Cellular and developmental pathogenesis is the result of complicated cellular 2821-Pos Board B29 processes involving molecular interactions among cells and their intra- and Role of the Dileucine Motif in Nef-Induced Trimerization of the inter-cellular milieus. The characterization of molecular interactions in ARF1:AP-1 Clathrin Adaptor Complex different steps of disease-related metabolic pathways is necessary to under- Cosmo Z. Buffalo1,2, Kyle L. Morris1,2, Xuefeng Ren1,2, James H. Hurley1,2. stand how abnormalities in these interactions relate to disease and disorders, 1Molecular and Cell biology, University of California, Berkeley, Berkeley, and how to develop therapeutic strategies to overcome these abnormalities. CA, USA, 2California Institute for Quantitative Biosciences, Berkeley, CA, We focus on kynurenine 3-monooxygenase (KMO) of the kynurenine USA. pathway, which is a metabolic pathway leading to the production of nicotin- HIV Nef downregulates cell surface host proteins, including SERINCs, CD4, amide adenine dinucleotide from the degradation of tryptophan. KMO inhi- and MHC-I, by redirecting them into clathrin coated vesicles (CCVs) for lyso- bition is neuroprotective in studies on animal models, by balancing the somal degradation. Nef achieves this by hijacking the AP-1 and AP-2 clathrin neurotoxic and neuroprotective metabolites of the kynurenine pathway. adaptors. HIV-1 Nef cooperates with the host GTPase Arf1 to induce oligomer- KMO is a flavin adenine dinucleotide-dependent enzyme, that catalyzes ization and activation of AP-1, assembling as trimers in vitro. AP complexes’ the insertion of molecular oxygen into the aromatic ring of L-kynurenine normal function is to sort two main classes of cargo proteins into CCVs. These to produce 3-hydroxy-L-kynurenine. Several kinetic and structural studies cargo classes are defined by the presence of a tyrosine-based YXXF motif or an have been performed on KMO as an attractive target for neurodegenerative, acidic-dileucine motif [DE]XXXL[LI]. Nef-induced downregulation of MHC-I neuroinflammatory and immunological diseases. Atomistic-level understand- involves the hijacking of both the tyrosine and dileucine cargo binding sites of ing of KMO’s catalytic and inhibition mechanism is still lacking but will be AP-1. Arf1 promotes dimerization of AP-1, while in the presence of Arf1 and crucial for further advances. Herein, we performed molecular dynamics sim- Nef together, AP-1 trimers predominate. The combination of dimer and trimer ulations to gain insight into the conformational changes and molecular inter- interfaces lead to formation of a hexagonal lattice. In order to determine the actions in KMO as a free enzyme and in complex with substrate, product, contribution of the Nef dileucine motif to trimerization, a cryo-EM analysis and inhibitor. We integrate simulation data with experimental findings to of the AP-1:Arf1:NefLLAA complex was carried out. From this analysis, we interpret the totality of our results and arrive at a framework to gain insight conclude that the dileucine motif of Nef is important for promoting trimeriza- into the conformational dynamics and plasticity of KMO as a free-form

BPJ 8720_8723 Wednesday, February 21, 2018 571a enzyme or a ligand binding complex. The outcome leads to an atomistic from FXN, which leads to iron-sulfur cluster assembly. We also have eluci- insight into the enzyme conformational dynamics and ligand-binding inter- dated the mechanism by which the variant ISCU(M108I) bypasses the actions which will be crucial in designing novel and more effective thera- requirement for FXN. peutic strategies. 2828-Pos Board B36 2825-Pos Board B33 Structure and Membrane Binding of R9AP, a Membrane-Anchor Protein Investigating the Role of Thrombomodulin in the Activation of Sarah Bernier, Marc-Antoine Millette, Line Cantin, Christian Salesse. Complement CUO-recherche, Hoˆpital du Saint-Sacrement, Centre de recherche du CHU Julia R. Koeppe, Gary Ellis, Nicholas Joannides, Vanessa Wiltsie. de Quebec, Departement d’ophtalmologie, Facultedemedecine and Chemistry, State University of New York at Oswego, Oswego, NY, USA. PROTEO, Universite Laval, Quebec, QC, Canada. Complement component 3 (C3) is at the junction of three different comple- Visual phototransduction involves many proteins. Mutations in their coding ment activation pathways (classical, lectin, and alternative). Activation to sequence cause serious eye diseases. Mutations in R9AP (RGS9-1-Anchor- C3b is a key step in the innate immune response that allows for the formation Protein) lead to a visual disease called bradyopsia. R9AP anchors a protein of important multi-protein complexes that ultimately participate in pathogen complex to disk membranes of photoreceptor outer segments. Ultimately, clearance. When misregulated, however, complement can lead to inflamma- R9AP allows to indirectly inactivate phosphodiesterase and visual photo- tory disease and autoimmune disorders. Several regulatory proteins for C3b transduction. This binding of R9AP to photoreceptor membranes was postu- are known, but the molecular details of interactions between these proteins lated to occur through its C-terminal domain. However, membrane binding have not yet been elucidated. Thrombomodulin (TM), and specifically its and structural data are still lacking for this particular protein. This work N-terminal lectin-like domain (TMD1), has been identified as a possible regu- was thus aiming to gather structural information on R9AP and to determine lator of complement through interactions with C3 or C3b, and the known the extent of its membrane binding as well as the importance of its C-termi- regulator CFH may also be required. We have used fluorescence-based assays nal domain in this binding. The present study was thus performed using 1) and hydrogen/deuterium exchange mass spectrometry (HDXMS) to study the full-length R9AP, 2) truncated R9AP without its C-terminal domain as interaction of TMD1 with C3 and C3b. We have found that TMD1 interacts well as 3) its C-terminal domain. Results have allowed to postulate an with C3b, and there is a lesser interaction with C3. TMD1 tends to make C3b important role of the C-terminal domain in the binding and the solubility more accessible to deuterium exchange, while C3 tends to be less accessible of R9AP. Indeed, the truncated protein is much more soluble than the to deuterium exchange in the presence of TMD1. This difference suggests a full-length protein and the C-terminal segment is only soluble in particular possible role for TMD1 in regulating a key step along the complement hydrophobic solvents. Infrared spectroscopy as well as circular dichroism pathway. measurements have allowed determining that the truncated protein as well as the C-terminal domain adopt an a-helical structure. Langmuir monolayer 2826-Pos Board B34 measurements revealed that the C-terminal peptide has a clear affinity for Host-Pathogen Interactions via Ubiquitination Pathways most of the phospholipids typical of photoreceptor membranes. In contrast, 1 1 2 3 Kathy Wong , Guennadi Kozlov , John D. Perpich , Miroslaw Cygler , the truncated protein showed a low affinity for these phospholipids except Yousef Abu Kwaik2, Kalle Gehring1. 1 2 for unsaturated phosphoserine. Spectrofluorometric measurements using Biochemistry, McGill University, Montreal, QC, Canada, Microbiology wild-type R9AP and two mutants where each of the two individual trypto- and Immunology, University of Louisville, Louisville, KY, USA, 3 phans of R9AP have been replaced by a phenylalanine provided information Biochemistry, University of Saskatchewan, Saskatoon, SK, Canada. on the specific environment of these amino acids. Ankyrin B (AnkB) is a bacterial protein that plays an essential role in the intracellular proliferation of Legionella pneumophila, the causative agent 2829-Pos Board B37 of Legionnaires’ disease. It collects proteins to target for degradation into Structure-Function Studies of the Hypoxia-Inducible Proyly Hydroxylases free amino acids, generating a source of carbon and energy, and preventing Pamela N. Gallo, Kayla Schardien, Taylor Keagy, Nathaniel V. Nucci. a starvation response. AnkB contains two eukaryotic-like domains, the com- Molecular and Cellular Biosciences, Rowan University, Glassboro, NJ, USA. bination of which have never been found in the same eukaryotic protein. The Prolyl-hydroxylase domain (PHD)-containing proteins are the primary oxygen N-terminal F-box domain allows mimicry of host F-box proteins for interac- sensors in the cell and have been identified as important potential targets for tion with the host’s ubiquitination pathway via Skp1 of the SCF (Skp1- treatment of ischemic events including heart attack and stroke. There are three Cullin-F-box) E3 ubiquitin ligase complex. The C-terminal ankyrin repeats known PHD isoforms that differ significantly in size and activity. We are per- allow AnkB to selectively bind targets for degradation. Here, we report forming structure-function studies of the PHD-3 (27.3 kDa) and the catalytic the crystal structure of full length AnkB in complex with host Skp1. We domain of PHD-2 (27.5 kDa) using fluorescence-based assays and nuclear mag- found that AnkB contains an enlarged the substrate-binding site with three netic resonance (NMR). These two isoforms are the most active in maintaining ankyrin repeats rather than the two expected based on its sequence. Struc- cellular oxygen levels. A protocol for refolding from inclusion bodies and pu- tural analyses and mutational studies have identified key residues respon- rification for both proteins will be presented. Because the known structure of sible for decorating the Legionella-containing vacuole with ubiquitin and trPHD2 shows that the catalytic center is buried, an important role for protein for the replication of the pathogen during infection.Our study provides the dynamics in enzymatic turnover has been proposed. Molecular dynamics sim- first structural insights into the structural mimicry that allows the bacterial ulations of the trPHD2 protein in various ligand-bound forms are being used to virulence factor, AnkB, to associate with the host ubiquitination complex investigate the functional relevance of internal motions. NMR experiments and select substrates. investigating the slow motions of these proteins provide experimental evidence for the locations of motions with potential functional relevance, and new inhib- 2827-Pos Board B35 itory molecules are being developed as potential new tools for understanding Interactions of Frataxin with ISCU and Ferredoxin on the Cysteine this important pathway and as possible new therapeutic leads. This project Desulfurase Complex Leading to Fe-S Cluster Assembly was funded by the New Jersey Health Foundation. Kai Cai. Biochemistry, UW Madison, Madison, WI, USA. 2830-Pos Board B38 Frataxin (FXN) is involved in mitochondrial iron-sulfur (Fe-S) cluster Understanding AAV Packaging biogenesis and serves to accelerate Fe-S cluster formation. FXN deficiency Vishaka Santosh. is associated with Friedreich ataxia, a neurodegenerative disease. We have Physiology and Biophysics, Virginia Commonwealth University, Richmond, used a combination of isothermal titration calorimetry, chemical cross- VA, USA. linking with analysis by LC/MS/MS, multinuclear NMR spectroscopy, and Adeno-associated virus (AAV) has been utilized as a gene therapy vector, but biochemical assays to investigate interactions among the components of the biology of the virus is unknown. One of the mysteries of AAV is how the biological machine that carries out the assembly of iron-sulfur clusters exactly the virus packages the genome. That being said, the non-structural in human mitochondria. We have constructed a structural model of the Rep proteins appear to be necessary and sufficient for proper AAV packaging. core of this machine by combining homology modeling with docking con- The small Rep proteins serve as the molecular motor to package the DNA into straints derived from NMR chemical shift perturbations and chemical the viral capsid, but the large Rep proteins’ role has not been clarified. cross-linking studies. We show that the machinery operates through dynamic Furthermore, there is a consensus all Rep proteins should have the same heli- interactions among its components and have identified interactions relevant case activity, since they have the same helicase domain, and therefore should to the cysteine desulfurase reaction, which generates S, and iron transfer serve the same role. That actually isn’t the case since only Rep40 appears to

BPJ 8720_8723 572a Wednesday, February 21, 2018 be necessary for this task, so helicase assays were conducted to assess the ac- 2833-Pos Board B41 tivity of each of the Rep proteins. We found that Rep40 has a higher helicase Dissecting the Divergent Functions and Dynamics of ZAP-70 and SYK activity compared to Rep68 implying its role as the molecular motor as Helen T. Hobbs1, Neel Shah2, Susan Marqusee2, John Kuriyan1. logical. Since Rep68 doesn’t unwind DNA nearly as well as Rep40, we hy- 1Chemistry, University of California-Berkeley, Berkeley, CA, USA, pothesized another role that is linked to its oligomeric capacity. Using 2Molecular and Cell Biology, University of California-Berkeley, Berkeley, AUC, we have discovered Rep68’s ability to not only form higher order CA, USA. molecular complexes on its own, but depending on the DNA substrate, the Activation of signaling pathways in immune cells can be initiated by binding of molecular weight of the DNA/protein complex can change. In the presence membrane proteins, known as B-cell receptors (BCRs) or T-cell receptors of the inverted-terminal repeats (ITR), it forms a structure that is smaller (TCRs), to fragments of an antigen. The proteins spleen tyrosine kinase and distinct from the structure formed with ssDNA and Rep68. Currently, (Syk) and zeta-chain associated protein kinase 70 (ZAP-70) are among the first we have cryo-EM data on the ssDNA/Rep68 complex and we have negative proteins to be activated upon engagement of the BCR or TCR.Recruitment of stain data on the ITR/Rep68 complex. All together, Rep68’s flexibility ap- these proteins to phosphorylated immunoreceptor tyrosine activation motifs pears to imply a more structural role for Rep68 to help hold the DNA in place (ITAMs) associated with the TCR or BCR activates the kinase whereupon it as Rep40 pushes the DNA into the capsid. initiatesdownstream signaling pathways, eventually leading to an immune response. Syk and ZAP-70 are paralogs, with Syk being present in B-cells 2831-Pos Board B39 and ZAP-70 in T-cells. Additionally, ZAP-70 and Syk share the domain archi- Simultaneous Real-Time Observation of DNA Unwinding and Nuclease tecture of tandem SH2 domains (tSH2) linked to a kinase domain. The tSH2 is Domain Activation in Cas9-RNA-DNA Complex via Three-Color Single responsible for binding the phosphorylated ITAMs. Despite similarities in the Molecule FRET structure and function of these two kinases, there are key differences in their Yanbo Wang1, Digvijay Singh1, John Mallon2, Boyang Hua1, intrinsic activity and specificity. Using the technique of hydrogen exchange Scott Bailey1,2, Taekjip Ha1,3. by mass spectrometry(HX/MS) we have identified the Inter-SH2 linker as being 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins 2 significantly different in ZAP-70 compared to Syk. HX/MS experiments University School of Medicine, Baltimore, MD, USA, Bloomberg School of indicate that the Inter-SH2 linker of the Syk tSH2 is very flexible and poten- Public Health, Johns Hopkins University School of Medicine, Baltimore, 3 tially disordered unless bound to a phosphorylated ITAM. This is not the MD, USA, Howard Hughes Medical Institute, Baltimore, MD, USA. case for the ZAP-70 tSH2, which shows protection even in the apo state. These S. pyogenes Cas9 is an RNA-guided DNA endonuclease that has been widely findings suggest that the intrinsic flexibility of these proteins may be the cause used for genome engineering applications, but its specificity remains to be a of some of the observed differences in auto-inhibition, activity, and specificity. challenge. To gain mechanistic insight into the specificity, many single mole- cule approaches, in particular single molecule fluorescence resonance energy 2834-Pos Board B42 transfer (smFRET), have been employed. Previous studies have shown that Using pH Changes to Obtain Time-Resolved Crystallographic Structures the degrees of Cas9-induced DNA unwinding, RNA-DNA heteroduplex exten- of HMG-CoA Reductase sion, and movements of Cas9 domains (REC2, REC3 and HNH) are critical Vatsal Purohit1, Tony Rosales2, Chandra Duncan1, Calvin Steussy1, checkpoints for activating DNA cleavage by Cas9. But in these studies, each Cynthia Stauffacher1. aspect was studied separately using two-color FRET and therefore, only limited 1Biology, Purdue University, West Lafayette, IN, USA, 2Chemistry, information on allosteric communication between them is available. Moreover, University of Notre Dame, South bend, IN, USA. it is likely that Cas9-RNA-DNA complex passes through multiple intermedi- HMG-CoA reductase (HMGR) is a key enzyme in the mevalonate pathway and ates and different parts may move only in semi-coordination. We have is involved in the biosynthesis of cholesterol. It catalyzes the conversion of employed three-color FRET to observe the degree of DNA unwinding and HMG-CoA into Mevalonate using a double-hydride transfer using 2 equiva- HNH domain movement simultaneously and determined how they are related lents of NAD(P). This pathway is also critical for the survival of antibiotic to each other. resistant pathogenic bacteria. Understanding the mechanism of bacterial HMGR can provide us with structural information that can be used to develop 2832-Pos Board B40 novel antibacterials against such bacterial infections. Structural and Kinetic Characterization of High-Affinity Lead(II)-Synap- HMGR from pseudomonas mevalonii, with the help of large solvent channels totagmin I Interactions which provide access to the active site has been shown to be active inside Sachin Katti1, Bin Her1, Atul Srivastava1, Alexander B. Taylor2, crystals and undergo large conformational changes that accompany the reaction P. John Hart2, Steve Lockless3, Tatyana Igumenova1. in the crystal. Therefore, the HMGR in pseudomonas mevalonii is a suitable 1Biochemistry & Biophysics, Texas A&M, College Station, TX, USA, 2 system to conduct time-resolved studies of the reaction, which can help under- Department of Biochemistry and Structural Biology and the X-ray stand its mechanism in pathogenic bacteria. We have obtained diffracting crys- Crystallography Core Laboratory, University of Texas Health Science Center 3 tals of HMGR from P.Mevalonii with Mevalonate, CoA and NAD bound at the at San Antonio, San Antonio, TX, USA, Department of Biology, Texas active site. However, we do not see any reaction taking place inside the crystal. A&M, College Station, TX, USA. The possibility is that the reaction does not proceed in the crystal environment Lead is an environmental toxin that is known to disrupt Ca2þ-mediated signal 2þ at the crystallization buffer pH which is 6.7. A 4-fold increase in activity is seen transduction and neurotransmitter release. Although several Ca sensor pro- at pH 9 in the crystallization buffer solution. Preliminary studies show the for- teins involved in signaling are proposed as the molecular targets of Pb2þ, 2þ mation of HMGCoA, when the pH of the crystal is slowly changed to 9. how they are affected at the atomic level by Pb exposure is not fully under- In this study, using soaking techniques at different pH conditions between stood. Using biophysical techniques such as X-ray crystallography and NMR 2þ 2þ 6.7 - 9 that are suitable for providing a crystal environment where the reac- spectroscopy, we have identified distinct Pb binding sites on the two Ca - tion progresses slow enough to be frozen along different time-points, we aim binding C2 domains (named C2A and C2B) of Synaptotagmin I (SytI), a major 2þ to record x-ray diffraction data along the reaction pathway and observe the Ca sensor of evoked neurotransmitter release. Thermodynamic analysis of formation of the various intermediates and the structural changes that facil- these sites using isothermal titration calorimetry revealed that the affinity of itate their formation in the crystal. Pb2þ for the C2 domains exceeds those of the native ligand Ca2þ by 2-3 orders of magnitude. We used NMR ZZ exchange spectroscopy to determine the 2835-Pos Board B43 kinetics and activation parameters of Pb2þ association with the C2 domains. Role of Export Chaperones in Regulation of Flagellum Assembly We found that although the on-rate constants for Pb2þ and Ca2þ both reflect Nandish K. Khanra, Paolo Rossi, Charalampos Kalodimos. a diffusion-limited binding, the off-rate constant for Pb2þ is two orders of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, magnitude slower than Ca2þ. Furthermore, we observed that the C2 domains USA. complexed with Pb2þ at the high-affinity sites have severely reduced affinity The bacterial flagellum is one of the most sophisticated nano-machines in na- towards Ca2þ, despite the existing coordination vacancies. This observation ture. The majority of flagellum stays outside the cell exterior, therefore, to build provides one of the potential mechanisms by which Pb2þ could disrupt the flagellum bacteria develops a dedicated flagellar export system to transport function of Ca2þ sensor proteins at concentrations several-fold lower than flagellar proteins to the distal growing end of a flagellum. Among the key Ca2þ. Taken together, our atomic level characterization of the SytI-Pb2þ asso- export associated proteins, there are flagellar chaperones which stabilize the ciation provides an insight into the potential modes of Pb2þ toxicity. [Acknowl- export substrates in the cytoplasm, prevents their undesired interaction, and tar- edgement: This research is supported by the funds from Welch Foundation gets them to the export gate. Flagellum assembly is strictly regulated, and grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 export chaperones play crucial roles in regulating export order. FliT, FliS, GM108998.] FlgN are export chaperones for export substrates, FliD, FliC, FlgK, and

BPJ 8720_8723 Wednesday, February 21, 2018 573a

FlgL, respectively. FliJ is a general chaperone that assists and regulates Omega loops are known to play an important role in controlling the structure the export process. FlhA and FlhB constitute the export gate and determines and function of proteins. In this work, we have studied the role of omega the hierarchy of the export process. Despite detailed understanding of the loop mutants of human cytochrome c in controlling its structure and function. morphology of flagellum, flagella export process is poorly understood. We We generated two mutants using site-directed mutagenesis in the omega loop at have determined the solution structure of FliT in its apo form along with its positions 44 and 51 by replacing alanine to cysteine residues. Protein samples complex with FliD, FliI, and FliJ. Solution structure of FliT differs from its purified from the E. coli expressing the wild-type and the mutants were crystal structure and explains the regulatory role of FliT. FliT stays in an compared using in vitro caspase-3 activity assay. Differential scanning calo- auto-inhibitory form in the absence of substrates to avoid any unwanted inter- rimetry data of these proteins suggests that the A44C and A51C mutants are action with export gate. Substrate binding activates FliT-substrate complex to less stable than the wild type protein. Lowering of the Tm for both mutants indi- bind to the export gate. This targeting mechanism of FliT is appeared to be cate a decline in their stability. Additionally, the preliminary results from the in shared among all export chaperones, FlgN and FliS. We further characterized vitro cell free caspase 3 activity assay indicates enhanced caspase activity of the interactions among export chaperones and showed that strict binding A44c compared to wild type. To decipher what structural changes underlying sequence among chaperones determines chaperone recycling process and regu- this altered activity we used isotopic labeling and the use of 2D nuclear mag- late subsequent export events. netic resonance (NMR) spectroscopy, analysis of the structure of A44C. Both1H-15N heteronuclear single quantum correlation (HSQC) and backbone 2836-Pos Board B44 15N relaxation experiments were conducted on the A44C mutant. The resulting Effects of Natural Polymorphisms of non-B HIV-1 Protease on Protein signal data were compared to previously obtained data from the known struc- Conformations ture of wild type protein. Our results indicate a structural alteration in the A44C Trang Tran, Zhanglong Liu, Gail Fanucci. protein which could explain its enhanced apoptotic activity. Chemistry, University of Florida, Gainesville, FL, USA. HIV-1 protease (PR) is essential to the maturation of HIV-1 virus as it post- translationally cleaves the viral polyproteins gag and gag-pol. Inhibition of Posters: Protein Structure, Prediction, and HIV-1 PR lead to non-infectious immature virus. This makes HIV-1 PR a Design II drug target for HIV infection. The flaps of HIV-1 PR play an important role in its catalytic activity as they control access of substrate as well as inhibitor 2839-Pos Board B47 to the catalytic pocket of the protease. The flaps of HIV-1 PR adopt different PredMP: A Web Resource for Computationally Predicted Membrane conformations: closed, semi-open, wide-open, and curled-open [1]. Our previ- Proteins via Deep Learning ous study suggests natural polymorphisms can affect conformational ensembles Sheng Wang1, Shiyang Fei2, Wang Zongan3,YuLi1, Feng Zhao2, Xin Gao1. of the protease [2]. In this study, double electron-electron resonance (DEER) is 1Computational Bioscience Research Center (CBRC), King Abdullah utilized to study how natural polymorphisms affect protein conformational en- University of Science and Technology (KAUST), Thuwal, Saudi Arabia, sembles in non-B HIV-1 PR variants including subtypes D, H, and F, and CRFs 2Prospect Institute of Fatty Acids and Health, Qingdao University, Qingdao, AC, AG, and BF. Most of these variants showed conformational ensembles China, 3Department of Chemistry, University of Chicago, Chicago, IL, USA. similar to that of multidrug resistant variant. Darunavir, the most potent prote- Experimental determination of membrane protein (MP) structures is chal- ase inhibitor to date, was unable to shift flap conformation of CRF_AC to lenging as they are often too large for nuclear magnetic resonance (NMR) closed conformation which is indicative of drug resistance. experiments and difficult to crystallize. Currently there are only about 510 non-redundant MPs with solved structures in Protein Data Bank (PDB). To 2837-Pos Board B45 elucidate the MP structures computationally, we developed a novel web Structural and Mechanistic Insight into how Pathogenic Bacteria resource, denoted as PredMP (http://52.87.130.56:3001/#/proteinindex), that Assemble Adhesive Surface Pili via Isopeptide Bonds delivers one-dimensional (1D) annotation of the membrane topology and sec- Scott A. McConnell. ondary structure, two-dimensional (2D) prediction of the contact/distance map, Chemistry & Biochemistry Dept, University of California, Los Angeles, Los together with three-dimensional (3D) modeling of the MP structure in the lipid Angeles, CA, USA. bilayer, for each MP target from a given model organism. Infections caused by antibiotic-resistant bacterial pathogens are a significant The precision of the computationally constructed MP structures is leveraged by þ threat to public health. Many species of Gram bacteria display pili (hair- state-of-the-art deep learning methods as well as cutting-edge modeling strate- like filaments that protrude from the cell wall) to form biofilms and to adhere gies. In particular, (i) we annotate 1D property via DeepCNF (Deep Convolu- to host tissues and cells. Pili are constructed on the cell surface by sortase pilin tional Neural Fields) that not only models complex sequence-structure polymerases, which link pilin subunits together via isopeptide bonds. Although relationship but also interdependency between adjacent property labels; (ii) pilus assembly has been studied at a cellular level, the mechanism of sortase- we predict 2D contact/distance map through Deep Transfer Learning which catalyzed isopeptide bond formation is poorly understood. Here we report learns the patterns as well as the complex relationship between contacts/dis- the integrated use of x-ray crystallography, NMR, biochemistry, biophysics tances and protein features from non-membrane proteins; and (iii) we model and cellular biology methods to explore the mechanism of assembly of the 3D structure by feeding its predicted contacts and secondary structure to the archetype SpaA-pilus from Corynebacterium diphtheriae. The crystal structure Crystallography & NMR System (CNS) suite combined with a membrane of the pilin polymerase, SrtA, reveals that it is held in an inactive state by an burial potential that is residue-specific and depth-dependent. auto-inhibitory ‘‘lid’’ structure. We demonstrate that targeted mutations intro- PredMP currently contains more than 2,200 multi-pass transmembrane proteins duced into the lid activate the enzyme and enable in vitro pilus assembly. (length<700 residues) from Human. These transmembrane proteins are classi- Phylogenetic comparisons, amino acid mutagenesis, NMR and a novel in vitro fied according to IUPHAR/BPS Guide, which provides a hierarchical organiza- assay define unique enzyme features required for isopeptide polymerization ac- tion of receptors, channels, transporters, enzymes and other drug targets tivity and provide insight into the structure of the reaction intermediate that is according to their molecular relationships and physiological functions. Among formed during repeated polymerization cycles. Finally, we present progress to- these MPs, we estimated that our approach could predict correct folds for ward determining the solution structure of the basal SpaB pilin subunit, which 1,345-1,871 targets including a few hundred new folds, which shall facilitate is required for efficient cell wall anchoring of the completed pilus polymer. The the discovery of drugs targeting at MPs. structure and mechanistic studies of SpaB incorporation into the completed pilus provide insight into the mechanism of termination. The pilus assembly re- 2840-Pos Board B48 action studied here is highly conserved and can be important for bacterial viru- Homology Modelling of Sodium Iodide Symporter lence, thus the results of this work are broadly applicable and may facilitate the Hristina R. Zhekova1, Igor Zdravkovic1, Sergei Yu Noskov1, design of pilus assembly inhibitors with useful therapeutic properties. Toshie Sakuma2, Susanna C. Concilio3, Ryan Johnson2, Stephen J. Russell3, Kah-Whye Peng2. 2838-Pos Board B46 1Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Imanis Solution NMR Study of a Cytochrome C Mutant(A44C) with Enhanced Life Sciences, Rochester, MN, USA, 3Department of Molecular Medicine, Apoptotic Activity Mayo Clinic, Rochester, MN, USA. Manoj Saxena1,2, Johnathan Dallman1,3, Ana B. Castaner1,3, Sodium Iodide Symporter (NIS) is a protein involved in the transport of iodide, Marvin J. Bayro1,3, Kai Griebenow1. necessary for synthesis of the thyroid hormones, through the cellular membrane 1Chemistry, University of Puerto Rico Rio Piedras, San Juan, PR, USA, of the thyrocites. It can be found in a number of other tissues (e.g. lactating 2Chemistry, Molecular Science Research Center, San Juan, Puerto Rico, breast, stomach, testicles) where its function is not fully understood. Impor- 3Molecular Science Research Center, San Juan, Puerto Rico. tantly, it is expressed in various cancers and this opens the possibility for

BPJ 8720_8723 574a Wednesday, February 21, 2018 practical application of NIS in cancer imaging and treatment through transport porates native contact predictions in a probabilistic manner. We show that of 125I- and other radioactive isotopes. Development of NIS mutants which MELD successfully folds a number of nonthreadable proteins. could translocate efficiently the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization 2843-Pos Board B51 of NIS and its relation to the anion transport. There are no known crystal struc- Efficient Sampling for the Prediction of Long and Multidomain Protein tures of NIS in any of its conformational states. Instead, the structural underpin- Structures 1 1 1 2 nings of NIS transport have been probed with mutagenesis studies and through Clare E. West , Saulo H.P. de Oliveira , Eleanor C. Law , Sebastian Kelm , 2 1 parallels with other similar transporters. Homology modelling can be very Jiye Shi , Charlotte M. Deane . 1Department of Statistics, University of Oxford, Oxford, United Kingdom, effective in such situations, however the sequence identity between NIS and 2 the relevant secondary transporters with available experimental structures is Department of Informatics, UCB Pharma, Slough, United Kingdom. very low, which makes the choice of a template and the validation of sequence We aim to improve the prediction of large and multidomain protein structures, alignments and generated 3D models non-trivial. which are biologically crucial but experimentally difficult to determine. There Here we report a homology model of NIS in its occluded state, based on 6 is experimental evidence that protein folding can occur via an ordered process different templates (LeuT, vSGLT, Mhp1, AdiC, BetP and dDAT). Our model of foldon-determined steps, in which small units (foldons) fold sequentially and was validated on the basis of important mutations occurring in all 6 templates guide the folding of subsequent units. Inspired by this folding pathway, we and additional proteins found in human cells (hSGLT, hSERT, hDAT). Some divide protein structures into two smaller, semi-stable segments, and predict important binding and gating residues, which emerged from the 3D structure of these individually in succession. Preliminary results indicate that individual the model and from preliminary molecular dynamics simulations and free en- ‘foldons’ can be predicted without the presence of the rest of the protein, and ergy calculations of ion-protein binding, were identified and subjected to that building the second foldon from a correct model of the first enables the pre- further mutagenesis and anion uptake tests. diction of previously intractable targets. 2844-Pos Board B52 2841-Pos Board B49 Novel Coarse-Graining Approaches for Large Scale Protein Modeling De Novo Protein Structure Prediction by Combining Replica Exchange Aleksandra E. Dawid, Andrzej Kolinski, Dominik Gront. Simulations with Coevolutionary Data Faculty of Chemistry, University of Warsaw, Warsaw, Poland. Arthur Voronin1, Alexander Schug2,3. 1 Coarse-grained modeling of biomolecules plays very important role in molec- Physics Department, Karlsruhe Institute of Technology, Karlsruhe, 1 2 ular biology. Realistic coarse-grained protein models, assuming a small num- Germany, Steinbuch Centre for Computing, Karlsruhe Institute of ber (2-4) of pseudo-atoms (or ‘‘united atoms’’) representing single amino acid Technology, Karlsruhe, Germany, 3John von Neumann Institute for € € units, proven to be very productive. Depending on structure representation, Computing, Julich Supercomputer Centre, Julich, Germany. force field definition, sampling schemes and some programming details popular Proteins perform important tasks in every living organism and are an essential coarse-grained simulation schemes are about 3 orders of magnitude faster than part of life. Studying the structure of proteins helps to understand certain inter- classical molecular dynamics. Such speed-up of simulations is still insufficient actions in a biological system and can be applied to other fields, like improving to solve many of real problems, for instance to make structure prediction of not drug design. Despite incredible progress in experimental techniques, protein too small protein by modeling its folding process. Qualitatively different ap- structure determination is arduous. Here, we suggest a complementary new proaches seem be necessary. SURPASS (Single United Residue per Pre- method of de novo protein structure prediction. Direct coupling analysis Averaged Secondary Structure fragment) model2 of proteins presented here (DCA) quantifies coevolution of amino acid pairs in large sequence alignments, surpasses some limitations of coarse-grained models, enabling efficient where high scoring pairs can be interpreted as spatially adjacent. Regular mo- modeling of long time dynamics and structures of large proteins and (after sim- lecular dynamics simulations are computationally too costly to identify the ple extensions) multi-domain proteins and protein complexes. The model as- native conformation in straightforward simulations. One reason is entrapment sumes single center of interaction per residue, accounting for pre-averaged in one of the many local minima. By integrating DCA-derived contacts as con- effects of four adjacent residues. Knowledge-based statistical potentials encode straints into molecular simulations we smoothen the energy landscape and complex interactions of these fragments. The SURPASS approach can be guide structure prediction. Additionally, any residual entrapment will be over- incorporated into efficient multiscale molecular modeling of large systems. come by the technique of replica exchange. With this combination of tech- At the second stage of modelling a high-resolution coarse-grained model is niques, it should be possible to predict the native structure de novo, i.e. used to optimize3 low-resolution structures. Finally, an all-atom stage is em- without prior knowledge of structural elements, in a single simulation run. ployed for final model refinement and evaluation. To study our methods performance we investigate proteins of different com- 1 Coarse-Grained Protein Models and Their Applications Kmiecik, S; Gront, plexities by using various numbers and quality of constraints. D; Kolinski, M; et al. Chem. Rev. 116 7898-7936 2016 2 submitted 2842-Pos Board B50 3 Optimization of protein models Gront, D et al. WIREs Computational Molec- Meld Folds Nonthreadable Proteins ular Science 2 479-493 2012 James Robertson, Alberto Perez, Ken Dill. Laufer Center for Physical and Quantitative Biology, Stony Brook 2845-Pos Board B53 University, Stony Brook, NY, USA. Compensatory Mutations in Protein Sequences from Big-Data Protein structure prediction is important because not all protein structures are Kejue Jia, Robert L. Jernigan. easily determined from experiment and being able to accurately and reliably Biochemistry, Biophysics and Molecular Biology, Iowa State University, predict structure from sequence allows for understanding structure-function re- Ames, IA, USA. lationships and provides structures for structure-based drug design. The state of Proteins are the central players in biology, and a deeper understanding of their the art in protein structure prediction is to use bioinformatics-based methods behaviors will facilitate the more meaningful interpretation of genome data. such as threading. This method is suitable for approximately 85% of the human Rapid progress in genome sequencing has already provided hundreds of mil- proteome, but poses a problem for the remaining 15% of proteins that remain lions of protein sequences, and similar advances in structural biology now pro- inaccessible, or ‘‘nonthreadable’’. Bioinformatics algorithms fail to predict vide over 100,000 protein structures, which are deemed to be a nearly complete nonthreadable proteins because the required fragment structures are missing set of characteristic structures (folds). The present work utilizes the Big Data of from the databases that threading relies upon to build an accurate protein protein sequences and structures, which have grown rapidly, to develop a more model. When databases lack the necessary information to solve the problem, reliable way to link between sequences and phenomes. Previously statistical methods that rely on databases have no way to come up with a correct solution. methods were developed that infer correlation signals from protein multiple An alternative approach is needed to solve nonthreadable protein structures. A sequence alignments, and these were applied successfully to protein structure physics-based (and database-free) method using atomistic molecular dynamics prediction. However these methods still suffer from high false-positive rates. simulations should accurately predict these structures. Additionally, physics- The indirect correlations and the direct correlations mixed together, plus the based methods are advantageous because, in addition to providing native state noise in the raw data, make it difficult to remove false-positive results. By care- protein structures, they provide dynamics and folding pathways; the simula- fully combining the information from 84,287 protein structures and 22,346,130 tions also obey Boltzmann’s law and provide populations, thus giving access sequences from 7,990 protein folds, our approach greatly improves the accu- to folding free energies, which can be used to further refine our physics- racy of selecting the true residue correlations, from which the patterns of based approach. Physics-based atomistic simulations have been limited by compensatory mutations are extracted the patterns of residue interactions re- the computational expense of folding proteins, but MELD greatly accelerates vealed. Many of the amino acid substitutions are unexpected when compared folding simulations by using replica exchange molecular dynamics that incor- to the substitutions from the BLOSUM series of substitutions. We derived a

BPJ 8720_8723 Wednesday, February 21, 2018 575a new substitution matrix that includes the compensatory mutations. Compari- guided iterative Rosetta-MD protocol which shows great promise in the refine- sons with results from BLOSUM62, demonstrate a significant gains in ment of membrane protein structures. The best models are identified based on a sequence matching, significantly increasing sequence similarity and reducing combination of their score and fit-to-density during the Rosetta model selec- gaps. The matrix is also able to align sequences of 269 similar structures that tion. By doing so, we have been able to pick superior models to those where did not have similar sequences with the use of BLOSUM62. These are major selection was based on Rosetta score only. The method was tested with five gains in sequence matching across diverse proteins. The outcome enables a membrane spanning protein structures. By applying density-guided Rosetta- significantly more reliable distinction between similar/different sequences MD iteratively we were able to refine the predicted structures of these mem- and will enhance identifying links between sequences and phenomes. brane proteins to atomic resolutions. We also showed that the resolution of the density maps determines the improvement and quality of the refined 2846-Pos Board B54 models. By incorporating high resolution density maps ( 4A˚ ) we were able Protein Structure Refinement via Molecular Dynamics Simulations to more significantly improve the quality of the models than when medium res- Michael Feig, Lim Heo. olution maps (6.9 A˚ ) were used. Beginning from an average starting structure Biochemistry & Mol. Biol., Michigan State University, East Lansing, MI, root mean square deviation (RMSD) to native of 4.66 A˚ , our protocol was able USA. to refine the structures and bring the average refined structure RMSD to 1.66 A˚ Computational protein structure prediction has long been an important alterna- when 4 A˚ density maps were used. The protocol also successfully refined the tive to experimental structure determination. Comparative modeling ap- HIV-1 CTD guided by an experimental 5 A˚ density map. proaches can often generate approximate models but generating highly accurate models that are comparable to experimental structures has remained 2849-Pos Board B57 a significant challenge. Refinement methods via molecular dynamics simula- Computational Design of High-Resolution Protein Crystals tions are an avenue for bridging this gap. MD-based protocols that provide Jeliazko R. Jeliazkov1, Aaron C. Robinson2, James M. Berger3, consistent but still modest improvement of initial models are presented based Bertrand Garcı´a-Moreno E.2, Jeffrey J. Gray4. on results from recent rounds of CASP. Furthermore, new detailed analysis 1Program in Molecular Biophysics, Johns Hopkins University, Baltimore, of microsecond simulations combined with Markov state modeling was used MD, USA, 2T. C. Jenkins Department of Biophysics, Johns Hopkins to construct energy landscapes that connect initial homology models to native University, Baltimore, MD, USA, 3Department of Biophysics and structures. These maps provide insight into the key kinetic barriers and ener- Biophysical Chemistry, Johns Hopkins University School of Medicine, getics along transition pathways from homology models towards the native Baltimore, MD, USA, 4Department of Chemical and Biomolecular state and inform the design of more effective structure refinement protocols Engineering, Johns Hopkins University, Baltimore, MD, USA. that can truly reach the native state from initial comparative models. X-ray crystallography can reveal intricate details that underlie protein function. 2847-Pos Board B55 However, the information contained in a crystal structure is dependent on its From Single Structures to Ensembles: Application of the Galaxy Program resolution: high-resolution structures can reveal individual atoms whereas Suite to Ubiquitin, Cyclophilin a and PTP1B low-resolution structures may not show side chains or small-molecule ligands Gyu Rie Lee1, Chaok Seok1, Matthias Buck2. in their entirety. The inability of crystal structures to resolve these crucial ele- 1Seoul National University, Seoul, Republic of Korea, 2Case Western ments limits our understanding of catalytic mechanisms, protein-protein inter- Reserve University, Cleveland, OH, USA. actions, and drug-protein interactions. Using Rosetta, we have developed a It is now widely accepted that an accurate representation of a protein structure computational design method to stabilize low-energy interfaces from low- should incorporate a description of internal as well as loop protein dynamics. resolution protein crystals and subsequently enhance resolution. By studying Previously has been suggested that protein structures can be refined by NMR existing crystal structures and past attempts at rational protein-crystal design, derived parameters, including them as an additional restraint in the refinement we found that one key determinant of resolution that we could design was pro- protocol [1]. More recently it has become clear that the amplitude of local mo- tein crystal contact interaction strength. We tested six protein design strategies tions can be predicted with considerable accuracy by considering the packing on published rational design results to identify the optimal approach. We found environment they may provide. Nevertheless, the prediction of loop motions, that design strategies with large backbone or rigid-body motions yielded a if not distinct loop conformations has remained a challenge. lower success rates (5-12%), while design strategies with only side-chain re- We apply here the Galaxy Suite of Programs (GalaxyLoop and GalaxyVoyage) packing followed by minimization yielded moderate success rates (36-50%). [2] to several test-cases: Ubiquitin, which has been extensively characterized We applied our design strategy to a model protein (Staphylococcal nuclease) by NMR spectroscopy well as by molecular dynamics simulations (e.g. and experimentally characterized our designed variants. We attempted to crys- [3,4]). Ubiquitin motions emphasized small displacements, whereas Cyclophi- tallize our variants with the standard hanging-drop vapor diffusion technique, lin A [5] and Protein Tyrosine Phosphatase 1B [6], both have loops near their using wildtype-like conditions for the reservoir solution. We found that half catalytic sites which undergo extensive motions; interestingly, these motions of the variants crystallized and the rest yielded amorphous aggregate. We that are communicated through a significant part of the structure. These exam- will present results from determining the crystal structures and resolution. In ples suggests that the implementation of ensemble refinement options into the general, our method can be applied to any protein target with a pre-existing Galaxy Suite of Programs should be successful. low-resolution crystal structure. References: [1] Lindorff-Larsen K, Best RB, Depristo MA, Dobson CM, Ven- druscolo M. Nature. 2005 433:128-32. [2] Park H, Lee GR, Heo L, Seok C. 2850-Pos Board B58 PLoS ONE. 2014 9: e113811. [3] Lange OF, Lakomek NA, Fare`s et al.,. Sci- Relative Contribution of the Refinement Steps to the Protein-Protein ence. 2008 320:1471-5. [4] Shaw DE, Maragakis P, Lindorff-Larsen K, et Docking Success Rate al.,. Science. 2010 330:341-6. [5] Chi CN, Vo¨geli B, Bibow S, Strotz D, Orts Taras Dauzhenka, Ivan Anishchenko, Petras J. Kundrotas, Ilya A. Vakser. J, Guntert€ P, Riek R. (2015) Angew Chem Int Ed Engl. 54:11657-61. [6] Center for Computational Biology, University of Kansas, Lawrence, KS, Choy MS, Li Y, Machado LE, Kunze MB, Connors CR, Wei X, Lindorff- USA. Larsen K, Page R, Peti W (2017) Mol Cell. 65:644-658. Structural modeling of protein-protein complexes is a challenging task as pro- teins may undergo significant conformational changes upon binding. Flexible 2848-Pos Board B56 docking of proteins typically consists of low-resolution rigid-body global dock- Iterative Molecular DynamicsRosetta Membrane Protein Structure ing search, followed by the structural refinement that takes into account confor- Refinement Guided by Cryo-EM Densities mational flexibility. The refinement typically involves consecutive steps Sumudu Leelananda, Steffen Lindert. associated with different weakly coupled sets of degrees of freedom (DOF). Ohio State University, Columbus, OH, USA. Previously, we developed a docking refinement pipeline consisting of three Knowing atomistic details of proteins is essential for the understanding of steps: (i) rigid-body adjustment of the protein position, which minimizes a protein function and drug development. Experimental methods such as X-ray semi-empirical atom-atom contact potential (rigid-body rotational/translational crystallography, NMR and cryo-EM are the preferred form of protein structure DOF), (ii) conformer selection procedure applied to the backbone loops at the determination. Computational methods may be an alternative when experi- interface, to maximize the Ramachandran probability (backbone loops mental techniques fail. However, computational methods are limited when it torsional DOF), and (iii) repacking of the interface side chains using a library comes to predicting large macromolecule structures with little sequence simi- of rotamers, to minimize the steric clashes (side-chain torsional DOF). In this larity to known structures. The incorporation of experimental restraints in study, we investigate relative contributions of these steps to the refinement suc- computational methods is becoming increasingly important to more reliably cess, in terms of the fraction of the native contacts, ligand RMSD, and interface predict protein structure. One such experimental input used in structure predic- RMSD, for bound and unbound proteins in the Dockground X-ray docking tion/refinement is cryo-EM densities. We developed a novel cryo-EM density- benchmark set 4.0 (http://dockground.compbio.ku.edu).

BPJ 8720_8723 576a Wednesday, February 21, 2018

2851-Pos Board B59 a thorough comparative analysis of FSA and PSA docking methodologies Structure Modeling of Disordered Protein Interactions based on the docking predictions for bound and unbound proteins from the Lenna X. Peterson1, Amitava Roy1,2, Charles Christoffer3, Genki Terashi1, Dockground benchmark set 4 (http://dockground.compbio.ku.edu). Using the Daisuke Kihara4. unbound structures in docking (i.e. accounting for conformational changes 1Biological Sciences, Purdue University, West Lafayette, IN, USA, upon binding) decreases the docking performance only slightly for both 2Bioinformatics and Computational Biosciences Branch, Rocky Mountain methods, compared to the docking of the bound structures (by 0.3% in top Laboratories, Montana, CO, USA, 3Computer Sci, Purdue University, West 10 predictions). FSA selects better templates for proteins with different func- Lafayette, IN, USA, 4Biological Sciences/Computer Sci, Purdue University, tion, whereas PSA provides better templates for multi-domain proteins in West Lafayette, IN, USA. which only one domain participates in the interaction (e.g., enzyme- Disordered protein-protein interactions (PPIs), those involving a folded protein inhibitor). Template structures that have matching CATH annotations with and an intrinsically disordered protein (IDP), are prevalent in the cell, including the target yield better docking performance (the success rate 65% and 67% important signaling and regulatory pathways. IDPs do not adopt a single domi- in top 100, for FSA and PSA, respectively, as opposed to 44% and 46% without nant structure in isolation but often become ordered upon binding. To aid un- the CATH annotation). Significant improvement in the unbound docking suc- derstanding of the molecular mechanisms of disordered PPIs, it is crucial to cess rate (from 30 to 60% in top 10) is observed when the same template obtain the tertiary structure of the PPIs. However, experimental methods is selected by both PSA and FSA. By adding templates identified by sophisti- have difficulty in solving disordered PPIs and existing protein-protein and cated sequence/evolutionary based methods, we were able to build near-native protein-peptide docking methods are not able to model them. Here we present models for 90% of complexes in our target set. a novel computational method, IDP-LZerD, which models the conformation of a disordered PPI by considering the biophysical binding mechanism of an IDP 2854-Pos Board B62 to a structured protein, whereby a local segment of the IDP initiates the inter- Predicting the Effect of Mutations in the KRas/c-Raf-RBD Protein-Protein action and subsequently the remaining IDP regions explore and coalesce Interface Anna Lowegard1,2, Marcel Frenkel3, Bruce Donald2,3. around the initial binding site. On a dataset of 22 disordered PPIs with IDPs 1 up to 69 amino acids, successful predictions were made for 21 bound and 18 Program in Computational Biology & Bioinformatics, Duke University, Durham, NC, USA, 2Department of Computer Science, Duke University, unbound receptors. The successful modeling provides additional support for 3 biophysical principles. Moreover, the new technique significantly expands Durham, NC, USA, Department of Biochemistry, Duke University, Durham, the capability of protein structure modeling and provides crucial insights into NC, USA. the molecular mechanisms of disordered PPIs. This work was published on KRas is a small GTPase commonly implicated in several difficult-to-treat can- PLoS Computational Biology, 13: e1005485, 2017. cers such as pancreatic ductal adenocarcinoma (PDAC). KRas normally cycles between an active, GTP-bound form and an inactive, GDP-bound form. Active 2852-Pos Board B60 KRas functions by forming protein-protein interactions (PPIs) with multiple A Novel Set of Quantitative Rules for Biological or Biochemical Self- effector proteins in order to regulate various important signal transduction path- Assembly in Water ways. However, when KRas is mutated it is constitutively active which leads to Xian Cheng, Irina Shkel. signal transduction pathway dysregulation that subsequently increases and sus- UW-Madison, Madison, WI, USA. tains tumorigenicity and invasiveness. KRas has long been considered an ‘‘un- Quantitative information about amide interactions in water is needed to under- druggable’’ target due to its picomolar affinity for its substrate. However, stand their contributions to protein folding and amide effects on aqueous pro- blocking the PPIs between KRas and its effectors eliminates harmful down- cesses, and to compare with computer simulations. Here we report free energy stream effects. The tightest known binder of KRas is c-Raf, an enzyme in derivatives (dm2/dm3 = m23) quantifying preferential interactions of urea, alky- the ERK1/2 pathway. The Ras-binding domain (RBD) is the minimal binding lated ureas and other amides with one another by osmometry and interactions domain of c-Raf that selectively binds to active, GTP-bound KRas. Previous with nucleobases and other aromatics by solubility. Using an established addi- work has measured Kd for various mutations in the KRas/c-Raf-RBD interface tive analysis based on water-accessible surface areas (ASA), these m23-values [1, 2]. We use OSPREY [3] (Open Source Protein REdesign for You), a state- are interpreted to obtain contributions from interactions of each alkylurea of-the-art software package for computational structure-based protein design and its unified atoms (amide sp2O, sp2N, sp2C; aliphatic sp3C) with the unified (CSPD), along with K* [4], an algorithm that estimates the binding constant atoms of amide compounds, nucleobases and aromatic hydrocarbons (amide for a given protein complex, to computationally predict the effect of these mu- sp2O, sp2N, sp2C and aliphatic sp3C atoms of amides; carbonyl sp2O, ring tations. We compared our computational predictions to the experimental mea- sp2C and sp2N, amino sp3N and methyl sp3C of nucleobases). Strengths of surements and found that we can accurately predict the effect of these interaction per unit ASA of one or both partners (a-values) demonstrate both mutations. These results validate the accuracy of CSPD with OSPREY to target chemical and biophysical significance. Values of m23 for all interactions inves- protein-protein interfaces and give us confidence that we can accurately tigated in the large training sets are well predicted using these smaller sets of a- redesign the KRas/c-Raf-RBD interface for future work towards targeting ‘‘un- values and ASA information, validating the hypothesis of additivity and the use druggable’’ proteins. of ASA. [1] M. Fridman, et al. Journal of Biological Chemistry, 275(39):30363-30371, We quantify for the first time the favorable preferential interactions (per unit of 2000. ASA) of sp2N and sp3N with sp2O (hydrogen bonds), of sp2C and sp3C with [2] C. Kiel, et al. Journal of Biological Chemistry, 284(46):31893-31902, 2009. other sp2C and sp3C (the hydrophobic effect and/or CH-p and p-p interac- [3] P. Gainza, et al. Methods in enzymology, 523:87-107, 2013. tions), of sp2O with sp2C (n-p* interaction) and favorable sp3C-sp2N/sp3N in- [4] I. Georgiev, et al. Journal of computational chemistry, 29(10):1527-1542, teractions, as well as unfavorable sp2O-sp2O, sp2O-sp3C, and sp2N/sp3N -sp2C/ 2008. sp2N/sp3N interactions. The success of this analysis demonstrates that strengths of atom-atom interactions increase in proportion to the product of accessible 2855-Pos Board B63 surface areas of the two atoms. The method developed in my research provides Small Angle X-ray Scattering for Data-Assisted Structure Prediction in a novel set of quantitative rules for biological/biochemical self-assembly in CASP12 with Prospects to Improve Accuracy water. Tadeusz L. Ogorzalek1, Greg L. Hura1, Andriy Kryshtafovych2, John A. Tainer3, Krzysztof Fidelis2, Susan E. Tsutakawa1. 2853-Pos Board B61 1Lawrence Berkeley National Laboratory, Berkeley, CA, USA, 2Protein Comparison of Full and Interface Structure Alignment in Template-Based Structure Prediction Center, Genome and Biomedical Sciences Facilities, Protein Docking University of California, Davis, Davis, CA, USA, 3Department of Molecular Devlina Chakravarty. and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center for Computational Biology, University of Kansas, Lawrence, KS, Center, Houston, TX, USA. USA. Experimental data offers empowering constraints for structure prediction. Template-based protein docking, based on sequence/structure similarity to co- These constraints can be used to filter equivalently scored models or more crystalyzed complexes complements ab initio docking in predicting the struc- powerfully within optimization functions toward prediction. In CASP12, Small ture of protein-protein complexes. When highly homologous templates for the Angle X-ray Scattering (SAXS) data measured on an exemplary set of novel entire target complex are not available, docking by interface or partial structure fold targets, were provided to the CASP community of protein structure predic- alignment (PSA) may provide an adequate solution. Earlier studies indicated tors. As high throughput, solution-based techniques, SAXS can efficiently mea- that PSA docking can also be used to further refine docking models generated sure structural information on a protein in its native solution conformation and by the full structure alignment (FSA) of the target and the template. We present assembly. Here, we consider the SAXS data collected, the information in these

BPJ 8720_8723 Wednesday, February 21, 2018 577a solution measurements, and the challenges in incorporating them into compu- folding distributions were observed. However, multiple peaks were seen in tational prediction. As improvement opportunities were only partly realized in the unfolding force distribution, consistent with multiple parallel unfolding CASP12, we provide guidance on how data from the full-length biological unit pathways or multiple folded states. We then performed long duration fixed and the solution state can better aid prediction of the folded monomer or sub- trap measurements while slowly stepping the applied force. We were thereby unit. We furthermore describe strategic integrations of solution measurements able to directly measure folding and unfolding rate constants versus force un- with computational prediction programs with the aim of substantially der near equilibrium conditions. All molecules showed the same folding improving foundational knowledge and the accuracy of computational algo- behavior, consistent with a simple two-state Bell’s formula. However, these rithms for biologically-relevant structure predictions for proteins in solution. same molecules showed one of two unfolding behaviors at low forces, either ‘fast’ and force independent, or ‘slow’ and strongly force dependent. Equilib- 2856-Pos Board B64 rium fixed trap observations of individual molecules for over tens of minutes Mimicking Microbial Rhodopsin Isomerization show no switching between unfolding mechanisms amid hundreds of transi- Alireza Ghanbarpour, Muath Nairat, Meisam Nosrati, Elizabeth Santos, tions. However, both unfolding mechanisms are observed for the same indi- Chrysoula Vasileiou, Babak Borhan, James Geiger. vidual molecules using the force ramp method, indicating that out of Chemistry, Michigan State University, East Lansing, MI, USA. equilibrium pulling may be necessary to switch individual molecule unfold- Photoreceptor proteins, like microbial rhodopsins, play crucial roles in sensing ing behavior. Taken together, these measurements indicate that protein G and responding to light. The microbial rhodopsins all contain a retinylidene unfolds on a more complex energy landscape containing multiple pathways chromophore bound via Schiff base to a lysine.1 Upon absorption of light, and intermediate states. retinal isomerizes from all-trans to 13-cis, driving a protein conformational change; then the all-trans retinal is rapidly and thermally regenerated at the 2859-Pos Board B67 end of the photocycle. The rhodopsin must: channel the photo-isomerization Mechanical Unfolding and Folding of a Slipknot Protein Observed by to the C13-C14 double bond exclusively; overcome the energy barrier for using Optical Tweezers photo-isomerization; and induce the rapid, thermal return to its original all Han Wang1, Chengzhi He1,2, Chunguang Hu2, Hongbin Li1. trans form.1,2 Research investigating this isomerization pathway has involved 1Chemistry, University of British Columbia, Vancouver, BC, Canada, either chemical modification or mutational studies. An alternative approach 2Tianjin University, Tianjin, China. would be to engineer a system from scratch that recapitulates all of the func- Slipknot is one of the unique structures found in some proteins, and how tions of a microbial rhodopsin, which would result in a much deeper under- such proteins overcome the topological barrier to fold remains unclear. standing of the protein/chromophore interactions required to make a To understand the mechanical unfolding and folding mechanisms of slip- rhodopsin a reality. Herein we report just such a system using human Cellular knotted proteins, here we used a combination of optical tweezers and Retinoic Acid Binding Protein II (hCRABPII) as a template. In this system, the steered molecular dynamics (SMD) simulation to investigate the mechani- photo-isomerization of retinal from all-trans to 13-cis 15-syn was demonstrated cal unfolding and refolding processes of a model slipknot protein, both in solution and in protein crystals, where atomic resolution structures after Pyruvoyl-dependent Arginine Decarboxylase (PADC). Our results reveal photo-isomerization showed both quantitative and specific photo-isomerization that upon stretching, the slipknot protein can be successfully untied into of the 13- bond and subsequent thermal relaxation exclusively to the all-trans a linear polypeptide via multiple unfolding pathways, including two-state isomer, fully recapitulating the microbial rhodopsin photocycle. In addition, the pathway and multi-state pathways involving various intermediates. SMD crystal structure of a 13-cis retinal-bound protein complex was determined at simulations revealed unfolding pathways that are similar to that observed atomic resolution for the first time, showing how proteins can stabilize this experimentally, and provided a possible mechanistic explanation of the un- isomeric form of retinal. folding mechanism of PADC. Upon relaxation of the stretching force, the 1-Ernst, O. et al. Chemical Reviews 2014, 114, 126. unfolded slipknot can refold back to its native state. Distinct refolding 2-Sudo, Y. et al., Journal of Biological Chemistry 2011, 286, 5967. pathways were observed, suggesting the complexity in the refolding pro- cess of such unique structures. Our results demonstrate the unique utility of optical tweezers in revealing the folding and unfolding mechanism of Posters: Protein Stability, Folding, and slipknotted proteins. Chaperones III 2860-Pos Board B68 2857-Pos Board B65 An Evolutionary Trend towards Kinetic Stability in the Folding Trajec- Structural-Elastic Determination of the Lifetime of Biomolecules Under tory of Ribonucleases H 1 2 3 4 Force Shion A. Lim , Eric R. Bolin , Kathryn M. Hart , Michael J. Harms , Susan Marqusee1. Shiwen Guo, Jie Yan. 1 National University of Singapore, Singapore, Singapore. Molecular & Cell Biology, University of California Berkeley, Berkeley, CA, USA, 2Biophysics, University of California Berkeley, Berkeley, CA, USA, Several recent experiments have suggested that the structural-elastic properties 3 4 of the native and the transition states of biomolecules are a key determinant of Chemistry, Williams College, Williamstown, MA, USA, Chemistry and their mechanical stability. However, most of the current theoretical models Biochemistry, University of Oregon, Eugene, OR, USA. were not derived based on the structural-elastic properties of the molecules. Proper folding of proteins is critical for producing the biological machinery Here, based on the Arrhenius law and taking into consideration of the essential for cellular function. Over the course of evolution, the rates and en- structural-elastic properties of the native state and the transition state, we ergetics of a protein’s folding landscape must be maintained such that the pro- derived a simple analytical expression for the force-dependent lifetime of the tein folds and remains folded throughout its biological lifetime. Developing a native state of the molecules. We show that this model is able to fit a wide scope comprehensive understanding of how a protein’s folding process is modulated of experiments, and explain a variety of complex force-dependent transition ki- during evolution is critical to our understanding and engineering of protein netics observed in recent experiments. The results highlight a previously biophysical properties. In this study, we characterized the folding trajectories largely unrecognized structural-elastic determinant of the lifetime of biomole- of ancestral proteins of the ribonuclease H (RNase H) family by using ances- cules under force, and provide a new theoretical framework that can inform us tral sequence reconstruction to access the evolutionary history between the structural-elastic properties of the molecules. RNases H from mesophilic and thermophilic bacteria. We find that the overall folding pathway of RNase H is preserved over billions of years of evolution. 2858-Pos Board B66 Although thermodynamic stabilities diverge between the mesophilic and ther- Equilibrium and Non-equilibrium Studies of Protein G using High- mophilic lineages, kinetic stability increases along both, with the last common Resolution Optical Trapping Unveil Heterogeneous Unfolding Pathways ancestor folding and unfolding faster than the modern descendants. The Yujie Chen, Dena Izadi, Miles L. Whitmore, Joseph D. Slivka, conserved folding intermediate permits this paradoxical uncoupling of ther- Lisa J. Lapidus, Matthew J. Comstock. modynamics and kinetics, and allows for the folding landscape to indepen- Physics and Astronomy, Michigan State University, East Lansing, MI, USA. dently respond to different selective pressures on global stability and Recent advances in spatial and temporal resolution of biophysical measure- kinetic barriers. Additionally, further characterization of the RNase H folding ments have revealed ‘beyond two-state’ complexity in protein folding, pathway by fragment models and hydrogen-exchange mass spectrometry even for small, single-domain proteins. In this work, the folding/unfolding (HX-MS) reveals distinct changes in folding pathway for the RNase H family kinetics of the B1 domain of inmmunoglobulin-binding protein G, a well- in the mesophilic and themophilic lineages, highlighting how subtle sequence studied model system, was investigated using high-resolution optical traps. changes can alter the structure of partially folded intermediates over the For non-equilibrium force ramp measurements, simple force-dependent course of evolution.

BPJ 8720_8723 578a Wednesday, February 21, 2018

2861-Pos Board B69 2864-Pos Board B72 Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Examination of the Effect of a Histidine Tag on the Energy Landscape of Destabilization of Collagen’s Triple Helix ACBP Michael W.H. Kirkness1, Nancy R. Forde2. Jamie Stankiewiz. 1Department of Molecular Biology and Biochemistry, Simon Fraser University of California, Berkeley, Berkeley, CA, USA. University, Burnaby, BC, Canada, 2Department of Physics, Simon Fraser Proteins have complex energy landscapes that can be altered by perturbations like University, Burnaby, BC, Canada. force, mutation, or changes in environment. Exploring these landscapes gives us Force plays a key role in regulating dynamics of biomolecular structure and in- insight to the folding and stability of a given protein in different environmental teractions, yet techniques are lacking to manipulate and continuously read out conditions. A 6 Histidine residue tag, is often attached to the end of a protein, this response with high throughput. We present an enzymatic assay for force- strictly for use in protein purification. It is interesting and necessary to determine dependent accessibility of structure that makes use of a wireless Mini-Radio if the His-tag has an effect on protein folding and stability. Experiments with Acyl Centrifuge Force Microscope (MR.CFM) to provide a real-time readout of ki- CoA binding protein (ACBP) reveal that effects of chemical denaturation, muta- netics. The microscope is designed for ease of use, fits in a standard centrifuge tion, and force can depend on the presence of a His-tag. To characterize how the bucket, and offers high-throughput, video-rate readout of individual proteolytic His-tag affects the protein energy landscape, we study the effects of mutations on cleavage events. Proteolysis measurements on thousands of tethered collagen ACBP stability and folding kinetics with and without a His-tag. molecules were used to determine how the triple helix responds to force. As the primary load- and tension-bearing protein in vertebrates, the physical 2865-Pos Board B73 properties of collagen are of significant biomedical interest. How collagen’s tri- Knob-Socket Predictions of Alpha-Helical Stability ple helix responds to applied force is controversial, with different studies infer- Taylor R. Rabara, Joshman Singh, Danielle MacArt, Shivarni Patel, ring incompatible outcomes: overwinding, unwinding, shearing, or maintaining Hyun Joo, Jerry Tsai. its zero-force structure. Because proteolytic cleavage requires a locally unwound University of the Pacific, Stockton, CA, USA. triple helix, our experiments reveal how local collagen structure changes in The novel knob-socket (KS) model provides a construct to interpret and analyze response to applied force. Our first results show a load-enhanced trypsin sensi- the direct contributions of amino acid residues to the stability in a-helical protein tivity, indicating destabilization of the triple helix. The generality of this result structures. Based on residue preferences derived from a set of protein structures, will be discussed in the context of collagen’s sequence heterogeneity. the KS construct characterizes intra- and inter-helical packing into regular pat- terns of simple motifs. Intra-helical interactions consist of a regular pattern of 2862-Pos Board B70 three residue triangular motifs called sockets, which contribute to helical stability. For inter-helical interactions, a single amino acid knob from one a-helix packs Comparison of the Stability and Reduction Potential of Cytochrome C6 Proteins from a Psychrophilic and a Mesophilic Diatom into a three amino acid socket within another a-helix. Therefore, sockets are Logan Tillery, Miranda Wilson, Nayandeep Parmar, Katherine Frato. defined in three categories: (1) free, unpacked and favoring intra-helical interac- Chemistry, Seattle University, Seattle, WA, USA. tions, (2) filled, packed and favoring inter-helical interactions, and (3) non, un- The eukaryotic, photosynthetic, unicellular diatom Fragilariopsis cylindrus is packed and disfavoring a-helical structure. The three amino acid socket described as a psychrophile because it thrives in marine environments at temper- composition serves as a code that can be used to predict protein packing and by atures near freezing point of water. The genome of F. cylindrus and a related, extension, can also be used to understand individual amino acid contributions mesophilic diatom Thalassiosira pseudonana both carry genes that are highly to helical stability. The KS model was used in the de novo design of an a-helical homodimer, KSa1.1. Using site-directed mutagenesis, KSa1.1 point mutants homologous to cytochrome c6, a heme-containing protein involved in electron transfer during photosynthesis. Since T. pseudonana and F. cylindrus are have been generated to correlate KS propensities with changes in a-helical struc- evolved to survive under very different temperature regimes, we are interested ture and stability. In the KS a-helical model, each point mutation affects six sur- rounding sockets by altering the free/filled propensity values. By analyzing the in the molecular-level adaptation strategies that allow cytochromes c6 in the two organisms to function at their physiological temperatures. In this study, changes in the propensities of these six sockets, KS based stability predictions we report the recombinant expression and purification of the predicted cyto- were made for each mutant. These predicted values are compared to the experi- mentally determined stability of each peptide from chemical and thermal denatur- chrome c6 proteins from T. pseudonana and F. cylindrus, as well as initial char- acterization of the thermal stability, chemical stability, and reduction potentials ation studies as measured by circular dichroism spectroscopy. This study serves as a starting point to reveal how residue packing contributes to protein stability. of the two homologous proteins. While the sequences of the two cytochromes c6 share a high percent identity (88%), they have distinct stability profiles, with the 2866-Pos Board B74 reduced state of F. cylindrus c6 unfolding at a temperature approximately 5 de- High Hydrostatic Pressure (HHP), Xenon as a Probe and spin Labelling grees lower than the melting temperature of the homologous T. pseudonana c6. of the N-Terminal Domain Reveal a Whole Variety of Conformational Chemical stability in the presence of guanidine hydrochloride follows a similar Transitions in the Full-Length Human Prion Protein trend; the reduced state of F. cylindrus c6 reproducibly unfolds at a slightly lower Werner Kremer. concentration of denaturant than the T. pseudonana c6. Initial experiments sug- University of Regensburg, Regensburg, Germany. gest that the midpoint potential of F. cylindrus c6 is consistently higher than Transmissible spongiform encephalopathies (TSEs) are a group of fatal neurode- T. pseudonana c6. Ongoing experiments are quantifying differences in the reduc- generative disorders associated with the accumulation of fibrils of misfolded tion potentials of the two proteins, with the goal of understanding how differing prion protein PrP. Conformational intermediates of the human prion protein reduction potentials at room temperature allow the two proteins to fulfill their huPRPc were characterized by a combination of either high hydrostatic pressure roles at differing physiological temperatures. (up to 200 MPa) as well as xenon gas pressure and spin labelling experiments at positons 93 or 107 with two-dimensional [1H, 15N]-HSQC NMR spectroscopy. 2863-Pos Board B71 Xenon binds to four transiently enlarged hydrophobic cavities located in the Parameter Optimization for a New Reaction Pathway Sampling Method: well-folded core of human PrP(23-230). A fifth xenon binding site is formed Action-CSA transiently by amino acids A120 to L125 of the N-terminal domain and by amino Naohiro Nishikawa, Juyong Lee, Bernard R. Brooks. acids K185 to T193 of the core. A model including a dynamic opening and clos- National Heart, Lung, and Blood Institute, National Institutes of Health, ing of the cavities allowed to derive the individual microscopic and macroscopic Rockville, MD, USA. dissociation constants. A structural coupling between the N-terminal domain and We have developed a new computational approach to sample multiple reaction the core domain can now be confirmed by high pressure NMR spectroscopy, pathways with fixed initial and final states through global optimization of xenon binding and spin labelling experiments. Onsager-Machlup action using the conformational space annealing (CSA) method. This method can sample not only the most dominant pathway but 2867-Pos Board B75 also other possible pathways without initial guesses on reaction pathways. Exploring and Enhancing Compartment-Specific Protein Disaggregases to The sampling ability of the method was assessed by the examples of alanine Combat Neurodegenerative Diseases dipeptide and hexane. The method also showed good agreement with recent ex- Ryan R. Cupo1, Emily Augustine2, James Shorter3. periments in the application to FSD-1 mini protein. However, there are several 1Pharmacology Graduate Group, University of Pennsylvania, Philadelphia, variable parameters in the method, and the parameter dependency of the PA, USA, 2University of Pennsylvania, Philadelphia, PA, USA, 3Department method is not revealed yet. Therefore, we applied the method to the system of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, of alanine dipeptide and nine-residue small peptide whose sequence is PA, USA. YQNPDGSQA by changing the parameters in order to optimize parameter Protein aggregates accumulate in several neurodegenerative diseases. Protein sets so that we can effectively use the method. disaggregases have great therapeutic potential as they can reverse toxicity

BPJ 8720_8723 Wednesday, February 21, 2018 579a caused by protein aggregates and soluble oligomers by disassembling these SPDB v4.10 based on published integrin aV crystal structure (PDBID 1JV2). structures and recovering natively-folded proteins. The yeast protein disaggre- The models were equilibrated through GROMACS v4.6.7 prior to analyses. gase, Hsp104, disassembles disease-associated protein aggregates and soluble Protein docking between integrin aV subunit and its heterodimeric pair, integ- oligomers to suppress toxicity. Hsp104 has a yeast mitochondrial homologue, rin b3, was performed through ClusPro 2.0. This revealed altered heterodimer namely Hsp78. I targeted mitochondrial (mt) Hsp78 (mtHsp78) to the cyto- formation in mutant aV-b3 structures compared to the wild type. Analysis of plasm (cHsp78) and potentiated its activity via homologous mutations that intermolecular interactions in the generated heterodimers showed diminished nonselectively potentiated Hsp104. Three cHsp78 variants selectively rescue salt bridges in the mutants relative to the wild type. In particular, G188TaV a-synuclein (aSyn), FUS, or TDP-43 toxicity in yeast. Three different mtHsp78 experienced the greatest decrease in salt bridge content (50% salt bridge variants rescue aSyn toxicity in yeast without affecting cytoplasmic aSyn ag- loss). Molecular dynamics simulations are currently underway to determine gregation. We are exploring the mechanism of rescue for each of these variants. and compare the binding strengths between each aV-b3 dimeric pair. Additionally, we are interested in the possibility of targeting non-native disag- gregases to other organelle such as the endoplasmic reticulum. The results 2870-Pos Board B78 further our understanding of the substrate and compartment specific demands Sequential Folding of Globular Protein Initiated by Fast Loop Closure of protein disaggregases in higher eukaryotes. Elisha Haas, Gil Rahamim, Dan Amir. Life Sciences, Bar Ilan University, Ramat Gan, Israel. 2868-Pos Board B76 Our understanding of the mechanism of ultrafast folding of globular protein de- Disulfide Transfer Enables Inverse-Prion Aggregation in Mixtures of pends on our ability to monitor transient sub-domain structural elements in a pro- Human Gamma-D Crystallin Variants tein molecule, in situ, during the fast folding transition Ultrafast Fo¨rster resonance Eugene Serebryany1, Jimmy Thai1, Jaie C. Woodard2, Shuhuai Yu3, energy transfer (FRET) based methods are ideal for characterization of the tran- Sunia A. Trauger4, Bogdan Budnik4, Eugene I. Shakhnovich1. sient ensembles of refolding molecules. However, each site specific labeling 1Chemistry and Chemical Biology, Harvard University, Cambridge, MA, modification might affect rates of folding of near neighbor structural elements USA, 2Biochemistry and Molecular Biology, Michigan State University, East and thus limit the ability to resolve fine differences in rates of folding of these Lansing, MI, USA, 3State Key Laboratory of Food Science and Technology, elements. Therefore, here we report the development of the ‘‘Transfer-Quench’’ Jiangnan University, Wuxi, Jiangsu, China, 4Science Core Facility, Harvard method for measuring the rate of formation of two structural elements using a sin- University, Cambridge, MA, USA. gle triple-labeled mutant. This method is based on FRET combined with fluores- How post-translational modifications alter the structures and interactions of pro- cence quenching. We placed the donor and acceptor at the loop ends’ and a teins is of great interest for understanding proteomic changes during aging and dis- quencher at an a-helical element involved in the node forming the loop. Thus, ease. Oxidative modifications of the long-lived cysteine-rich lens g-crystallins are the kinetics of change of the acceptor emission intensity reports both folding strongly associated with their aggregation into light-scattering structures that events simultaneously. The method was applied in a study of the folding mecha- result in cataracts - the leading cause of age-related vision loss. How oxidation nism of the B domain of staphylococcal protein A (a three helix buldle). We found leads to aggregation is not well understood. Our previous computational and that the folding pathway start in loop L2 closure and the helix H3 formation which experimental work showed that formation of a particular non-native intramolecu- are very fast, followed by a slower helix H2 formation which is prior to the closure lar disulfide bond in cataract-associated W42Q/R human gD-crystallin variants of loop L1. These results show the power of the ‘‘Transfer-Quench’’ method for trapped a partially unfolded intermediate state prone to aggregation. Surprisingly, unraveling hidden folding pathway elements in a protein that was considered as a it also revealed that the wild-type protein was able to specifically promote aggre- two-state folder. The results support the hypothesis that early formation of loops gation of these variants without itself aggregating. The search for a biochemical are essential in the early phase of the folding pathway. mechanism behind this unprecedented ‘‘inverse- prion’’ interaction has now re- vealed that human gD-crystallin exhibits oxidoreductase activity. This activity de- 2871-Pos Board B79 pended on formation of a specific internal disulfide bond, which we mapped by A Right-Handed Coiled Coil Tetramer to Induce Cell Arrest in Prostatic LC/MS/MS and by comprehensive Cys mutagenesis. All-atom Monte-Carlo sim- Carcinoma Cells ulations with a statistical potential revealed conformational strain upon formation Francisco Padron1, JingJing Li2, Rihe Liu2. 1 2 of this disulfide, which was confirmed by differential scanning flourometry. Disul- University of Illinois at Chicago, Chicago, IL, USA, University of North fide exchange occurred among purified gD-crystallin molecules in solution. Both Carolina at Chapel Hill, Chapel Hill, NC, USA. the Cys-oxidized (disulfide-bonded) wild-type protein and the destabilized (Trp- A naturally occurring right-handed coiled tetramer protein was identified in oxidation mimicking) W42Q variant were highly soluble at physiological temper- Staphylothermus marnus, a thermophilic marine organism. Previous research ature and pH. When the two were mixed, however, the disulfide bond transferred shows that the hydrophobic interactions and network of salt bridges on the sur- from the WT to the mutant. Once oxidized, the mutant became aggregation-prone, face create an exceedingly stable tetrameric peptide (Stetefeld et al, 2000). its insolubilization helping drive the disulfide transfer. Destabilized or damaged Modification of the tetramer sequence was done to include a binding site to g- crystallins may act as oxidation sinks in the lens, forming light-scattering ag- inhibit a transmembrane protein that is upregulated in prostate cancer cells. gregates as a consequence. There is evidence that human gD-crystallin’s newly Currently, inhibition of the transmembrane protein, Prostate Specific Mem- found oxidoreductase activity is enzymatically regulated in vivo. brane Antigen (PSMA), is thought to induce apoptosis through the p38 MAPK pathway (Evans et al, 2016). After expression and purification of the 2869-Pos Board B77 modified tetramer protein, its presence was verified using liquid chromatog- Investigating the Effect of Cancer-Related Mutations on the Structure and raphy and gel electrophoresis. Binding analysis to Glucose-Regulated Protein Function of Integrin aVb3 Heterodimers through Molecular Dynamics (GRP) 78 as a non-specific binding measurement control using bio-layer inter- Simulations ferometry was done resulting in the expected negative results. Subsequent Rey Kristoffer V. Salinas, Raphael D. Caballes, Andre Rhey C. Haro, testing to the target biomarker PSMA using microscale thermophoresis also Neil Andrew D. Bascos. demonstrated negative results. Further testing using varying concentrations Protein Structure and Immunology Laboratory, National Institute of of ligand is necessary in order to establish reliable results. Molecular Biology and Biotechnology, Quezon City, Philippines. Metastasis is the tightly regulated development of secondary growths from a 2872-Pos Board B80 primary tumor site, and represents the biggest challenge in the treatment of can- EGCG Inhibits Fibrillation of Light Chain 6aJL2-R24G, Associated with cer. Metastasis requires several proteins working in concert to proceed, and Light Chain Amyloidosis among the primary proteins that govern its progression are the integrins. The Angel Enrique Pela´ez- Aguilar1, Lina Andrea Rivillas Acevedo1, integrins are heterodimeric cell surface proteins that serve as the major adhe- Leidys French Pacheco1, Gilberto Valdes Garcı´a1, Roberto Maya Martı´nez1, sion molecules in mammalian cells. They are known to control cell-cell as Nina Pastor Colo´n1, Carlos Amero Tello2. well as cell-ECM interactions, and regulate bidirectional signaling in cells. In- 1Centro de Investigacio´n en Dina´mica Celular, Universidad auto´noma del tegrins are frequently dysregulated or mutated in cancer, where they transduce estado de morelos, Cuernavaca, Mexico, 2Centro de Investigaciones extracellular cues from the tumor microenvironment, leading to changes in cell Quı´micas, Universidad auto´noma del estado de morelos, Cuernavaca, Mexico. shape, motility, survival, proliferation, and angiogenesis. While the mecha- Light chain amyloidosis (AL) is a deadly disease characterized by the deposition nisms underlying differential integrin expression in invasive tumors is well- of monoclonal immunoglobulin light chains as insoluble amyloid fibrils in characterized, the role of integrin mutagenesis in cancer progression remains different organs and tissues. Germ line l VI has been closely related to this con- largely unknown. This study thus aims to characterize three mutations in the dition; moreover, the R24G mutation is present in 25% of the proteins of this germ integrin aV subtype: G50T, G188T, and G252A. These mutants were obtained line in AL patients. In this work, five small molecules were tested as inhibitors of from mutational screens in cancer patients, as deposited in the CoSMiC data- the formation of amyloid fibrils from the 6aJL2-R24G protein. We have found by base. Molecular models for the ITGAV mutants were generated through thioflavin T fluorescence and transmission electron microscopy that EGCG

BPJ 8720_8723 580a Wednesday, February 21, 2018 inhibits 6aJL2-R24G fibrillogenesis. Furthermore, using nuclear magnetic reso- unique folding landscape of aLP arose over an evolutionary process provides in- nance spectroscopy, dynamic light scattering, and isothermal titration calorim- sights into how the amino acid sequence can modulate kinetic barriers and ther- etry, we have determined that the inhibition is due to binding to the protein in modynamic stabilities of the protein energy landscape. its native state, interacting mainly with aromatic residues. 2876-Pos Board B84 2873-Pos Board B81 Thermodynamics of a Coiled-Coil Protein Structure A Helix 1 Peptide from UBA(1) Provides Evidence for Tertiary Interac- Mojtaba Jokar, Korosh Torabi. tions in the Denatured State of UBA(1) Chemical Engineering and Materials Science, Wayne State University, Dustin C. Becht, Bruce E. Bowler. Detroit, MI, USA. University of Montana, Missoula, MT, USA. A coiled-coil protein structure consists of two or more interacting a-helical Studies over recent decades have shown that the denatured state ensemble (DSE) - strands that together form a supercoil structure. Coiled-coil structures entail the collection of all random-coil conformations of a denatured protein - exhibits unique mechanical properties critical to the function and integrity of various mo- significant conformational bias that deviates from theoretical random coil tor proteins, cytoskeletal filaments and extra-cellular matrix proteins. The main behavior. This inherent bias is thus integral to the initial stages of protein folding objective of our work is to develop a statistical mechanical model to predict and can give insight into unstructured proteins, such as intrinsically disordered the propensity of a protein sequence to form an isolated coiled-coil structure proteins (IDPs). In this work, we probe the denatured state of a three-helix bundle and to study its structural response to the mechanical load. We develop a partition protein termed the ubiquitin-associated domain, UBA(1), derived from the DNA- function for coiled-coil dimerization to estimate the probability of a given amino excision repair protein HHR23A. UBA(1) is strongly denatured using guanidine acid sequence to form various dimeric coiled-coil structures. We use our model’s hydrochloride (GdnHCl) and investigated using circular dichroism (CD) spec- predictions to reexamine the assumptions of the thermodynamic models em- troscopy. Under moderate to strongly denaturing conditions, UBA(1) demon- ployed to obtain various free energy terms from the experimental measurements. strates persistent a-helical content, eventually forming significant polyproline- The model parameters will be optimized based on comparisons with our experi- II (PII) helical structure at 7M GdnHCl. Isolation of the first helix of UBA(1) mental data of coiled-coil forming sequences designed to quantify various free via insertion of a stop codon at R177 reveals substantially decreased per- energy effects. We will combine the above coiled-coil dimerization model with residue helicity at all concentrations of denaturant when compared to full protein. our previously developed model on the tensile mechanics of a-helical polypep- This indicates that full-length UBA(1) maintains transient tertiary contacts under tide. This results in a model that predicts the structural response of a given strongly denaturing conditions. We have also acquired high resolution NMR coiled-coil motif to a mechanical tension. (15N-HSQC, HNCO, and HNCA) spectra of UBA(1) to obtain chemical shifts of the backbone atoms for each residue in denaturant. Our data provide 2877-Pos Board B85 residue-specific secondary structure information and indicate that UBA(1) forms Examining the Effect of Ubiquitination on the Energetics of Substrate Proteins transient native-like helices of nearly equal proportion in the DSE. These data will Emma Carroll, Susan Marqusee. be compared to backbone chemical shifts of the isolated first helix of UBA(1) to Molecular and Cell Biology, UC Berkeley, Berkeley, CA, USA. further elucidate contributions of transient tertiary contacts to residual helical Ubiquitination is a common protein posttranslational modification in which the content in the first helix of UBA(1) in the DSE of full length UBA(1). protein ubiquitin is attached to the primary amine of lysine (K) residues on the target protein. Ubiquitination is canonically associated with targeting proteins 2874-Pos Board B82 to the proteasome for degradation; however, it is also involved in many cellular Primary and Secondary Structure Preference of Hydrogen Exchange processes, and the cell must carefully regulate which ubiquitinated proteins Boshen Wang, Jie Liang. should be degraded. While proteasomal degradation is dependent on ubiquitin University of Illinois at Chicago, Chicago, IL, USA. chain length and topology, it has also been shown that the conformational fea- Hydrogen exchange experiments can provide important information towards tures of the tagged protein can play a role in proteasomal engagement. How- understanding the dynamics of protein structure. Linderstrom-Lang hypothe- ever, these biophysical factors driving the in vivo proteasomal degradation sized that only the amide in backbone will exchange hydrogen. In addition code remain largely unexplored. These types of studies are hampered by the to the intrinsic amide capability, previous experiments also show that the co- difficulty of characterizing the energetics and dynamics associated with prob- operativity of protein affects hydrogen exchange process. We examine the pri- ing macromolecular complexes such as ubiquitinated proteins. I will present mary and secondary structure preference of hydrogen exchange from our approach and results on a model system for characterizing the stability Start2fold database. Our results shows hydrogen exchange prefers ALA, and dynamics of proteins with and without defined ubiquitin modifications. CYS, ILE, LEU, PHE, TRP, VAL. In contrast, ASN, ASP, GLU, GLY, We are using both ensemble and single molecule approaches to characterize PRO, SER are unfavorable for hydrogen exchange. Among different second- the effect of ubiquitin on the energetics and kinetics of a variant of barstar. ary structures, loop region is unfavorable for hydrogen exchange. In contrast, helix and sheet region have high propensity for hydrogen exchange.In general, 2878-Pos Board B86 hydrogen exchange prefers hydrophobic residues and hydrogen bonding Identification of Segments in Variable Domains of IG Light Chains that riched secondary structure. These results suggest that cooperativity plays a Drive Formation of Amyloid Fibrils important role in hydrogen exchange. Shannon R. Esswein, Boris Brumshtein, David S. Eisenberg. University of California, Los Angeles, Los Angeles, CA, USA. 2875-Pos Board B83 Systemic light chain amyloidosis is a disease characterized by the overex- Using Ancestral Proteins to Probe the Thermodynamic and Kinetic Prop- pression of immunoglobulin light chains. Full-length light chains or their erties of the Alpha-Lytic Protease Family variable domain fragments aggregate into amyloid fibrils, which cause organ Charlotte Nixon1, Shion A. Lim1, Zachary Sailer2, Michael Harms2, failure. We previously showed that dimers of variable domains of light Susan Marqusee1. chains first disassociate into monomers before unfolding to form amyloid fi- 1 2 Molecular and Cell Biology, UC Berkeley, Berkeley, CA, USA, Institute of brils. However, the particular amino acid segments of the variable domain Molecular Biology, University of Oregon, Eugene, CA, USA. responsible for forming the beta-sheet spine of amyloid fibrils remained un- The linear sequence of a protein defines its entire energy landscape, which in- clear. Here we identify multiple segments of variable domains that indepen- cludes all of the accessible conformations of the polypeptide chain, their relative dently drive amyloid fibril formation in both lambda and kappa subtypes of stabilities, and dynamics. Although small changes in the sequence do not alter light chains. These findings provide insight into the molecular pathogenesis the native fold of a protein, they invariably alter the energetics and dynamics of amyloid fibril formation, may explain the difficulty in treating this and of the landscape, and thus, the function and fitness of the protein. Through their other amyloid diseases, and inform therapeutic strategies that target the am- effect on fitness and function, these changes drive evolution. We can use these yloid fibril spine. evolutionary sequence changes to probe the code between sequence and the en- ergy landscape. Here we use the technique of Ancestral Sequence Reconstruc- 2879-Pos Board B87 tion (ASR) to probe the biophysical properties of the kinetic barrier to protein An in Silico Investigation of Amyloid Beta with a Focus on N-Terminus: folding through the lens of evolutionary history. We use ASR to study the protein from Structure to Amyloid Inhibitor Design alpha-lytic protease (aLP) and its unusual kinetic stability to investigate the na- Payel Das1, Srirupa Chakrabarty1, Anita Chacko1, Brian Murray2, ture and origin of kinetic stability and conformational barriers in general. This Georges Belfort2. protease has an unusual energy landscape: the native state of aLP is not its 1IBM T J Watson research center, Yorktown heights, NY, USA, 2RPI, Troy, most thermodynamically stable state. Instead, aLP requires its own pro-region NY, USA. chaperone to fold, and the native state of aLP is kinetically trapped, preventing The aggregation of intrinsically disordered amyloid-beta (Ab) peptides unfolding at biologically relevant time scales. Thus, understanding how this plays a crucial role in the etiology of Alzheimer’s disease (AD). Recently,

BPJ 8720_8723 Wednesday, February 21, 2018 581a two familial mutations in Ab, namely A2T and A2V, have been reported to 2882-Pos Board B90 offer protection from AD in the heterozygous state. Characterization of Field Flow Fractionation Characterization of the NIST Monoclonal these natural Ab variants thus offers an intriguing approach to understand Antibody Standard RM 8671 the molecular mechanism of AD. In this talk, I will describe atomistic Robert Reed1, Soheyl Tadjiki1, Thorsten Klein2. simulation results of the Ab1-42 N-terminal variants, which suggest altered 1Postnova Analytics Inc., Salt Lake City, UT, USA, 2Postnova Analaytics misfolding into disease-implicated b-hairpin structures during the early Inc, Salt Lake City, UT, USA. stage of aggregation. We further characterize binding of N-terminal hexa- The NIST monoclonal antibody (mAb) standard reference material providesa plat- peptide variants with wild-type Ab1-42. Those peptide fragments exhibit form for evaluating methods used to characterize physicochemical and biophysical a sequence-dependent ability to reconfigure the Ab folding landscape attributes of other mAbs. In this study, the NIST mAb was used to evaluate sepa- away from the disease-implicated structures. Our simulations thus offer ration parameters for asymmetrical flow field flow fractionation (AF4) analysis of novel insights onto the sequence-structure-function relationship of disor- mAbs, especially in comparison to size exclusion chromatography (SEC). Results dered amyloidogenic peptides, which can further guide mechanism-based will be shown for AF4 and SEC, both coupled with refractive index and multi- therapeutic design for devastating protein aggregation diseases. angle light scattering detectors for measuring molecular weight. For AF4, both a References: 1. Das, P., Murray, B., & Belfort, G. Biophysical Journal, 2015, standard (33.5 cm) and micro channel (8 cm) were used, to evaluate separation pa- 108, 738-747. 2. Murray, B., et al. Proteins, 2016, 84, 488-500. 3. Das, P., rameters such as analysis time, resolution, and recovery. The NIST mAb was Chacko, A., & Belfort, G. ACS Chem Neurosci, 2016, 8(3), 606-618. 4. Chak- agitated at room temperature to induce aggregation, and the ability of both AF4 raborty, S. & Das, P. Sci Rep, 2017 (accepted). and SEC to measure the full distribution of aggregated species was evaluated.

2880-Pos Board B88 2883-Pos Board B91 Use of Fluorescence A-Teems to Track Insulin Solubility and Solvent Resurrecting a Desiccation-Inactivated Enzyme Environment in Micelle Solutions Samantha Piszkiewicz, Aakash Mehta, Kenny Nguyen, Ashlee M. Propst, Karen E. Gall. Gary J. Pielak. Fluorescence, Horiba Scientific, Edison, NJ, USA. Chemistry, UNC Chapel Hill, Chapel Hill, NC, USA. Human recombinant insulin protein is used to show that the method of simul- For decades, the osmolyte and disaccharide trehalose has been FDA-approved as taneous absorbance and fluorescence excitation emission matrices (A-TEEM) an excipient to stabilize pharmaceuticals, including biologic drugs. The protein is a technique suitable to fingerprint the solubility state of proteins. Insulin is a human serum albumin is also a useful excipient. However, their mechanisms of action remain controversial. We show that trehalose and the sucrose polymer protein therapeutic, used by those with type 1 diabetes (T1D) to regulate the Ô amount of glucose in the bloodstream. Insulin is also a relatively insoluble Ficoll 70 , which both contain non-reducing linkages, protect the enzyme lactate protein at biological pH and commercial formulation is carefully tailored to- dehydrogenase against desiccation-induced inactivation, but sugars containing wards improved solubility and decreased aggregation at high concentrations. reducing linkages are not protective. Trimethylamine N-oxide, but not the other We use the intrinsic tyrosine fluorescence of insulin to follow the solvent osmolytes tested, shows protective properties similar to those of trehalose. We environment of the protein in solutions of varied concentrations of surfactant also identified both globular and disordered proteins with robust protective prop- polymer, Pluronic(R) F88, an amphiphilic triblock copolymer, which forms erties against desiccation-induced inactivation. Unlike osmolytes, these protec- micelles at elevated concentrations and temperature. The intrinsic fluores- tive proteins have no known biological function in desiccation tolerance. cence of insulin shifts the A-TEEM fingerprint of insulin in micelle solution Polyethylene glycol 3350 lacks a protective effect, suggesting that protection from that of less solubilized insulin in low polymer concentrations or buffer/ does not stem from simple hard-core repulsions. Of the molecules that protect water alone. Simultaneous A-TEEM measurements are non-destructive and lactate dehydrogenase against desiccation-induced inactivation, the proteins beneficial for characterization of high protein concentration solutions due to were more effective at protecting the desiccated enzyme against heat-induced the ability to correct for high absorbance levels and the resulting primary inactivation. In addition, desiccation-inactivated lactate dehydrogenase was res- and secondary inner-filter effects on reported fluorescence. Protein local sol- urrected by re-suspension in dilute protein solutions, a phenomenon not observed vent environment and especially solubility are important aspects of formu- with osmolytes. This finding suggests that properly folded and disordered protein lating of protein therapeutics and the A-TEEM method is shown to crowders may facilitate refolding of unfolded or misfolded lactate dehydrogenase characterize this type of solution. upon rehydration rather than protect the enzyme against desiccation-induced inactivation. The fact that proteins with no known link to desiccation tolerance 2881-Pos Board B89 protect and resurrect lactate dehydrogenase activity also suggests that these phe- Activation Mechanism of Cocoonase nomena result from non-specific protein-protein interactions. Nagisa Tajima1, Mitsuhiro Miyazawa2, Shigeru Shimamoto1, Yuji Hidaka1. 2884-Pos Board B92 1 2 Kindai University, Higashi-osaka, Japan, Institute of Agrobiological Molecular Recognition Mechanism of Hematopoietic Prostaglandin Sciences, National Agriculture and Food Research Organization, Tsukuba, D Synthase with Cofactor and its Substrate Japan. Shigeru Shimamoto, Keisuke Asada, Yuji Hidaka. Cocoonase, a protein produced by the silkworm (Bombyx mori), consists of 226 Kindai University, Higashi-osaka, Japan. amino acid residues and three intra-molecular disulfide bonds and is thought to Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is a Sigma class mem- specifically digest the sericin protein which is a coating protein of fibroin of ber of the glutathione S-transferase (GST) superfamily, which requires gluta- silkworm cocoons. However, the specificity of the protease activity and its cat- thione (GSH) as a cofactor. H-PGDS catalyzes an isomerization of alytic structure have not yet been determined in detail. Therefore, to further prostaglandin H2 (PGH2) to prostaglandin D2 (PGD2), which acts as allergic investigate the biological activity of cocoonase, we prepared recombinant co- and inflammatory mediator. Overproduction of PGD2 by H-PGDS causes allergic coonase using an E. coli expression system. and inflammatory reactions in the necrotic muscle fibers of Duchenne muscular The cDNA sequence of cocoonase has recently been determined. Therefore, dystrophy (DMD) patients. Therefore, H-PGDS inhibitors are expected for to investigate the activation and folding mechanism of cocoonase, recombi- drug therapy of DMD. The catalytic mechanism of H-PGDS remains still unclear nant cocoonase (CCN) and pro-cocoonase (pro-CCN) were expressed using a since essential information, such as the binding affinity and stoichiometry of GSH chemically synthesized cDNA, in which the codon bias was optimized, in and PGH2 to H-PGDS, have not exactly determined yet. Therefore, to clarify the E. coli BL21(DE3) cells. CCN and pro-CCN were successfully expressed cofactor and substrate recognition of H-PGDS, we investigated the interaction be- as inclusion bodies by E. coli. The refolding reactions were examined in tween H-PGDS and PGH2 in the presence and absence of GSH.For this purpose, the presence of 2 mM GSH/1 mM GSSG and forms of both proteins pro- isothermal titration calorimetry (ITC) measurements were carried out using vided soluble proteins after the refolding reactions. During the reactions, GSH, a cofactor mimetic peptide Glu-Cys-Gly (ECG), and a substrate mimetics the pro-peptide region of pro-CCN was automatically processed by itself. (U-44069) in the place of unstable PGH2. Recombinant H-PGDS was prepared However, only the recombinant CCN derived from pro-CCN showed prote- using an E. coli expression system, and purified by several types of chromatog- ase activity. raphy, and applied for the ITC measurements. The results showed that GSH To obtain structural information regarding the propeptide-assisted folding of bound to the recombinant H-PGDS while ECG did not, suggesting that the local- cocoonase, CD measurements of the refolded CCN proteins were carried out. ization of NH3 group or it positive charge on GSH play an important role for the The results suggested the refolded CCN derived from pro-CCN consisted of binding to H-PGDS. In addition, U-44069 was able to bind to H-PGDS in the pres- alpha-helical and beta-sheet structures. ence of GSH but the binding was not observed in the absence of GSH. Therefore, These results indicated that the pro-peptide region of cocoonase plays an these results suggest that the GSH binding promotes the interaction between important role in the construction of the native conformation required for its H-PGDS and PGH2, and that GSH plays an important role, not only for the cat- enzyme activity. alytic reaction but also the substrate binding.

BPJ 8720_8723 582a Wednesday, February 21, 2018

Posters: Enzyme Function, Cofactors, and Post- that impact its multiple growth factor, integrin, and ECM protein binding do- mains. Fibronectin is a unique ECM protein in that it contains 4 free cysteine translational Modifications residues that are protected in cryptic sites, but that can be exposed under force. S-glutathionylation is a post-translational modification of cysteines that occurs 2885-Pos Board B93 in oxidative environments like the extracellular space during pathologies like Structural Analysis of Bifunctional Enzyme Obc1 for Oxalogenesis cancer and fibrosis. Here, we show for the first time that fibronectin’s cysteines Juntaek Oh, Sangkee Rhee. can be glutathionylated when exposed either through chemical unfolding in its School of Agricultural Biotechnology, Seoul National University, Seoul, soluble form or mechanical unfolding in its fibrillar form (both on the single Republic of Korea. fiber and matrix level). Moreover, we proved that S-glutathionylation impacts Bacterial quorum sensing (QS) controls the gene expression for bacterial the mechanical properties of fibronectin fibers. Specifically, our results reveal population-wide characteristics, including bioluminescence, motility, and that glutathionylated fibronectin fibers are more extensible and less stiff virulence-related factors. QS provides further benefits at the population level compared to unmodified fibronectin. This is true for both single fibronectin fi- by regulating the production of public goods, the function of which could be bers and fibronectin matrices. Thus S-glutathionylation of mechanically beneficial to all members of the group. In Burkholderia species, oxalic acid exposed cysteines on fibronectin may act as a mechanism of mechanical mem- was recently identified as an excreted public good for the QS-dependent ory within fibronectin-rich ECMs, regulating subsequent mechanotransduction. growth. In these species, oxalogenesis via the oxalate biosynthetic component One possible realism of this mechanism could be the enhancement fibronectin’s (obc) is a cellular event indispensable for the survival of bacteria in the station- integrin binding mechanoswitch due to fibronectin fibers being more easily ary phase. In B. glumae, obc consists of two genes encoding ObcA and ObcB stretched under lower force regimes. for coordinating the production of oxalic acid, as well as acetoacetate and CoA, by using oxaloacetate and acetyl-CoA as substrates. Unlike B. glumae, a bi- 2888-Pos Board B96 functional enzyme Obc1 from B.thailandensis carries out oxalogenesis. In Tuning a Prolyl Cis/Trans Molecular Switch that Regulates lateral Root fact, Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in Development in Rice its C-terminal domain despite no sequence homology with ObcB. In the N- Lucila A. Acevedo, Linda K. Nicholson. domain, oxaloacetate serves as a nucleophile by forming an enolate intermedi- Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA. ate mediated by Tyr322 as a general base, which then attacks the thioester Negative feedback cycles are processes in biology where a component of the carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two cycle plays the role of inhibitor and output. Recently, the cis-trans isomeriza- substrates. In the C-terminal domain, there is an a/b hydrolase fold that has tion of X-Pro has emerged as an important molecular timer in diverse biological a catalytic triad for oxalate production and a novel oxyanion hole distinct processes, which are regulated by Peptidyl Prolyl Isomerases (PPIases). One from the canonical HGGG motif in other a/b hydrolases. Mutagenesis studies example of a negative feedback cycle in which a PPIase plays a key role is suggested that His-934 is an additional catalytic acid/base for its lyase activity the auxin-responsive transcription regulation circuit in plants (Auxin circuit). and liberates two additional products, acetoacetate and CoA. This work was OsIAA11 and LRT2 are proteins involved in lateral root development in supported by Next Generation BioGreen 21 program of Rural Development rice. OsIAA11 is a transcription repressor that is expressed in response to Administration (Plant Molecular Breeding Center) of Republic of KOREA. Auxin, and by repressing its own promoter OsIAA11 plays a negative feedback 2886-Pos Board B94 role for expression of its own and other genes that initiate lateral root formation. Mapping and Analysis of S-Palmitoylation Sites on RPE65 Protein OsIAA11 belongs to the Aux/IAA repressor protein family that contains a ‘‘de- Sheetal Uppal, Eugenia Poliakov, Susan Gentleman, T. Michael Redmond. gron’’, a short sequence that targets the proteosomal degradation machinery. Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, OsIAA11 binds to its cognate E3 ligase in the cis conformation of the 104W- National Institutes of Health, Bethesda, MD, USA. P105 peptide bond in the degron, resulting in cis specific ubiquitination and sub- S-palmitoylation, a post-translational lipid modification of cysteine thiol sequent proteosomal degradation. LRT2 is a PPIase classified as a cyclophilin. groups is the major form of protein acylation in eukaryotic cells. It is reversible LRT2 is known to catalyze isomerization of 104W-P105 in a peptide represent- and highly dynamic in nature, and has been shown to regulate protein function ing the degron of OsIAA11. Flanking regions of the degron have been shown to and localization via modulating protein’s membrane affinity and stability. increase the rate of Aux/IAA degradation. Here we observe that by including Several hundred mammalian proteins have been reported to undergo S-palmi- these flanking regions the isomerization rates of 104W-P105 peptide bond in- toylation with the current simple and robust methodology to detect S-palmitoy- crease, suggesting that the isomerization rate directly governs the degradation lated proteins. Retinal pigment epithelium specific-65 kDa (RPE65) protein is a rate. Additionally, we have identified LRT2 mutants that change the cis/trans trans- to cis- retinoid isomerase, indispensable for regenerating the visual chro- isomerization rate. Introduction of these mutants into rice will test whether mophore of rhodopsin, 11-cis retinal. Previous biochemical studies have shown the prolyl cis-trans molecular switch in OsIAA11 acts as a timing device in that RPE65 associates with membrane even though it lacks any membrane tar- auxin circuit dynamics and sets the timing of lateral root development. geting motifs. It has been proposed that reversible post-translational S-palmi- toylation of RPE65 controls its membrane association. However, despite 2889-Pos Board B97 detailed understanding of RPE65 physiological function, the existence and Investigating the Conformational Dynamics of Plant Protein Kinases 1 2 3,4 role of RPE65 palmitoylation still remains a matter of debate. To gain insight Alexander S. Moffett , Kyle W. Bender , Steven C. Huber , 5 into RPE65 palmitoylation, we employed biochemical methods including Diwakar Shukla . 1Center for Biophysics and Quantitative Biology, University of Illinois at Acyl-Resin assisted capture (RAC) and Acyl-biotin exchange (ABE) assays, 2 in combination with mass spectrometry techniques. We also performed exten- Urbana-Champaign, Urbana, IL, USA, The Sainsbury Laboratory, Norwich, United Kingdom, 3Department of Plant Biology, University of Illinois at sive site-directed mutational analysis to map the S-palmitoylation sites on 4 RPE65 and to investigate how palmitoylation contributes to RPE65 membrane Urbana-Champaign, Urbana, IL, USA, Global Change and Photosynthesis Research Unit, United States Department of Agriculture - Agricultural association. Our results demonstrate that RPE65 palmitoylation is dynamic in 5 nature and that three out of RPE65’s 12 cysteines are potential sites for palmi- Research Service, Urbana, IL, USA, Department of Chemical and toylation. Additionally, experiments in HEK293F cells showed reduced levels Biomolecular Engineering, University of Illinois at Urbana-Champaign, of RPE65 palmitoylation when co-expressed with LRAT, a palmitoyl acyl- Urbana, IL, USA. transferase, whereas co-expression with less active LRATC161S mutant did The receptor-like kinases Brassinosteroid Insensitive 1 (BRI1) and BRI1- not result in any significant decrease in RPE65 palmitoylation levels. This Associated Kinase 1 (BAK1) are critical to plant growth and development result clearly suggests that palmitate shuttles between RPE65 and LRAT and signaling, serving as co-receptors for brassinosteroid hormones. Possibly thus regulates the dynamics of RPE65 palmitoylation, warranting further inves- because of their importance in these pathways, BRI1 and BAK1 are both highly tigations, currently underway in the laboratory. regulated. Upon brassinosteroid binding, the BRI1 and BAK1 kinase domains (KDs) phosphorylate and activate one another, while among other negative reg- 2887-Pos Board B95 ulatory mechanisms, BAK1 is deactivated in vitro by S-glutathionylation in S-Glutathionylation Influences the Property of Fibronectin oxidative conditions. However, the molecular mechanisms of BRI1 and Wei Li, Thomas Barker. BAK1 regulation and activation remain unclear. In order to investigate the dy- University of Virginia, Charlottesville, VA, USA. namics of fully phosphorylated BRI1 and BAK1 KDs and to understand the Fibronectin is a key extracellular matrix (ECM) protein that plays a role in cell mechanism of BAK1 deactivation by S-glutathionylation, we performed exten- growth, migration and differentiation through its engagement of cell surface re- sive all-atom molecular dynamics simulations on the BRI1 and BAK1 core ceptors, known as integrins, in a spatiotemporal manner. Fibronectin is a me- KDs along with BAK1 in all viable singly glutathionylated forms. In non- chanically sensitive protein displaying force-induced conformational changes glutathionylated BRI1 and BAK1, we found considerable disorder in the aC

BPJ 8724_8726 Wednesday, February 21, 2018 583a helix, a common regulatory domain in protein kinases (PKs) which must be organizational state of chromatin. We focus on a particular modification, folded in an active conformation. In order to validate our findings, we per- methylation lysine-9 of histone H3, which is involved in one of the most repre- formed circular dichroism spectroscopy experiments on the BRI1 aC helix pep- sentative and critical epigenetic processes that affects chromatin structure and tide, yielding results consistent with our simulations. Using disorder prediction gene expression. Epigenetic methylation plays an essential role in heterochro- software on all Arabidopsis thaliana PK sequences, we found that aC helix dis- matin formation and transcriptional repression of genes and is characterized by order may be a common feature in plant kinomes. From our simulations of glu- two key features. First, epigenetic methyl marks interact with the protein HP1, tathionylated BAK1, we found that S-glutathionylation of Cys408, neighboring which oligomerizes when bound to adjacent nucleosomes, leading to conden- the aC helix, destabilized active-like BAK1 conformations, while modification sation. Second, methyltransferases spread methyl marks to neighboring nucle- of other Cys residues had little effect. Using Kullback-Leibler divergence, we osomes in spatial proximity. found that Cys408 S-glutationylation had long-range effects on individual res- Our model combines the effect of segregation and condensation on chromo- idue conformations. Our results suggest that additional factors beyond phos- somal organization with the effect of the interaction between HP1 and methyl- phorylation are required for BRI1 and BAK1 activation, while supporting an transferases on methyl spreading. The result is a fully predictive framework allosteric mechanism of BAK1 deactivation by Cys408 S-glutathionylation. that describes the heritability of the methylation sequence. First, we utilize To our knowledge, our simulations represent the first insight into atomistic experimental measures of methyl spreading around a nucleation site in undif- plant PK dynamics. ferentiated stem cells to define the function of the methyltransferase. Our model shows good agreement with the degree of spreading observed experimentally 2890-Pos Board B98 5 and indicates that in the absence of higher order organization, methyl marks Crystal Structure and Characterization of D -3-Ketosteroid Isomerase are confined to a local domain. Using our predictive model for heterochromat- from Mycobacterium Strain HGMS2GL in/euchromatin condensation, we extend our methylation model to address the Fei Peng1, Fei Yang1,2, Xiyao Cheng1, Zhengding Su1. 1 2 reestablishment of the methylation sequence following DNA replication. Our Hubei University of Technology, Wuhan, China, Wuhan University, model reliably maintains methylation over several generations, thereby recapit- Wuhan, China. ulating the robustness of the epigenetic code. 4-androstene-3,17-dione (4-AD) is an important pharmaceutical intermediate and is produced by biotransformation of phytosterols using Mycobacteria 2893-Pos Board B101 through a series of enzyme-catalyzed reactions. 5-androstene-3,17-dione (5- Investigating the Effect of Alpha-Synuclein Post-translational Modifica- AD) is its first immediate. However, the biotramsformation can be jeopardized 5 tions on Synaptic Vesicle Trafficking by D -3-Ketosteroid isomerase (KSI) which transforms of 5-androstene-3,17- Buyan Pan, James Petersson, Elizabeth Rhoades. dione (5-AD) to 4-AD. Here we report the crystal structure and enzymological Chemistry, University of Pennsylvania, Philadephia, PA, USA. characterization of KSI from Mycobacterium HGMS2GL. The enzyme was Post-translational modifications (PTMs) are covalent modifications made to overexpressed in E. coli BL21 (DE3) and purified by Ni-NTA affinity chroma- proteins that give rise to the complexity and diversity of proteomes. PTMs tography and size exclusion chromatography. The purified protein exhibited a can regulate protein activity, folding, stability, and localization, playing a single band of molecular mass 15.7 kDa on SDS-PAGE gels. The enzyme spe- key role in biological processes. Many PTM sites have been identified and cific activity was 9.1 U mg1 using 5-AD as substrate, and its optimal pH and studied in alpha-synuclein (a-syn), an intrinsically disordered protein temperature for activity was 7.0 and 40 C,respectively. The amino acid believed to mediate synaptic vesicle trafficking. Abnormalities in a-syn’s sequence of KSI has 69% of similarity to that from Mycobacterium tubercu- interactions with lipid vesicles and membranes lead to the formation of losis. The crystal structure data showed that the KSI had a large difference non-functional, misfolded fibrils, which are implicated in Parkinson’s disease. with the reported structure of KSI from Mycobacterium tuberculosis. Although phosphorylation is the most common PTM, a variety of modifica- 2891-Pos Board B99 tions such as nitration, arginylation, and N-terminal acetylation also affect Crystal Structure and Characterization of 3-Ketosteroid-D1-Dehydroge- the structure and behavior of a-syn and its interactions with other biomole- nase from Mycobacterium Strain HGMS2GL cules. To illustrate - the phosphorylation of a tyrosine on a-syn has been found Hongwei Wang, Fei Yang, Xiyao Cheng, Yongqi Huang, Zhengding Su. to alter the protein’s helical conformation state when bound to lipid vesicles, Hubei University of Technology, Wuhan, China. while nitration of tyrosine residues in a-syn has been linked to its accumula- The androst-1,4-diene-3,17- dione (ADD) is an important drug intermediate of tion in filamentous structures. N-terminal acetylation and arginylation of the pharmaceutical steroids. Currently, it is produced as a by-product during the glutamate residues have also been identified in a-syn. We combine protein biotransformation of phytosterols into 4-androstene-3,17-dione (4-AD) using semi-synthesis and single molecule fluorescence spectroscopy techniques to Mycobacteria. Through genomics analysis of Mycobacterium strain determine the effect of various PTMs on the ability of a-syn to mediate HGMS2GL, we identified that the 3-ketosteroid-1-dehydrogenase (KsdD) in vesicle fusion. Using peptide synthesis, recombinant production of proteins Mycobacterium strain HGMS2GL could catalyze the conversion of 4-AD to with unnatural amino acids, and native chemical ligation, we produce modi- ADD. In this study, we reported crystal structure and characterization of fied, fluorescently labeled a-syn. We then determine changes in its binding af- KsdD from mycobacterium strain HGMS2GL. The gene encoding KsdD finity for lipid vesicles through Fluorescence Correlation Spectroscopy and from Mycobacterium strain HGMS2GL was subcloned into pET28b vector validate the structural changes induced by the PTMs via single molecule and the KsdD was expressed in Escherichia coli BL21 (DE3) strain. Cell FRET in the presence of lipid vesicles. We examine the effects of these struc- extract was used for catalyzing the reaction of AD to ADD. The reaction mix- tural and interactional changes on protein function by assaying the fusion of tures were extracted with the same volume of ethyl acetate. The organic phases fluorescently labeled vesicles mediated by modified a-syn. Our study pro- were collected and concentrated for thin layer chromatography (TLC) analysis. vides insight into the impact of PTMs on protein structure, function, and po- The KsdD enzyme activity was spectrophotometrically determined at 30 C. In tential contribution to disease. order to improve KsdD activity for the efficient conversion of 4-AD into ADD, we performed random mutation on the KsdD gene. A KsdD mutant with high 2894-Pos Board B102 activity was crystallized and analyzed, revealing the active center that is Active Site Dynamics and Substrate Permissiveness of Hydroxylcinna- different from its homologues. Thus, our results not only provide a reliable ba- moyltransferase (HCT) sis for understanding the catalytic mechanism for the KsdD enzyme, but also Ying-Chih Chiang1, Olesya Levsh2,3, Chun Kei Lam1, Jing-Ke Weng2,3, provide an applied process for the conversion of AD to ADD. Yi Wang1. 1Physics, Chinese University of Hong Kong, Shatin, N.T., Hong Kong, 2892-Pos Board B100 2Whitehead Institute for Biomedical Research, Cambridge, MA, USA, Physical Modeling of the Spreading and Maintenance of Epigenetic Mod- 3Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. ifications through DNA Looping and Condensation Substrate permissiveness has long been regarded as a facilitator in the evolution Sarah Sandholtz1, Quinn MacPherson2, Andrew Spakowitz3. of new enzymatic functions. In land plants, HCT is an essential enzyme 1Chemistry, Stanford University, Stanford, CA, USA, 2Physics, Stanford involved in the phenylpropanoid metabolism. Unlike some other essential en- University, Stanford, CA, USA, 3Chemical Engineering, Stanford University, zymes that are diffusion-limited and evolutionarily perfected, HCT exhibits Stanford, CA, USA. inferior kinetic parameters and can utilize a variety of non-native substrates. We have developed a comprehensive model of the physical mechanisms that To uncover the structural basis of their substrate permissiveness, here, we govern the spreading of epigenetic modifications and the heritability and main- report molecular dynamics (MD) simulations performed on the specialized ma- tenance of these marks. chine Anton of HCTs from five land plants. Through altogether 22-ms simula- Core histone proteins are responsible for the local packing of DNA, and chem- tions, we demonstrate the prevalent swing motion of an arginine handle, which ical modifications on the tails of these proteins control both the local and global adopts an external conformation in the apo-state of the enzymes, and ‘swings’

BPJ 8724_8726 584a Wednesday, February 21, 2018 to an internal conformation in the presence of the native substrate. We discuss population, which negatively impacts their ability to maintain normal work the role of a previously unidentified salt bridge in such motion as well as the and social schedules. Given the importance of PER2 phosphorylation for implication of the remarkable flexibility of the arginine handle. Through com- circadian timekeeping, we are interested in understanding the molecular de- bined analysis of the Anton trajectories and hot spot calculation via FTMAP, tails of the CK1d-PER2 interaction. We have used time-resolved NMR to we further reveal how a non-native substrate can bind HCTs at sites distinct monitor phosphorylation of a peptide containing the FASP phosphorylation from that of the native substrate and radiate from these sites to encounter its sites by CK1d at single-residue resolution. Upon addition of CK1d and reaction partner as well as key catalytic residues of the enzyme. Finally, by ATP, we observed an exponential decrease in the intensities of resonances solving the Smoluchowski equation numerically, we discuss how a weak bind- from residues that encompass the sequential phosphorylation sites, as well ing site shifted away from the catalytic center may affect the reaction efficiency as the appearance of additional peaks that increase in intensity over time to of the non-native substrates of HCTs. report on the kinetics of phosphorylation. Additional studies explore the mechanism by which the kinase is stably anchored to PER2 and maintained 2895-Pos Board B103 in an active state. Elucidating the kinetics and mechanism of PER2 regulation A Novel Biological Nanopore for Active DNA Transport and Detection by CK1d will bring greater insight into their powerful role in controlling the Ke Sun, Yuejia Chen, Xialin Zhang, Changjian Zhao, Jia Geng. timing of circadian rhythms. State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China. As reported, scientists have found many kinds of biological pores to study the 2898-Pos Board B106 stochastic sensing. Nanopore sensing has been developed as a platform technol- Expression, Purification, and Crystallization of the Human Oxido- ogy and those biological porins have been reengineered with enormous proba- reductase, Pyrox-D1: A New Described Cause of Early-Onset Myopathy bility for single-molecule DNA/RNA sequencing, such as a-Hemolysin in Humans channel and mycobacterial porin. Here we report a novel porin having a narrow Isaac L. Scott. diameter of 1.3-nm with a transmembrane region. This porin can be recon- Cell Physiology and Molecular Biophysics, Texas Tech University Health structed into lipid bilayer and transport ions, small molecules, DNA and Sciences Center, Lubbock, TX, USA. RNA. Interestingly, we find that both single-stranded and double-stranded nu- Mutations in the oxidoreductase, Pyrox-D1, have been linked to a newly cleic acids can be translocated. The average dwell time for 48-nt ssDNA was 2- described, early-onset recessive myopathy from five families with four 5 4 mS, and the blockade was 80% 5%. We also find its DNA helicase activity different recessive variants. This new myopathy presents with a histopathol- fueled by ATP hydrolysis. This nanopore has the potential application in active ogy that is distinctive in that it combines multiple pathological hallmarks DNA transport and sequencing. characteristic of different myopathies; central and minicore disease, centro- 2896-Pos Board B104 nuclear, myofibrillar and nemaline myopathy. Patients present in infancy Effects of Crowding on Alkaline Phosphatase Kinetics as Seen through the with slowly progressive proximal and distal weakness, facial weakness, Lens of Small-Molecule Inhibition nasal speech, swallowing difficulties,andmildtomoderatelyelevated Oksana Yavorska1, Lukas Syriste2, Maryam Yaqoob3, Chantal du Plessis4, serum Creatine Kinase (CK) levels. The link between the mutations in Kyle Poffenroth5, John Chik5. the Pyrox-D1 gene and the disease phenotype are not yet understood. 1Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, This project involves expression, purification, and crystallization of Canada, 2Microbiology, Immunology and Infectious Diseases, University of Pyrox-D1 for eventual characterization by X-ray diffraction. Following Calgary, Calgary, AB, Canada, 3University of Calgary, Cumming School of crystallization, preliminary characterization will involve spectroscopic Medicine, Calgary, AB, Canada, 4Nursing and Midwifery, Mount Royal and enzymatic analysis to determine the co-factors used by this enzyme, University, Calgary, AB, Canada, 5Chemistry and Physics, Mount Royal and eventual structure determination by X-ray crystallography. Understand- University, Calgary, AB, Canada. ing the co-factors and biophysical characteristic of this protein could help in Many enzymes have evolved to function in aqueous environments filled with developing new treatments in the future for this form of myopathy. To date, many other solutes. In addition to specific interactions, these co-solutes may we have purified and crystallized Pyrox-d1. Going forward, the goal is to also indirectly modify enzyme behaviour through various means generally determine the molecular structure of Pyrox-D1, which will help elucidate referred to as crowding. Using UV-visible spectrometry, we have measured the protein’s function. The structure and function can be utilized to develop the kinetics of para-nitrophenol phosphate (PNPP) hydrolysis catalyzed by pharmaceutical treatments for patients presenting with this idiopathic dys- bovine intestinal alkaline phosphatases in the presence of various solutes trophy. Currently we know that a cofactor is present in Pyrox-D1, and we such as betaine, sucrose, triethylene glycol and polyethylene glycols. The ef- will use an FAD assay to further understand the structure. A potential ho- fects of these solutes were quantitated using the ‘‘classical’’ small-molecule mologous structure has been created for Pyrox-D1. Two mutations have language of competitive and uncompetitive inhibition. Nearly all the solutes been identified in idiopathic muscular dystrophy patients, which we believe increased the difficulty of enzyme/substrate formation. In other words, they will be found in Pyrox-D1 once we have determined the molecular acted as competitive inhibitors. But at the same time, these same solutes structure. showed a variety of uncompetitive effects ranging from a significant decrease in the rate of product formation rate using sucrose, to noticeable 2899-Pos Board B107 increases in the same product formation rate with polyethylene glycol Enhancing Enzymatic Activity for Cellulose Degradation 1000 and 8000 molecular weight. Using the classical language of small- Hengameh Shams, Mohammad R.K. Mofrad. molecule inhibition provides new perspectives on the effects of crowding University of California, Berkeley, Berkeley, CA, USA. on enzyme kinetics. Cellulose is converted to combustible fuels via cellulolytic enzymes (CE). These enzymes contain carbohydrate-binding modules (CBM) that tune enzy- 2897-Pos Board B105 matic affinity for cellulose in plant walls. Understanding the role of specific Kinetics of Multisite Phosphorylation in the Circadian Clock using Time- residues in binding various CBMs to cellulose can lead to recognizing site- Resolved NMR directed point mutations in wild type CBMs that can assist engineering en- Sabrina R. Hunt, Carrie L. Partch. zymes with desired targeting capabilities. Here, we investigated the mecha- Chemistry and Biochemistry, University of California Santa Cruz, Santa nism by which direct interactions are formed between cellulose binding Cruz, CA, USA. modules of CE and cellulose. After finding all key residues involved in the The mammalian circadian clock is a transcription-translation feedback loop CE-cellulose complex formation, further mutagenesis studies revealed muta- that governs a variety of behavioral and physiological processes with approx- tions that may potentially increase the efficiency of enzymatic activity by re- imately 24-hour periodicity. One clock protein, Period (PER2), functions as a inforcing its anchorage point, which may in turn influence the rate of fuel repressor of its own gene expression. The concentration and subcellular local- production. Our results indicated that most CBMs aligned their aromatic res- ization of PER2 oscillate over the course of the day, regulated in part by dif- idues flat against the hydrophobic surface of cellulose. Conversely, family 3 ferential phosphorylation by casein kinase 1 delta (CK1d). Phosphorylation at CBM did not show such behavior and its contact surface with cellulose was S478 (mouse PER2) promotes proteasomal degradation of PER2, whereas relatively curved. We tried mutations that could change the curvature of the phosphorylation of a competing site, S659, results in the initiation of multisite CBM surface and examined their effect on the cellulose binding affinity. phosphorylation that stabilizes PER2. The S659G mutation blocks down- Interestingly, simulations showed that robust secondary structure of CBM stream phosphorylation to favor S478 phosphorylation and destabilize limited its flexibility for adapting to the cellulose surface. Furthermore, we PER2, which shortens the circadian period and leads to familial advanced investigated the mechanism by which CBMs were driven towards the cellu- sleep phase (FASP) syndrome in humans. People with FASP syndrome are lose surface mainly through entropic forces. The molecular dynamics simula- intrinsically driven to wake up and fall asleep much earlier than the general tion revealed distinct binding mechanisms of different CBM families, which

BPJ 8724_8726 Wednesday, February 21, 2018 585a were governed by both the type of aromatic residues that directly contributed 2903-Pos Board B111 to cellulose association as well as groups of polar residues proximal to the Influence of Conserved Structural Elements of the Proximal Pocket in binding site. HEME-Thiolate Enzymes on Oxygen Insertion Reactions David C. Chatfield, Alexander N. Morozov. 2900-Pos Board B108 Chemistry & Biochemistry, Florida International U., Miami, FL, USA. The Kinetics of Hepatitis C Virus RNA-Dependent RNA Polymerase Inhi- In many heme-thiolate enzymes, the chemical transformations of substrates bition by Nucleoside Analogues occur in a binding site located on the distal side of the heme. The opposite Brian Villalba, Jiawen Li, Kenneth A. Johnson. (proximal) side of the heme is characterized by conserved structural elements Molecular Biosciences, University of Texas at Austin, Austin, TX, USA. that vary subtly from enzyme to enzyme. These structural elements include The Hepatitis C Virus (HCV) currently infects an estimated 71 million peo- hydrogen bonds to the proximal sulfur and the dipole moment of an alpha helix ple worldwide. Currently, there is no vaccine available to treat or prevent that terminates in the proximal cysteine ligand. We present research on two HCV infections. Nucleoside analogues (NA) are a group of drugs that target heme-thiolate enzymes, chloroperoxidase and cytochrome P450CAM, that the HCV RNA-dependent RNA polymerase, NS5B. These compounds work shows that these structural elements have significant influence on a wide range by mimicking natural nucleotides and competing for binding and incorpora- of reactivities (formation of compound I, epoxidation and allylic hydroxylation tion during the replication of the HCV genome. Once incorporated they act of alkene substrates, regioselectivity and even enantiospecificity). The fine- as chain terminators by preventing further elongation of the nascent RNA tuning of these structural motifs can select among closely related modes of strand. Recently, it has been shown that NA can be excised by a reactivity. nucleotide-mediated excision reaction which rescues RNA synthesis. There- fore, in this study, we use transient-state kinetics to measure the ability of 2904-Pos Board B112 three CTP analogues and a UTP analogue to bind and incorporate, as well Insights on Viral DNA Packaging Motor Mechanisms from the Effects of as the ability for the NA to act as a chain terminator. Furthermore, we mea- Motor Residue Changes on Single-Molecule Packaging Dynamics sure NA excision and RNA synthesis rescue by NS5B nucleotide-mediated Douglas E. Smith1, Mariam Ordyan1, Damian delToro1, Jean Sippy2, excision. These results explain the effectiveness of Sofosbuvir in treating Michael Feiss2. 1 HCV infections. Physics, University of California, San Diego, La Jolla, CA, USA, 2Microbiology and Immunology, University of Iowa, Iowa City, IA, USA. 2901-Pos Board B109 Many dsDNA viruses utilize a molecular motor to translocate DNA into pre- Evidence for the Deregulation of Protein Turnover Pathways in ATM-Defi- assembled viral prohead shells. We use optical tweezers to measure the dy- cient Mouse Cerebellum: An Organotypic Study namics of packaging of single DNA molecules by the bacteriophage Catherine Kim. lambda motor. We used site-directed mutagenesis to investigate the roles of Louisiana State University Health Sciences Center, New Orleans, LA, USA. various motor protein residues in translocation function. We found that resi- Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is constitu- due changes in the proposed Walker A and Walker B ATP binding motif and tively elevated in cultured cells and brain tissues obtained from ataxia telangi- catalytic glutamate cause varying impairments, ranging from partial impair- ectasia (A-T) patients. Previous studies from our lab illustrate that elevated ment to total abrogation of DNA translocation activity. Altogether, we studied ISG15 inhibits the canonical ubiquitin pathway, and causes a compensatory in- 23 mutants and identified 11 with no detectable translocation and 12 having crease in basal autophagy for protein turnover in A-T cells. Additionally, gen- varying impairments. The results support the proposed motif assignments. otoxic stress, such as ultraviolet radiation, led to aberrant degradation of Observed changes in motor velocity and pausing and slipping for the partly ubiquitylated proteins due to the overactivation of autophagy in A-T cells. impaired mutants implicates residues involved in ATP binding and posi- More recently, we demonstrated that ISG15 expression is elevated in the cere- tioning, coupling between ATP binding and DNA gripping, and catalysis of bellum, the area of the brain primarily affected in A-T, and other tissues ob- hydrolysis. tained from Atm-deficient mice. ISG15 expression levels were 3-fold higher in the Atm-deficient cerebellum compared to the cerebrum from the same Poster: Intrinsically Disordered Proteins (IDP) mice. Furthermore, similar to the in vitro A-T cell culture model, UV induces and Aggregates III aberrant degradation of ubiquitylated proteins and autophagy in Atm-deficient cerebellar brain slices compared to Atm-proficient (wild-type) cerebellar brain 2905-Pos Board B113 slices. We conclude that the ex vivo organotypic A-T mouse brain culture Interactions between Calcineurin, Tau, and RCAN1-1: A Disordered Trio model mimics the human cell culture model. This model can consequently Trevor P. Creamer, Amanda Wilburn, Daryn Smith. be used to study the role of ISG15-dependent proteinopathy in cerebellar neuro- Molecular and Cellular Biochemistry, University of Kentucky, Lexington, degeneration in A-T. KY, USA. Calcineurin (CaN) is a highly-conserved, heterodimeric Ser/Thr phosphatase 2902-Pos Board B110 that plays vital roles in memory development and retention, cardiac growth, Optimizing Tetrazine Amino Acid Size and Reactivity for Efficient Protein and immune system activation. Alterations in the regulation of CaN have Labelling been noted to contribute the development of Alzheimer’s disease (AD). CaN Subhashis Jana. is known to dephosphorylate the microtubule-binding protein tau, helping to Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA. regulate stabilization of microtubules. There is evidence that Rcan1-1, an Bioorthogonal ligation reactions are essential for labelling biomolecules and endogenous inhibitor of CaN, is overexpressed in AD, suppressing CaN activ- interrogating biomolecular function in vivo and in vitro. Ideally bioorthogonal ity and potentially contributing to the hyperphoshorylation of tau that is a hall- reactions work inside cells, have fast reaction kinetics to compete with biolog- mark of the disorder. Interestingly, CaN contains an essential intrinsically ical process at low concentrations, and show selectivity and stability under disordered region, tau is known to be an intrinsically disordered protein biological conditions. The inverse-electron demand Diels Alder (IED-DA) (IDP), and Rcan1-1 appears to be an IDP. Here we present our initial studies cycloaddition reaction between 3,6-disubstituted-s-terazene and strained cy- of the interactions between CaN and tau, and how these are disrupted by clooctene (sTCO) has recently shown to match ideal biorthogonal reaction Rcan1-1. characteristics. Exploiting this chemistry, we have developed tetrazine func- tionalized non canonical amino acids (ncAA’s) for site-specific incorporation 2906-Pos Board B114 into proteins using genetic code expansion. Maintaining high biorthogonal re- Manipulation of Tau Oligomerization and Aggregation Characterized by action rates with undetectable side-reactions using small tetrazine-ncAA that Time-Resolved FRET don’t compromise protein structure is challenging. To overcome these diffi- Chih Hung Lo1, Tory Schaaf2, Benjamin Grant3, Colin Kin-Wye Lim1, culties, we have developed synthetic routes to a family of tetrazine-ncAA, David Thomas2, Jonathan Sachs1. including the smallest possible Tetrazine-ncAA, and characterized their 1Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA, reactivity with sTCO. To illustrate the utility of these new ncAA for labelling 2Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, proteins we have genetically incorporated a family of Tetrazine-ncAA Minneapolis, MN, USA, 3Fluorescence Innovations, Minneapolis, MN, USA. site-specifically into proteins via genetic code expansion with orthogonal We have used a novel time-resolved fluorescence resonance energy transfer Tet-synthetase/tRNACUA pairs (Tet-RS/tRNA). The new tetrazine-ncAA con- (FRET) assay to detect and manipulate tau oligomers and aggregates that taining protein which shown efficient reactivity and selectivity with sTCO and play a significant role in tauopathy, which belongs to a class of neurodegen- will advance our ability to label proteins for protein engineering and biolog- erative diseases that includes Alzheimer’s disease and Pick’s disease. Tauop- ical studies in vivo. athy is associated with the formation of neurofibrillary tangles (NFTs)

BPJ 8724_8726 586a Wednesday, February 21, 2018 caused by the accumulation of hyperphosphorylated tau. Although many was observed with changing pH in the range 3—11, which correlated with believe that NFTs are the main histopatholgocial hallmark of these diseases, changes in secondary structure of PEST fragment. Importantly, absorbance at recent studies suggested that NFTs do not play the main role as toxic entities 280 nm, which is often employed as a measure of protein concentration, was leading to progression of disease. Instead, the tau oligomer, an intermediate profoundly altered by changes in ProCharTS intensity in response to changing form of tau prior to NFTs formation, has been proposed to be the true species pH in Dehydrin. ProCharTS intensity was sensitive to temperature induced leading to toxicity. However, the mechanisms by which tau oligomers changes in the secondary structure of PEST fragments between 25—85oC. trigger neurodegeneration remain unknown. In this study, we have engi- Finally, increase in ProCharTS absorbance with time, in HEWL at pH 2, neered a cell-based tau FRET biosensor to study tau-tau interactions with directly correlated with growth of HEWL aggregates and amyloid fibrils as an emphasis on elucidating the mechanistic contribution of different struc- confirmed by rising thioflavin T fluorescence. Taken together, our work high- tural states of the protein to cell cytotoxicity. As an initial control to validate lights the utility of ProCharTS absorption as a novel label-free intrinsic probe to our approach, we monitored tau-tau interactions in the absence and presence monitor changes in protein charge, structure and oligomeric state. of aggregation inducers such as glycogen synthase kinase 3b, forskolin, and okadaic acid, to obtain a FRET signal characteristic of oligomers and aggre- 2909-Pos Board B117 gates. We confirmed the structural states of tau proteins by thioflavin S stain- Prying into Hydration Water in Amyloidogenic Intrinsically Disordered ing and tested their correlations with toxic effects in cells with cell Proteins 1 2 3 cytotoxicity assays. Our time-resolved FRET measurements are used to Samrat Mukhopadhyay , Shruti Arya , Karishma Bhasne , 2 4 4 4 investigate any potential connection between cell-level observation and the Priyanka Dogra , Avinash K. Singh , Tuhin Khan , Anindya Datta , Payel Das5. biophysics of tau oligomerization and aggregation. This work was supported 1 by NIH grant (R01AG053951). Centre for Protein Science, Design and Engineering and Departments of Biological & Chemical Sciences, Indian Institute of Science Education and 2907-Pos Board B115 Research (IISER), Mohali, Mohali, India, 2Department of Chemical A Temperature-Controlled Stopped-Flow Droplet-Based Microfluidic Sciences, Indian Institute of Science Education and Research (IISER), Reactor for Fast Biomolecular Kinetics Mohali, Mohali, India, 3Department of Biological Sciences, Indian Institute Tianjin Yang, Stavros Stavrakis, Paolo Arosio, Andrew deMello. of Science Education and Research (IISER), Mohali, Mohali, India, Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland. 4Department of Chemistry, Indian Institute of Technology Bombay, Powai, Several important biomolecular reactions, including folding/unfolding, aggre- Mumbai, India, 5Data Science Department, IBM Thomas J. Watson Research gation, and binding, occur on a time scale of sub-seconds or seconds. One rele- Center, Yorktown Heights, NY, USA. vant example is represented by microscopic reactions underlying, the formation The hydration water layer, termed as ‘biological water’, present at the protein- of amyloid fibrils from normal soluble proteins and peptides, which is associ- water interface shares an intimate relationship with protein functions. Howev- ated with the onset and progression of Alzheimer’s and Parkinson’s diseases. er, little is known about hydration water in intrinsically disordered proteins The protein aggregation process consists of a network of several microscopic (IDPs) that are capable of adopting a multitude of conformational states. steps of nucleation and growth. Each individual step has different contributions Recent studies have revealed that many IDPs, due to their astonishing confor- to the formation of the final fibrils and of the soluble intermediates generated mational plasticity, can readily convert into highly ordered amyloid aggre- during the reactions, which are currently thought to represent the most toxic gates that are associated with several deadly human diseases. For instance, species. The characterization of individual reactions in a sub-second time scale a-synuclein is an IDP present in nerve cells and aggregates into amyloids is challenging with the current available biophysical techniques. In this work, via toxic oligomeric intermediates in Parkinson’s disease. To address the we leverage the power of droplet-based microfluidics to quantitatively explore role of water in the pathological conversion of a-synuclein, we utilized a com- aggregation kinetics within a time window of milliseconds to seconds. Herein bination of ultrafast measurements and all-atom molecular dynamics simula- we present a universal droplet-based microfluidic platform for rapid and high- tions. We created a number of single cysteine mutants and labeled these precision kinetics using stroboscopic illumination and fluorescence detection variants using environment-sensitive thiol-active fluorophore. We then moni- within a single experiment. The microfluidic platform is also integrated with tored the solvation dynamics using ultrafast fluorescence spectroscopy that is a heating element at the reactor zone, which is isolated from the droplet gener- capable of capturing the femtosecond time-resolved snapshots of water mol- ation and mixing part. The droplets can be heated up rapidly to a set tempera- ecules around the polypeptide chain. These site-specific ultrafast studies al- ture (within a range of 24 to 90C) once they enter the heating zone enabling lowed us to construct the hydration map of a-synuclein. Our studies fast biomolecular kinetics to be extracted at different temperatures. We prove revealed the presence of three distinct types of water: bulk-, hydration- and the potential of this platform with two systems: the denaturation kinetics of hen confined water. Atomistic molecular dynamics simulations supported our egg white lysozyme under various temperatures, and the elongation rate of fi- experimental observations and provided insights into the molecular origin brils composed of the peptide Abeta42, the peptide associated with Alzheimer’s of confined water. Our results from this experimental-simulation approach disease. reveal trapping of interfacial water near the amyloidogenic hotspot that trig- gers the conversion of IDPs into pathological amyloids via entropically favor- 2908-Pos Board B116 able water exclusion events during disorder-to-order amyloid transition. Our Protein Charge Transfer Absorption Spectra: An Intrinsic Probe to findings provide an experimental underpinning of significant water rearrange- Monitor Structural and Oligomeric Transitions in Proteins ments associated with both chain desolvation and water confinement upon 1 1 1 Mohd. Ziauddin Ansari , Amrendra Kumar , Dileep Ahari , amyloid formation from IDPs. I will also discuss our new results on the hy- 1 2 2 Anurag Priyadarshi , Padmavathi Lolla , Rashna Bhandari , dration dynamics of a diverse range of amyloidogenic IDPs possessing Rajaram Swaminathan1. 1 different conformational preferences due to varied chain-chain and chain- Biosciences and Bioengineering, Indian Institute of Technology Guwahati, solvent interactions. Guwahati, India, 2Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India. 2910-Pos Board B118 Protein Charge Transfer Spectra (ProCharTS) originate when charged amino/ Utilzing Forster Resonance Energy Transfer (FRET) and Photocrosslink- carboxylate groups in Lys/Glu, act as electronic charge acceptors/donors for ing to Visualize Conformational Changes of Alpha-Synuclein photoinduced electron transfer either from/to the polypeptide backbone or to John J. Ferrie1, Conor M. Haney1, Jimin Yoon1, Buyan Pan1, each other (Prasad et. al., 2017, DOI: 10.1039/c7sc00880e). The absorption Elizabeth Rhoades1, Abhinav Nath2, E. James Petersson1. band intensities in ProCharTS (250—800 nm) are dependent on 3D spatial 1Chemistry, University of Pennsylvania, Philadelphia, PA, USA, 2Medicinal proximity between charged functional groups across the protein. Intrinsically Chemistry, University of Washington, Seattle, WA, USA. disordered proteins (IDPs) are rich in charged amino acids, but lack structure Intrinsically disordered proteins (IDPs) make up an expanding category of pro- promoting intrinsic spectral probes like Tyr or Trp in their sequence, making teins defined by their conformational heterogeneity. Of these, a-Synuclein their structural characterisation difficult. Here, we exploit, the richness of (aS), like many other IDPs, has been recognized for its ability to self associate charged amino acid population among IDPs to sense structural transitions to produce cytotoxic oligomeric and fibrillar species, which have been shown to among multiple IDPs (like PEST fragment of human c-Myc, its mutant and De- play a causative role in Parkinson’s Disease (PD). Despite the recognized role hydrin from maize) using ProCharTS absorption spectra. Conformational of aS in PD, there has yet to emerge consensus models of the disordered changes induced in the IDP by altering pH/temperature of aqueous medium ensemble or fibrillar species of this protein. Although these structural studies was monitored by ProCharTS and confirmed by CD spectra. Further, utility have been dominated by the use of NMR, we focus on using techniques such of ProCharTS to detect protein aggregation was examined using Hen Egg- as Fo¨rster Resonance Energy Transfer (FRET) and photocrosslinking, which White Lysozyme (HEWL) protein. Significant changes in ProCharTS spectrum can be applied in both in vitro and in cell-based experiments. As a proof of

BPJ 8724_8726 Wednesday, February 21, 2018 587a concept, we have first directed our attention towards visualizing the compaction 2913-Pos Board B121 of aS induced by trimethylamine-N-oxide (TMAO). After generating a library Improved Structural Estimation of Disordered Proteins by CD Spectros- of analogs consisting of two probe pairs, sensitive to two different distance copy: Method Development and Application ranges, ensemble FRET measurements were performed to collect a set of intra- Andra´s Micsonai1, Nikoletta Murvai2,E´ va Bulya´ki1, Bea´ta Szabo´2, molecular distances. These distances were utilized as constraints within Monte Frank Wien3, Young-Ho Lee4, Matthieu Refregiers3, Yuji Goto4, Carlo simulations to generate models of the TMAO-compacted ensemble. Peter Tompa2, Kyou-Hoon Han5,A´ gnes Tantos2, Jo´zsef Kardos1. Furthermore, in separate experiments, we demonstrate that photocrosslinking 1Department of Biochemistry, ELTE Eo¨tvo¨s Lora´nd University, Budapest, can be effectively used to identify the binding sites of small molecules to the Hungary, 2Research Centre for Natural Sciences, Hungarian Academy of surface of aS fibril. Lastly, we highlight the use of these techniques within Sciences, Budapest, Hungary, 3Synchrotron SOLEIL, Gif sur Yvette, France, aS fibrils and demonstrate the use of these data in refining already existing fibril 4Institute for Protein Research, Osaka University, Osaka, Japan, 5Korea models. Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea. 2911-Pos Board B119 There is a growing body of evidences showing the importance of intrinsically High-Speed AFM to Characterize Nanoscale Dynamics of Cross-Linked unstructured proteins (IUPs) and disordered protein segments in a large variety ABETA42 Oligomers of processes of the living cell. Circular dichroism (CD) spectroscopy is an inex- Siddhartha Banerjee1, Zhiqiang Sun1, Eric Y. Hayden2, David B. Teplow2, pensive and fast technique for the study of protein secondary structure. It is Yuri L. Lyubchenko1. beneficial when the use of high-resolution techniques, such as X-ray or NMR 1Department of Pharmaceutical Sciences, University of Nebraska Medical 2 is difficult, such as the case of IUPs. However, the generally used CD spectrum Center, Omaha, NE, USA, University of California, Los Angeles, Los analyzing algorithms often overestimate the b-sheet content making the study Angeles, CA, USA. of disordered proteins problematic. Recently, we developed a new method Spontaneous aggregation of amyloid beta (Ab) proteins into oligomers and called BeStSel (http://bestsel.elte.hu) for the secondary structure estimation eventually into fibrils has been established as the hallmark for Alzheimer’s and fold recognition from the CD spectra (Micsonai et al., PNAS (2015) disease pathogenesis (1). Rather than fibrils, oligomers have been found out 112, E3095-E3103.), which provided superior results for any of the secondary to be most neurotoxic species (2). Even the dimers isolated from the brain structure elements compared to other methods. In the present work, we have showed the toxicity. As a consequence, detailed investigation of structure further developed this method for unstructured proteins. To extend the refer- and dynamics of the oligomers are essential to develop oligomer specific ence database, we collected CD spectra of disordered proteins and modelled therapeutic agents. The major problem in this regard remains the transient their structure by MD simulations. Cross-validated statistics proved that the nature of the oligomers due to their spontaneous aggregation into higher or- re-optimized algorithm can distinguish the otherwise spectrally similar disor- der oligomers. One approach to address this problem has been photo- dered structure from the highly twisted b-sheets. The method was applied for induced cross-linking of Ab42 peptides (3). This method provides isolated, the structural study of various IUPs and their interactions, e.g., a-synuclein var- stable oligomers in pure form, which can be used for further biophysical iants, p53 TAD. Acknowledgements: The work of J.K. and A.M. was supported studies. Here, we have investigated the structure of trimer, pentamer and by the Hungarian National Research, Development and Innovation Office heptamer with atomic force microscopy (AFM) imaging and probed their (grants K_120391, KH_125597 and TE´ T_16-1-2016-0134) and the Ja´nos Bo- dynamics by applying time-lapse high-speed AFM imaging. This technique lyai Research Scholarship program of the Hungarian Academy of Sciences. provides direct visualization of nanoscale dynamics of the oligomers at millisecond time scale. Results indicate that the pentamer and heptamer 2914-Pos Board B122 are dynamic species, spontaneously unfolding into lower order oligomers Measurements of Aggregation Propensities of Amyloid Peptides by Real like dimer/trimer and folds back into single globular assemblies, whereas Time Monitoring of Growth of the Aggregates using Total Internal Reflec- trimer remains in single unit compact structure. These dynamic features tion Fluorescence (TIRF) Microscopy of oligomers shed a new light on the role of oligomer sizes on their neuro- Subhas C. Bera, Shamasree Ghosh, Timir Baran Sil, Kanchan Garai. toxic properties. TIFR Centre for Interdisciplinary Sciences, Tata Institute of Fundamental Ref: (1) Semin. Cell Dev. Biol. (2004), 15, 3-16; (2) Nature (2002), 416 (6880), Research, Hyderabad, India. 535-9; (3) Anal Biochem (2017), 518, 78-85. Aggregation of amyloid proteins are involved in the pathology of multiple hu- This work is supported by National Institutes of Health grants GM096039 and man diseases. In vitro, growth of the amyloid fibrils has been shown to occur GM118006 to YLL. via multiple competing pathways such as primary and secondary nucleation, and by fragmentation. Knowles and collaborators have developed a theoretical 2912-Pos Board B120 framework for estimating the rate constants of these processes from the kinetics Examining the Nanosecond-to-Millisecond Dynamics of Sic1 by Fluores- of aggregation measured at different concentrations of the amyloid peptides. cence Techniques Here, we use an alternative method for estimation of the rate constants of ag- John Darvy M. Castroverde1,2, Taehyung Chris Lee1,2, gregation of amyloid-b (Ab) peptides, both Ab40 and Ab42, by real time moni- Gregory-Neal W. Gomes1,2, Julie D. Forman-Kay3,4, Claudiu C. Gradinaru1,2. 1 2 toring of the growth of the individual amyloid fibrils using TIRF microscopy. Physics, University of Toronto, Toronto, ON, Canada, Chemical and The aggregation experiments are performed on supported lipid bilayers pre- Physical Sciences, University of Toronto, Mississauga, ON, Canada, pared on glass coverslips to mimic surface of cellular membranes and to avoid 3Molecular Structure and Function Program, Hospital for Sick Children, 4 artifacts induced by glass surfaces. Fibrillar aggregates are visualized by fluo- Toronto, ON, Canada, Biochemistry, University of Toronto, Toronto, ON, rescence of thioflavin-T (ThT). We find that Ab40 forms primarily unbranched, Canada. long fibrils but Ab42 forms short but highly branched fibrils. Additionally, sur- As part of the cell cycle of the budding yeast Saccharomyces cerevisiae, the face of the Ab40 fibrils appear smooth but Ab42 fibrils show rough surfaces. cyclin-dependent kinase inhibitor Sic1 is degraded by Cdc4 of the SCF com- Analysis of the time dependent increase of the number, length and the number plex upon the phosphorylation of Sic1 on at least six sites (known as Cdc4 of branches of the fibrils indicates that the rate constant of elongation is higher phosphodegrons, or CPDs). More specifically, the binding affinity of Sic1 to for Ab40 but the rate constants of both primary and secondary nucleation are the WD40 binding domain of Cdc4 increases as the number of phosphorylated higher for Ab42. Additionally, rate constants of growth of different fibrils sites increases, especially after the sixth site. Experimental work is necessary to are found to be considerably heterogeneous even for the same peptide. No frag- study the structural dynamics of Sic1 as it undergoes phosphorylation and mentation of the fibrils was observed in our experiments most likely due to the binding. quiescent growth conditions. We conclude that TIRF microscopy is a powerful We used fluorescence techniques to study the dynamics of the disordered N- tool to understand mechanisms of growth of the amyloid peptides on lipid terminal targeting tail of Sic1 (residues 1 to 90), phosphorylated Sic1, and the bilayers. Sic1-WD40 dynamic complex. Using a combination of fluorescence correla- tion spectroscopy and time-resolved fluorescence anisotropy, we measure 2915-Pos Board B123 the chain dynamics of Sic1 in the nanosecond-to-millisecond timescales. Electrostatic Interactions to Guide the Self-Assembly of Highly Ordered Additionally, we perform these experiments on six different single cysteine Amyloid-Like Nanostructures Sic1 mutants to probe the segmental dynamics of the regions near the Ximena Zottig1, Soultan Al-Halifa1, Miche`le Auger2, Steve Bourgault1. CPDs. Data from these initial fluorescence experiments would form a basis 1Chemistry, UniversiteduQuebec a` Montreal, Montreal, QC, Canada, for the interpretation of subsequent single-molecule data to elucidate the phys- 2Chemistry, Universite Laval, Quebec, QC, Canada. ical basis for the ultrasensitive phosphorylation-dependent binding of Sic1 to The design of self-assembled biomolecular scaffold is of great interest for Cdc4. applications in various areas such as drug and gene delivery, vaccine

BPJ 8724_8726 588a Wednesday, February 21, 2018 design, tissue engineering, enzyme catalysis and biosensors. Polypeptides become significant at high temperature (350 K) at which most proteins un- that can self-assemble into non-covalent amyloid-like assemblies offer dergo thermal unfolding. At room temperature, these interaction energies many advantages to generate tailored biomaterials and nanoparticles are less pronounced. We utilized the obtained interaction parameters in an including; functionalization, mechanical resistance similar to spider silk, Ising-type approach to calculate the temperature dependence of the UV circu- biocompatibility and enzymatic stability. Nonetheless, the control over lar dichroism (CD) of the so-called MAX3 peptide that is predominantly built the self-assembly and the difficulty of predicting the final supramolecular by KV-repeats. The agreement between simulation and experimental data is architecture from the peptide sequence constitute major issues. Addition- very good. Finally, we analyzed the temperature dependence of the CD ally, polymorphism and structural heterogeneity that are endogenous char- spectra and selected 3J(HNHa ) parameters of the Ab(1-9) fragment. The re- acteristics of materials produced by a bottom-up process are key challenges sults corroborated the occurrence of anti-cooperative nearest neighbor interac- that remained to be addressed. In this study, we report the preparation of tions. A comparison of CD spectra of unfolded and intrinsically disordered rod-like nanoparticles based on an amyloidogenic peptide backbone with proteins with predictions of our model suggests that anti-cooperative interac- a high homogeneity of size and shape. Short and planar fibrils (average tions govern their experimentally obtained temperature dependence. Gener- length of 5 150 nm) were obtained from a building block strategy that ex- ally, our results suggest that unfolded peptides and proteins behave like ploits the intrinsic ability of an amyloidogenic core connecting to a charged antiferromagnetic systems in the presence of a magnetic field of intermediate amino acid by a GSGS tetrapeptide spacer. Amyloid nanoparticles were strength. characterized by transmission electron microscopy, atomic force micro- scopy and dynamic light scattering. Moreover, circular dichroism and 2918-Pos Board B126 Fourier-transform infrared spectroscopy revealed an overall parallel b-sheet Supercharging as a General Strategy for Making Proteins into Conforma- fibril structure. A strong diffraction pattern with characteristic signals at 4.2 tional Switches and their Use in Biosensing 1 2 2 A˚ and at 8.2 A˚ was observed by X-ray diffraction, confirming the cross-b- Peter J. Schnatz , Joseph M. Brisendine , Ronald L. Koder . 1The Graduate Center, CUNY and City College of New York, New York, sheets quaternary structure of the assemblies. This rod-like amyloid fibril 2 preparation with a unique low polydispersity opens new direction in the NY, USA, City College of New York, New York, NY, USA. application of protein nanoparticles in nanomedicine and biotechnology. Greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into a ligand-gated or counterion- 2916-Pos Board B124 gated conformational molecular switch. To demonstrate this we first modi- Effect of Neighbouring Residues in Conformational Plasticity of Intrinsi- fied the designed helical bundle hemoprotein H4 using simple molecular cally Disordered Proteins modeling to create a highly charged protein which both unfolds reversibly Sushmita Basu, Ranjit Prasad Bahadur. at low ionic strength and undergoes the ligand-induced folding transition Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India. commonly observed in signal transduction in biology. To demonstrate Effect of neighbouring residues in conformational plasticity of intrinsically this process using more complex proteins, we then modified green fluores- disordered regions. The concept of unstructured proteins has opened new cent protein and the cytochrome B562, using a combination of simple avenues in the field of structural biology. Intrinsically disordered proteins modeling and electrostatic calculations to create proteins that unfold revers- (IDPs) are the new class of proteins which have been found to be a major ibly with decreasing ionic strength. These simple model systems allow us to player in many significant cellular functions. IDPs have been characterised derive and then experimentally validate a mass-action model for the by its physicochemical properties as well as its molecular interaction coupled folding and binding behavior of ligand-gated conformational behaviour. Detailed study of IDPs can lead to a better understanding of pro- switches, establishing a set of engineering principles which can be used tein folding and its functioning. To understand the source of disorderedness to convert natural and designed soluble proteins into molecular switches in the disordered regions (IDRs) in IDPs, we studied how the sequence useful in biodesign and synthetic biology. We have applied these principles environment of a disordered region correlates to its randomness. Here, to a biosensing project in which supercharged IDPs are attached to nano- we analysed the physicochemical and structural features like amino acid structured gold surfaces and conformational changes are sensed using sur- propensities, net charge, hydropathy index, secondary structure propensity, face plasmon resonance. An increase of the refractive index at the gold relative surface accessibility, interaction density and H-bonds to character- surface is caused by the ligand induced conformational change and can ise the neighbours of the IDRs. Five residues, each towards N and C termi- be detected via transmission surface plasmon resonance (SPR) spectros- nal of the disordered region are considered as the neighbours of IDRs. copy. We calculate that the shift in the resonance wavelength of the surface These neighbouring residues are found to be enriched in disorder promoting plasmons is almost two orders of magnitude more than simple ligand bind- amino acids and have higher propensity to form loops than other secondary ing to an already folded protein. Continuing SPR studies provide practical structures. Solvent accessibility of neighbouring residues also showed insight into the use of our model as conformational switches for biosensing increasing trend as we move towards the IDRs. The variation of other pa- devices. rameters along with the above observation indicates that the neighbouring residues of IDRs induce a degree of flexibility to the adjoining IDRs. Based 2919-Pos Board B127 on our findings, we are designing an algorithm using random forest, which Advances in Quantitative Analysis of Intracellular Protein Phase Separa- shall predict the disordered region based on its neighbouring sequences. tion Kinetics and its Modulation by Cellular Interactions Ammon E. Posey1, Tejbir Kandola2, Rohit V. Pappu1, Randal Halfmann2. The information on IDRs and its neighbours can be useful for proteins to 1 be expressed or characterised for the first time. It can also provide a lead Department of Biomedical Engineering & Center for Biological Systems Engineering, Washington University in St. Louis, St. Louis, MO, USA, in understanding the molecular mechanism behind the polymorphic interac- 2 tions that are involved with IDPs. Stowers Institute for Medical Research, Kansas City, MO, USA. Flow cytometry-based Fo¨rster Resonance Energy Transfer measurements 2917-Pos Board B125 are a promising new approach for high-throughput measurements of the ki- Anti-cooperative Nearest Neighbor Coupling Determines the Statistical netics of intracellular phase behavior of proteins such as prions and other Coil State of Peptides and Proteins at High Temperatures nucleation-limited self-assembling systems in cellular environments. Chal- Reinhard Schweitzer-Stenner1, Siobhan E. Toal2. lenges arise in interpreting these data because experimental constraints 1Chemistry, Drexel University, Philadelphia, PA, USA, 2Chemistry, make it difficult to gather continuous kinetic traces or collect data under University of Pennsylvania, Philadelphia, PA, USA. conditions where the concentration of the protein of interest is held con- The interest in the unfolded state of peptides and proteins stems partially from stant, which is the domain of applicability of classical nucleation theories. the observation that conformational distributions of amino acid residues are Here, we present advances in experimental design, statistical analysis and more restricted and residue specific than expected for an ideal random coil. the development of a theoretical framework based on extending classical Moreover, multiple lines of evidence suggest that contrary to the isolated nucleation theory to overcome many of the aforementioned challenges pair hypothesis the conformational propensity of a distinct residue depends and enable extraction of meaningful insights regarding the nucleation and on the properties of its neighbors. We analyzed the recently reported confor- propagation of protein phases in yeast. These advances will enable future mational propensities of the two guest residues of GxyG peptides in terms of a systematic and quantitative comparisons of a variety of proteins, including statistical thermodynamics model that allows for cooperative as well as anti- prion-like domains and other aggregating proteins under a wide range of cooperative interactions between adjacent residues adopting either a polypro- cellular conditions. This offers a path forward for quantitative understand- line II (pPII) or a b-strand conformation. Our analysis revealed that the near- ing of the determinants of protein self-assembly, phase separation dy- est neighbor interactions between most of the central residues in the namics, and the impact of cellular factors as modulators of intracellular investigated GxyG peptides is anti-cooperative. Interaction Gibbs energies phase behavior.

BPJ 8724_8726 Wednesday, February 21, 2018 589a

2920-Pos Board B128 Some low molecular weight di- or tripeptides with aromatic residues and Charged Side Chain Mutations of CaMKII Peptide Alter Binding Affinity terminal groups have been shown to form gels. Contrary to expectations, for CaM through Creation of Non-local Alternate Binding Contacts we recently discovered that cationic glycylanalylglycine (GAG), a tripeptide Jacob Ezerski1,2, Pengzhi Zhang1, Margaret Cheung1,2. of low hydrophobicity, forms a gel in 55 mol% ethanol/45 mol% water at 1Physics, University of Houston, Houston, TX, USA, 2Rice University, room temperature if the concentration exceeds 200 mM. The underlying Houston, TX, USA. structure is comprised of unusually long crystalline fibrils (in the 10-5m Calmodulin (CaM) trapping is a phenomenon where the active and inactive range), which do not exhibit the canonical b-sheet structure. Rheological states of calmodulin dependent protein kinase II (CaMKII) produce drastically data and vibrational circular dichroism spectra suggest the existence of þ different affinities for Ca2 saturated CaM. The states of CaMKII are para- two different gel phases, one formed between 15 and 35Cwithlefthanded mount in the scheme of calcium ion signaling, which is an essential biological twisted fibrils and G’ values at ca. 2*104 Pa and another one formed below function whose underlying mechanism is largely unknown. Experimentally, a 15C with right handed twisted fibrils and G’ values close to 105 Pa. Results set of peptides modeled after CaMKII’s binding domain (293-312) were from DFT calculations indicate that the two phases might be underlied by created through systematic mutations of charged residues to mimic the two rather differently structured fibrils. The fluorescence kinetics probing the distinct affinity states for CaM and probe the mechanisms responsible for the incorporation of thioflavin T into the hydrophobic interior of fibrils indicate observed change in kinetics. Although a model was successfully created, the a retarded diffusion of the fluorophore into fibrils that formed rather quickly observed interactions could not be explained through current protein interaction after incubation above 15C, while fluorescence increase and gelation pro- models or electrostatic steering effects. We investigate the dynamics of this ceed on a similar time scale for the gel phase formed below this tempera- experiment through the use of all atom simulations, choosing three of the ture. Upon increasing the temperature, it can preserve this capability until mutant peptides with a length of 20 amino acids. We refer to these peptides the melting temperature is reached, which suggests that this gel phase has by the 296-298 residues, specifically RRK (wildtype), RAK (1-residue muta- all what it takes to function as a drug delivery system. We care currently tion) and AAA (3-residue mutation), and validate our simulation findings working on optimizing the gelation/melting conditions for specific biotech- through comparison with circular dichroism (CD) data. We demonstrate that nological applications of the gel. the large side chains present in the first 6 residues of each peptide interact with each other, and that the mutation of charged residues produce global 2923-Pos Board B131 changes in sidechain conformations and disordered regions. The specific Phase Separation of the Variable Domain of Dynamin Related Protein 1 in ensemble of secondary structure/rotamer conformations present in RRK, TMAO Suggests a Role in Assembly that are not observed in AAA, are essential for high affinity binding between Blake Hill1, Ammon Posey2, Mehran Bagheri3, Nolan Kennedy1, CaMKII and CaM. James Harden3. 1Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA, 2921-Pos Board B129 2Washington University, St Louis, MO, USA, 3Physics, University of Designing Ligands for Structure-Less Proteins Ottawa, Ottowa, ON, Canada. Anirban Das1, Anju Yadav1, Barun Kumar Maity1, Bappaditya Chandra1, The Dynamin superfamily is a class of large GTPases that perform essential Alexander Korn2, Juniane Adler2, Sri Rama Koti Ainavarapu1, membrane remodeling events via self-assembly stimulated GTP hydrolysis in Daniel Huster2, Sudipta Maiti1. a manner that involves the coordination of lipid-membrane interaction, high- 1Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India, order oligomerization, and GTP hydrolysis, however a clear understanding of 2Institut fu¨r Medizinische Physik und Biophysik, Universitat Leipzig, how these functions are coordinated has remained elusive. A ‘‘variable Leipzig, Germany. domain’’ (VD) is present in many superfamily members and may enable spe- Disordered proteins can play physiologically important roles, but can also cific targeting to sites of action, but the nature of VD function is unclear in lead to major diseases especially when they aggregate. Designing small most dynamins. Dynamin-related protein 1 (Drp1) is the primary mechanoen- molecule / peptide ligands for disordered proteins is a difficult challenge, zyme responsible for mitochondrial fission. We seek to identify the role of the since structure cannot be used as a guide. Here we demonstrate effective ap- Drp1 VD, or VD. We find that removal of the VD from Drp1 results in proaches for three major cases: 1) for partially disordered proteins, we iden- enhanced assembly and GTP hydrolysis, suggesting that the VD has an tify key structured portions and design ligands for disrupting those, 2) for auto-inhibitory role. We find that the VD is intrinsically disordered (ID) mostly disordered proteins, we measure spontaneous fluctuations and use and surprisingly, undergoes phase separation or coacervation under conditions that as an assay to quantify transient interactions with candidate ligands, that induce other ID proteins to fold. The same conditions that induce phase and 3) for unfolded states of well-folded proteins, we use the folding nucleus separation of the VD also enhance binding to cardiolipin, a lipid that is unique to design an artificially stabilized exo-nucleus which can induce folding. We to the mitochondrial membrane. Based on these findings we suggest a model demonstrate the first strategy with amyloid beta (Ab), whose aggregation is in which the VD inhibit Drp1 assembly by dynamically occluding assembly associated with Alzheimer’s disease. Using a combination of fluorescence interfaces, and that this auto-inhibition is relieved upon interaction of the correlation spectroscopy (FCS), Forster resonance Energy transfer (FRET) VD with cardiolipin at the mitochondrial membrane, involving the process and solid state NMR, we show that a small peptide designed to specifically of coacervation. This model may also be applicable to other members of disrupt a distal intra-molecular contact can drastically alter the membrane the dynamin superfamily, and intrinsically disordered membrane remodeling affinity of the Ab oligomers. We demonstrate the second strategy with proteins in general. IAPP (amylin), whose aggregation is associated with Type II diabetes. We show that ns-timescale spontaneous fluctuations of IAPP, measured using 2924-Pos Board B132 FRET and FCS, can provide a sensitive assay for interactions of IAPP olig- Prevention of Aggregation/Fibrillation of Human Serum Albumin by omers with small molecule drug candidates. The third case is demonstrated Surfactant and Anti-inflammatory Drug Under Physiological Conditions: by designing small peptides which can specifically perturb the folding ki- Biophysical Aspects netics of GdnHCl-unfolded ubiquitin. We construct a chemically stabilized Achal Mukhija, Nand Kishore. peptide fragment that mimics the folding nucleus of ubiquitin. We show Chemistry, Indian Institute of Technology, Bombay, India, Mumbai, India. that this peptide can accelerate the folding of ubiquitin. Overall, here we An anomaly in the protein folding process can lead to aggregation or fibrillation demonstrate ‘‘dynamics’’ based methods which can help in designing drugs of proteins which have been related to neurodegenerative and peripheral dis- for disordered proteins. eases. Therefore, it is important to understand the mechanism of prevention of aggregation/fibrillation and to design suitable inhibitors for this process. 2922-Pos Board B130 Literature information suggests that most of the work on these systems has Exploring the Tunability of the Aggregation and Gelation Process of been done on heat induced fibrils (57Cto60C). As a step ahead, in the present Tripeptides study, efforts have been made to understand inhibition process under physio- David DiGuiseppi1, Reinhard Schweitzer-Stenner1, Nicolas Alvarez2. logical conditions (37C, pH-7.4) which is more relevant to the fibrils formed 1Chemistry, Drexel University, Philadelphia, PA, USA, 2Chemical and under natural cellular environment. The qualitative and quantitative aspects of Biological Engineering, Drexel University, Philadelphia, PA, USA. the interactions of the surfactant sodium dodecyl sulfate and anti-inflammatory The self-assembly of biomolecules is an important issue explored in drug diclofenac sodium (DCF) with human serum albumin at different stages of biomedical, biophysical, and bio-material research. Understanding how the fibrillation process have been studied employing a combination of spectro- and why certain peptides/proteins prefer to self-assemble into larger net- scopic, calorimetric and microscopic techniques. Fibril formation understudied works can reveal the mechanism of amyloid formation and assist in conditions was confirmed by TEM images and Thioflavin T binding assay bottom-up designs of supramolecular structures like gels and nanotubes. along with DLS measurements. Energetics from isothermal titration

BPJ 8724_8726 590a Wednesday, February 21, 2018 calorimetry provided insights into the nature of interactions and mechanism of 2927-Pos Board B135 inhibition. We found inhibition efficiency in the order, micellar SDS > 45 mM Multi-site Phosphorylation Modulates the Conformation and Electrostatic DCF > monomeric SDS > 5 mM DCF. The energetics of interaction, corre- Response of Intrinsically Disordered Protein Brushes lated with the molecular structure of inhibitors provide guidelines for effective Ruoxing Lei. synthesis and design of inhibitors. ITC results have imparted important rela- Chemistry, University of California, Berkeley, Berkeley, CA, USA. tionship between inhibition efficiency and exothermicity of interactions and Intrinsically disordered proteins (IDP) lack 3D structure, which endows have demonstrated the significance of polar interactions in fibril prevention them with unique structural flexibility and facilitates their function as pro- by these inhibitors. Interestingly it was found that the micellar SDS not only tein interaction hubs. While IDP phosphorylation can induce functionally inhibits the process but also effectively disintegrates the formed fibrils. significant structural changes, the mechanistic relationship between phos- phorylation and IDP conformation remains unclear, particularly for multi- 2925-Pos Board B133 phosphorylated IDPs. Here we address this question by combining enzy- Dynamics Based Drug Design for Intrinsically Disordered Proteins matic phosphorylation in a model IDP with atomic force microscopy Barun K. Maity. (AFM) measurements of IDP conformation. We chose the recombinant Department of Chemical Science, Tata Institute of Fundamental Research, C-terminal sidearm domain of the neurofilament heavy subunit (rNFH- Mumbai, India. SA), which has a highly polyampholytic (zwitterionic) sequence but ac- How does one design a small-molecule ligand for a disordered protein quires significant polyanionic character through phosphorylation of its 50 (IDP)? Typical structure-based drug design approaches are not applicable serine residues, which is known to influence NF network assembly in to IDPs, since most regions of IDPs do not possess specific structures in vivo. To manipulate NFH-SA phosphorylation, we developed an in vitro physiological conditions. This is a critical problem, since many IDPs are phosphorylation strategy using mitogen-activated protein kinase 1 (ERK2) associated with serious human diseases (such as Alzheimer’s and Parkin- pre-activated by mitogen-activated protein kinase kinase (MKK). We son’s). Here we propose that drug discovery in such cases can rely on the confirmed a strong activity-dependent serine phosphorylation by SDS- intrinsic fluctuations of the disordered protein. Even transiently interacting PAGE and mass spectrometry, with some chains exhibiting the theoretical small molecules will likely leave their imprint on this dynamics, and the maximum of 50 phosphoserines. To test if phosphorylation could trigger degree of perturbation can be used as an assay for narrowing down the IDP conformational changes and if these changes depend on chain and sol- chemical search space for candidate drug molecules. We demonstrate this vent electrostatics, we immobilized rNFH-SA onto glass surfaces, phosphor- approach for hIAPP (amylin), an IDP associated with Type II diabetes, using ylated the protein brush in situ, and measured brush heights using AFM. a home-built fluorescence cross correlation spectrometer (FCCS). The After phosphorylation, rNFH-SA brush swelled significantly, with the extent intrinsic fluctuations can be studied using FRET using a donor (D) at strongly sensitive to pH and ionic strength. This phosphorylation-induced the C-terminal and a quencher (A) at the N-terminal. The donor blinks as swelling is fully reversible through alkaline phosphatase-mediated dephos- the D-A separation changes due to spontaneous fluctuations, and the time phorylation. These phosphate-dependent changes align with a mechanism 5 scale of this bright-dark transition is 137.5 5.1 ns. This varies linearly in which IDP conformation is governed by phosphate-phosphate electro- with the viscosity of the medium, as is expected for intra-chain diffusion. þþ static repulsion within and between adjacent chains. Furthermore, micro- Zn , which is known to interact with hIAPP, does not induce any change molar concentrations of divalent cations dramatically condensed in the dynamics. However, some other reagents which have been suggested phosphorylated rNFH-SA brushes, consistent with a classical chelation to interact with amylin (mentioned below), do alter the dynamics signifi- mechanism. This work is among the first efforts to systematically map cantly. Our fluctuation based scoring yields the following order for the IDP structure-phosphorylation relationships and demonstrates that multi- z > > strength of interaction: quercetin epicatechin EGCG congo red. site phosphorylation can induce significant conformational changes that We infer that it is possible to quantitatively measure the degree of transient are predictable from simple physical models. interactions of small molecules with an IDP, even when no particular struc- ture is formed as a result. 2928-Pos Board B136 Proton-Induced Switching of an Intrinsically Disordered Domain of a 2926-Pos Board B134 Melanosomal Protein into a Polymorphic Functional Amyloid Sequence-Encoded Charge Patterning of the Intrinsically Disordered Tail Priyanka Dogra1, Sourav Singha Roy2, Mily Bhattacharya1, of FtsZ Impacts Polymerization and Bacterial Cell Division Suchitra S. Prabhu2, Samrat Mukhopadhyay1. Megan Cohan1, Ammon Posey1, Anuradha Mittal1, Steven Grigsby2, 1Dept. of Chemical Sciences, Indian Institute of Science Education and Alex Holehouse1, Paul J. Buske2, Petra A. Levin2, Rohit V. Pappu1. Research Mohali, Mohali, India, 2Dept. of Biological Sciences, Indian 1Biomedical Engineering, Washington University in St. Louis, Saint Louis, 2 Institute of Science Education and Research Mohali, Mohali, India. MO, USA, Biology, Washington University in St. Louis, Saint Louis, MO, Pmel17, a melanosomal protein, forms a functional amyloid that plays an USA. essential role in melanosome maturation and melanin biogenesis in mam- Polymerization of FtsZ, a bacterial homolog of tubulin, is an essential step in mals. The luminal part of Pmel17 comprises an intrinsically disordered the assembly of the divisome (Z-ring) and the regulation of cell division in repeat domain that is critical for the generation of fibrillar matrix that pro- rod-shaped bacteria. The modular architecture of FtsZ includes a highly motes melanin biosynthesis in vivo. A growing body of evidences suggests conserved GTPase domain and an intrinsically disordered C-terminal that the repeat domain aggregates into amyloid fibrils under mildly acidic tail (CTT) that encompasses a poorly conserved polyampholytic linker conditions and constitutes the amyloid core. However, the molecular mech- (CTL) that is essential for Z-ring formation. Being a polyampholyte, the anism of amyloid formation as well as the organization of individual pro- conformational properties of the CTL are determined by the patterning of tein molecules within the supramolecular assembly remains elusive. oppositely charged residues quantified using a parameter k. Although the se- Using a diverse array of spectroscopic and imaging tools, we have em- quences of CTLs are poorly conserved, k is bounded within a narrow range barked upon studies aimed at elucidating the molecular mechanism of am- % % of 0.2 k 0.4. This suggests that sequence-encoded conformational yloid formation from the disordered repeat domain under various properties and interactions mediated by the CTL must be conserved. We physicochemical conditions. Our atomic force microscopy imaging in tested this hypothesis by scrambling the CTL of B. subtilis FtsZ to generate conjunction with multi-parametric fluorescence studies revealed that a CTT variants with k values that are within and beyond the observed bounds. mechanistic switch occurs in the aggregation pathway from an isodesmic FtsZ variants that lie within the bounds support ring-formation through a to a nucleation-dependent polymerization as a function of pH. This results GTP-dependent mechanism. However, as k increases beyond the bounds, in the formation of diverse nanoscopic dendritic and thread-like fibrils. Our ring formation is impaired and the CTTs promote an alternative tail- findings suggest that the shift in the mechanism that modulates the nano- mediated assembly. Sequence features within CTLs modulate the subtle scale morphology might play a pivotal role in the melanosomal maturation interplay amongst tail- and core-driven FtsZ assembly, high and low GTPase and melanin biosynthesis. Further, we characterized the aggregation process activity, and strong and weak lateral associations of FtsZ polymers. Our in the presence of various salts comprising different inorganic ions. These findings suggest that a ‘‘Goldilocks precept’’ i.e., the tail-mediated interac- studies indicated that amyloid formation follows a direct Hofmeister series. tions must not be too strong or too weak, might govern the evolution of Interestingly, the aggregation kinetics exhibited a biphasic transition in the FtsZ-CTLs. Sequence selection in accord with the Goldilocks precept re- presence of different ions and buffer conditions. We believe that the quires tail sequences with appropriately complex amino acid composition biphasic nature of the aggregation process is a consequence of a complex and optimal k-values. Interestingly, the principles we have uncovered for interplay between surface tension and Hofmeister effects. Additionally, I FtsZ appear to be relevant for understanding the evolution of a larger class will discuss our recent results on the role of membrane composition in of tailed or ‘‘bristled’’ NTPases. the regulation of melanogenesis.

BPJ 8724_8726 Wednesday, February 21, 2018 591a

2929-Pos Board B137 Canada, 3Molecular Structure and Function Program, Hospital for Sick Conformational Effects of Various Hydrophobic-to-Hydrophobic Substi- Children, Toronto, ON, Canada, 4Biochemistry, University of Toronto, tution Located at the Midpoint of the Intrinsically Disordered Region of Toronto, ON, Canada. ProBDNF Sic1 is a disordered cyclin-dependent kinase inhibitor which must be phosphor- Ruchi Lohia, Grace Brannigan. ylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its CCIB, Rutgers University, Camden, NJ, USA. recognition by the WD40 binding domain of Cdc4. The highly-cooperative Although the role of electrostatic interactions and mutations that change switch-like dependence on the number of phosphorylated sites on Sic1 cannot charge states in intrinsically disordered proteins (IDPs) is well-established, be accounted for by traditional thermodynamic models of cooperativity. many disease-associated mutations in IDPs are charge-neutral. Earlier, we Further experimental attention is necessary to determine the physico- studied the effects of the disease-associated Val66Met substitution at the chemical basis of its highly cooperative binding. midpoint of the prodomain of precursor brain-derived neurotrophic factor We used single molecule fluorescence techniques to study the dimensions (proBDNF) using fully atomistic molecular dynamics simulations. Val66Met and dynamics of Sic1’s N-terminal targeting region (residues 1-90, hence- substitution is found in 25% of the American population, which has been forth Sic1) and phosphorylated Sic1 (pSic1). A quantitative relationship widely studied for its association with aging-related and stress-related disor- between sequence properties and ensemble properties is a prerequisite ders, reduced volume of the hippocampus, and variations in episodic memory. for understanding IDP phosphorylation and its role in highly cooperative We found that the local secondary structure, transient tertiary contacts, and binding. Theories which seek a quantitative relationship between the pri- compactness of the protein are correlated to backbone configuration around mary amino-acid sequence of an IDP and its conformational properties pre- residue 66. The midpoint location and the substitution at the most highly dominantly consider sequences of IDPs that are not phosphorylated and charged region of the protein played a critical role in causing the conforma- which are deficient in proline residues. We find that Sic1’s global dimen- tional changes of Val66Met substitution. To gain further insight into the sions, such as the radius of gyration, are decoupled from electrostatic de- generalizability of the found mechanism with which a hydrophobic-to- terminants (phosphorylation, charge screening). We analyze the resulting hydrophobic substitution can impact the IDP’s conformational ensemble conformational properties using polymer theory sequence-ensemble and to further establish the significance of substitution location, we studied relationships. 5 more hydrophobic substitutions at residue 66. We report on fully- Furthermore, a quantitative relationship between sequence properties and atomistic temperature replica exchange molecular dynamics simulations of ensemble properties requires the accurate determination of ensemble dimen- the 90 residue proBDNF for Ala66, Ile66, Leu66, Phe66 and Tyr66 sequence. sions. We analyze our single molecule FRET data (smFRET) using homopol- Analyzing and comparing the residue level insight from all 5 simulations ymer models, and by comparison with NMR- and SAXS- restrained helped us in further establishing the significance of charge-neutral mutations ENSEMBLES. We find that homopolymer models fail to infer accurate radius in IDPs. of gyration in the presence of significant transient structure, though inferences of root-mean-squared end-to-end distance appear to be accurate. The ENSEM- 2930-Pos Board B138 BLES are directly compared to the smFRET data by comparing with the calcu- Insights into the Balance between Folding and Aggregation during a lated mean FRET efficiency. The consistency shown will allow future Protein’s Life ENSEMBLE calculations to use smFRET as a restraint. Matthew D. Dalphin1, Yoo Jin Song2, Rayna Addabbo1, Yue Liu2, Angela Varela1, Andrew Stangl2, Teddy Jennaro2, Silvia Cavagnero2. 2932-Pos Board B140 1Biophysics Graduate Degree Program, University of Wisconsin, Madison, Intrinsically Disordered Proteins PH-Induced Structural Transitions in Madison, WI, USA, 2Department of Chemistry, University of Wisconsin, Overcrowded Milieu Madison, Madison, WI, USA. Alexander V. Fonin1, Sitdikova K. Asiya1, Iuliia A. Gagarskaia1, Despite its fundamental importance for life, the delicate balance between Elena I. Kostyleva1, Maksim M. Karasev2, Olga I. Povarova1, protein folding and aggregation in the cellular environment is poorly under- Vladimir N. Uversky3, Irina M. Kuznetsova1, Konstantin K. Turoverov1,4. stood. This lack of knowledge poses serious challenges in basic science, 1Institute of Cytology of Russian Academy of Science, Saint-Petersburg, biotechnology and medicine, as inclusion body formation limits the efficient Russian Federation, 2University of Helsinki, Helsinki, Finland, 3University of overproduction of recombinant proteins and protein-based therapeutics. In South Florida, Tampa, FL, USA, 4Peter the Great St. Petersburg Polytechnic this work, we combine experimental and computational approaches to better University, Saint-Petersburg, Russian Federation. understand how proteins undertake and discriminate folding and aggregation A distinctive feature of cellular milieu is macromolecular crowding. Such pathways in the cell. A wide variety of proteins studied, including apomyo- conditions limit the number of possible polypeptide chain conformations it globin and the soluble E. coli proteome, are able to populate stable aggregate could promote polypeptide chain folding to compact structure, but also could states under physiologically-relevant conditions. The aggregate and native lead to protein aggregation and/or misfolding. Change of milieu conditions states of these proteins are found to be kinetically trapped relative to each (temperature, pH, salt concentration, etc.) could induce phase separation other over a wide range of low concentrations. Kinetic simulations modeling of macromolecules with formation of high functional dynamic structures apomyoglobin’s folding and aggregation landscape recapitulate these find- termed as membraneless organelles. Intrinsically disordered proteins ings. Surprisingly, our simulations also show that the addition of stable (IDPs) which could form multivalent interaction with their numerous part- pre-formed aggregated seeds, especially if small in size, facilitate the con- ners play crucial role in membraneless organelles formation. A study version of native proteins into aggregates on biologically-relevant time- of conformational transitions of macromolecular in crowding conditions scales. Further studies probing the earlier stages of a protein’s life, i.e. in vitro could clarify what’s happening in vivo. We studied pH-induced translation and ribosome release, unveiled that these two events are critical conformational transitions of homopolypeptides poly-L-lysine (poly-K) for directing the relative flux between competing protein folding and aggre- and poly-L-glutamic acid (poly-E), as well as IDPs linker histone H1 and gation pathways. Overall, we found that the ribosome serves as a solubility prothymosin alpha, whose sequences contain about 30% lysine and glutamic tag for nascent proteins that are not yet fully synthesized. In addition, acid, respectively. Intracellular milieu conditions were simulated by highly cotranslationally-active molecular chaperones help preventing longer concentrated polyethylene glycol (PEG) solutions. An increase of PEG con- nascent proteins from forming insoluble aggregates during the later stages centration and polymerization degree induces significant cooperativity of translation, prior to ribosome release. Ongoing studies are aimed at eluci- decrease and shift of transition between unfolded and ordered forms of dating the structural features and chaperone-dependence of cotranslationally- poly-K polypeptide to more acidic pH. The middle of coil-helix transition aggregated proteins. In all, these findings shed light on the cellular mecha- for poly-E does not depend on PEG concentration or polymerization degree nisms underlying the fidelity of protein folding upon ribosome release in but their increase induce significant growth of transition cooperativity. It is healthy cells. established that macromolecular crowding conditions have little effect on characteristics of pH-induced histone H1 transition, it promote only protein 2931-Pos Board B139 aggregation. However, the same conditions promote the increase of prothy- Global Dimensions are Decoupled from Electrostatics in the Intrinsically mosin alpha and poly-E transition cooperativity. Apparently, the different Disordered Protein Sic1 effects of highly concentrated PEG solutions on similar structural transitions Gregory Gomes1,2, Mickael Krzeminski3,4, Julie Forman-Kay3,4, of homopolypeptides and IDPs is due to different ratio between sizes Claudiu Gradinaru1,2. of PEG’s clusters and structurally organized domains of studied poly 1Physics, University of Toronto, Toronto, ON, Canada, 2Chemical and amino acids and IDPs. This work was supported in part by the Program Physical Sciences, University of Toronto Mississauga, Mississauga, ON, ‘‘MCB RAS’’ (KKT) and the RF President Fellowship (AVF).

BPJ 8724_8726 592a Wednesday, February 21, 2018

2933-Pos Board B141 reverse rotation might occur in two resolvable sub-steps. Finally, we find that Hidden Structural Codes in Protein Intrinsic Disorder while a strong mechanical barrier does not significantly decrease the EF-G Gonzalo de Prat Gay. dependent unwinding rate, it biases ribosomes into a kinetically altered, 10- Fundacio´n Instituto Leloir, Ciudad Autonoma de Buenos Aires, Argentina. fold slower pathway prior to translocation, resulting in a dramatic reduction Protein intrinsic disorder is a major structural category in biology yet its of the global translation rate. We propose that such a slower pathway could definition is often limited to the absence of folding. The explosion of infor- be used to tune translation rate for nascent chain folding. mation in the genomic era showed that it may account for over 30% of cod- ing regions across life domains, and it is particularly overrepresented in 2936-Pos Board B144 O viruses. Papillomaviruses are an unparalleled case for sequence to structure How 2’- -Methylation in mRNA Disrupts tRNA Decoding during Trans- correlation analysis because of the existence of hundreds of anciently lation Elongation evolved and stable virus types which are divergent enough in sequence, Junhong Choi1, Gabriele Indrisiunaite2, Hasan DeMirci3, Ka-Weng Ieong2, but conserving the function of each protein. E7, the main transforming on- Jinfan Wang4, Alexey Petrov4, Arjun Prabhakar4, Gideon Rechavi5,6, coprotein from human papillomaviruses, is a paradigmatic example of an Dan Dominissini6,7, Chuan He8,Ma˚ns Ehrenberg2, Joseph D. Puglisi4. 1Applied Physics, Stanford University, Stanford, CA, USA, 2Uppsala intrinsically disordered protein with pathological moonlighting activities 3 evolved for hijacking cell cycle control. Despite of being intrinsically disor- University, Uppsala, Sweden, SLAC National Accelerator Laboratory, Menlo Park, CA, USA, 4Stanford University, Stanford, CA, USA, 5Cancer dered, the N-terminal domain shows more conserved residues than the glob- 6 ular C-terminal domain. Mutation of five hyper conserved residues precisely Research Center, Chaim Sheba Medical Center, Tel-Hashomer, Israel, Tel Aviv University, Tel Aviv, Israel, 7Chaim Sheba Medical Center, Tel- distributed along the sequence lead to a marked increase in both a-helix and 8 ß-sheet structural content, reflected by drastic effects on equilibrium propen- Hashomer, Israel, University of Chicago, Chicago, IL, USA. sities and oligomerization kinetics. These results strongly suggest the exis- Chemical modifications of messenger RNA (mRNA) may regulate many as- tence of local nuclei, yet to be defined in structural terms, that oppose to pects of mRNA processing and protein synthesis. Recently, 2’-O-methylation canonical folding as expected for globular proteins. Moreover, direct (Nm) of nucleotides was identified as a frequent modification in translated re- coupling analysis pinpoint interactions surprisingly coincident with the pro- gions of human mRNA, showing enrichment in certain amino acid codons. posed nuclei. In addition, these match with regions of helix propensities Here, using single-molecule, bulk kinetics and structural methods, we show found in TFE/NMR experiments. that Nm within coding regions of mRNA disrupts key steps in codon reading during cognate transfer RNA (tRNA) selection. Our results suggest that Nm Posters: Ribosomes & Translation sterically perturbs interactions of ribosomal monitoring bases (G530, A1492 and A1493 of 16S ribosomal RNA) with cognate codon-anticodon helices, 2934-Pos Board B142 thereby inhibiting downstream GTP-hydrolysis by elongation factor Tu (EF- Allosteric Logic of the V. vulnificus Adenine Riboswitch Resolved by Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate Four-Dimensional Chemical Mapping aminoacylated-tRNAs in initial selection and proofreading. The observed ef- Rhiju Das. fect of monitoring bases in decoding accuracy mirrors the effect of disturbed codon-anticodon pairing, previously studied using N6-methyladenoside Physics and Biochemistry, Stanford University, Stanford, CA, USA. 6 The structural logic that define the functions of gene regulatory RNA molecules (m A) modified mRNA. Combined together, our findings highlight how chem- may be radically different from classic models of allostery, but the relevant ical modifications of mRNA tune the dynamics of protein synthesis at different structural correlations have remained elusive in even intensively studied steps of translation elongation. RNA model systems. Here, we present a four-dimensional expansion of chem- 2937-Pos Board B145 ical mapping called lock-mutate-map-rescue (LM2R), which integrates multi- Fluorescent Dye Dynamics Attached to EF-Tu and their Effect on a ple layers of mutation with nucleotide-resolution chemical mapping. This Hetero-FRET System technique resolves the core mechanism of the adenine-responsive V. vulnificus Senthilkumar Kailasam, Luc Roberts, Hans-Joachim Wieden. add riboswitch including its gene expression platform, a paradigmatic system Chemistry and Biochemistry, Alberta RNA Research and Training Institute, for which both Monod-Wyman-Changeux (MWC) conformational selection University of Lethbridge, Lethbridge, AB, Canada. models and non-MWC alternatives have been proposed. To discriminate Elongation Factor-Tu (EF-Tu) is an essential protein involved in the elonga- amongst these models, we locked each functionally important helix through de- tion cycle of the protein synthesis by delivering aminoacylated tRNAs to the signed mutations and assessed formation or depletion of other helices via high- ribosomal A-site. The active form of the protein has GTP bound to the G- throughput compensatory rescue experiments. These LM2R measurements domain. The G-domain along with two additional domains undergoes signif- give strong support to the pre-existing correlations predicted by MWC models, icant conformational transition to GDP-bound state, following GTP hydroly- disfavor alternative models, and reveal new structural heterogeneities that may sis and Pi release. We developed an intra-molecular hetero-FRET system be general across ligand-free riboswitches. using Elongation Factor-Tu variants to study the details of these structural rearrangements in real time. This intra-molecular FRET system will also 2935-Pos Board B143 allow for the elucidation of EF-Tu’s dynamic transitions during interactions Simultaneous Force and Fluorescence Measurements on Single Ribosomes with components of the bacterial translation machinery. Here we report mo- Demonstrate that mRNA Secondary Structures do not Restrict EF-G lecular dynamics simulation of the protein with covalently linked to the Catalyzed Translocation FRET-dye pair and compare the results with equilibrium and rapid kinetics Varsha P. Desai, Filipp Frank, Maurizio Righini, Antony Lee, fluorescence experiments. The flexibility of the dye pair and their orienta- Ignacio Tinoco, Carlos J. Bustamante. tions were analysed and related to FRET parameters. Our finding suggests University of California, Berkeley, Berkeley, CA, USA. that it is critical for the selection of dyes and their location on the molecular During protein synthesis, the ribosome translocates along a messenger RNA surface of the protein to assess their flexibility in the context of the respec- (mRNA) in one-codon steps catalyzed by the activity of a GTPase elongation tive protein. factor, EF-G. Secondary structures in an mRNA pose a mechanical barrier to translocation as the mRNA entry pore on the ribosome only accommodates 2938-Pos Board B146 single-stranded RNA, and thus they must be opened prior to translocation. Unfolding Intermediate of mRNA Pseudoknot Correlates with Ribosomal This opening is facilitated by several conserved positively charged amino Frameshifting acid side chains located at the entry pore. It is not clear, however, whether Kai-Chun Chang1, Po-Szu Hsieh1, An-Yi Lee1, Emmanuel Salawu2, the ribosome disrupts the secondary structure at the mRNA entry site prior to You-Hsin Lin1, Yu-Ting Chen1, Jin-Der Wen1. translocation or concomitantly with translocation. Here, we measure both 1Institute of Molecular and Cellular Biology, National Taiwan University, hairpin opening and EF-G binding to the ribosome using optical tweezers Taipei, Taiwan, 2Institute of Bioinformatics and Structural Biology, National with single molecule fluorescence capability. We find that EF-G arrival pre- Tsing Hua University, Hsinchu, Taiwan. cedes the opening of an mRNA hairpin and EF-G release occurs after the In many viruses and bacteria, stable mRNA structures are known to induce ri- hairpin is opened, providing direct evidence that the unwinding action of the bosomal frameshifting (FS) for synthesizing different proteins from the same ribosome is concomitant with translocation. Furthermore, we occasionally mRNA. However, what structural features (thermal stability, mechanical un- observe an unwinding intermediate where the hairpin is opened in two succes- folding force and/or structural plasticity) of an mRNA structure play dominant sive half-codon steps while EF-G remains bound to the ribosome. Current roles in determining FS efficiency remains controversial. Here we use a FS- models posit that translocation occurs through the reverse rotation of the small inducing pseudoknot derived from the human telomerase RNA (hTR-PK) as subunit head domain. Our results suggest that in the presence of a barrier, this our model system. The unfolding and folding dynamics of hTR-PK and its

BPJ 8724_8726 Wednesday, February 21, 2018 593a mutant derivatives are investigated with optical tweezers, steered molecular dy- 2941-Pos Board B149 namics (SMD) simulations and single-molecule FRET (smFRET). With Conservation of Folding Mechanism in Cotranslational Folding of smFRET, we show that base triples at one end (formed by loop 1 and stem Titin I27 2) of hTR-PK promote the formation of those at the distal end (formed by Pengfei Tian1, Annette Steward2, Jane Clarke2, Robert B. Best1. stem 1 and loop 2), and thereby limit the flexibility of the distal loop (loop 1National Institute of Health, Bethesda, MD, USA, 2University of 2). The coordination between base triples is therefore responsible for the Cambridge, Cambridge, United Kingdom. single-step unfolding event of the structure. By contrast, smFRET experiments Protein can fold into partially and fully structured states while they are synthe- identify a compact intermediate structure before hTR-PK is completely disrup- sized on the ribosome. Interactions between the nascent polypeptide and the ted by the ribosome. SMD simulations further reveal that as the hTR-PK loop 2 ribosome exit tunnel may also affect synthesis rates thus regulating gene attaches to the positively charged residues of ribosomal protein S3, base triples expression. Recently studies have shown that insertion of a stalling peptide facilitate formation of the unfolding intermediate of hTR-PK. When the base sequence (SecM17) upstream of a folding domain allows the force exerted triples are disrupted by mutations (the delta-triple mutant), the compact unfold- by the protein as it folds to be indirectly probed: this force will tend increase ing intermediate can no longer be seen in SMD simulations and smFRET ex- the yield of protein which is able to overcome the trapping. To gain mechanistic periments (that is, the mutant exhibits single-step unfolding). The delta-triple insight into such observations, we have used molecular simulations to study the mutation also results in a dramatic drop in FS efficiency from 50% to co-translational folding of the muscle protein titin I27. We can quantitatively 0%. Our study demonstrates the importance of a base triple-stabilized unfold- predict the fraction of folded protein based on the force measured in the simu- ing intermediate in PK-induced FS. lations at the arrest site, together with knowledge of the force-dependence of the escape rate using a kinetic model. The predicted fraction folded is in 2939-Pos Board B147 remarkable agreement with experiment, validating the simulation result. We Coupled Ribosome Conformational and Compositional Dynamics in the find that the generated mechanical force, and hence folding yield, is a trade- Mechanism of Release Factor 3 during Translation Termination off between the force exerted by the folding protein (highest at short linker Arjun Prabhakar, Joseph D. Puglisi. lengths) and population of the folded state (highest for long linkers). We also Structural Biology, Stanford University, Stanford, CA, USA. investigated the folding mechanism of Titin I27 arrested by SecM-stalled ribo- After the ribosome finishes translation of an mRNA during protein synthe- somes using a Bayesian method for testing the importance of specific contacts sis, the completed protein is released from the ribosome through the pro- in the folding mechanism, as well as by computing phi-values. This analysis cess of translation termination catalyzed by class I release factors (RFs). suggests that the mechanism is relatively insensitive to the presence of the ribo- The ribosome then needs to be recycled for the next cycle of translation. some, but that there are subtle shifts of critical contacts from the C- to the N- Despite their importance in biology, our current knowledge on the mech- terminus. anism of even the simple prokaryotic translation termination and recycling is incomplete. The gap in our knowledge is centered on RF3, a class II RF 2942-Pos Board B150 whose role in translation is still not well-defined. Biochemical studies have The Structural Basis for Initiation Factor 2 Activation during Translation revealed that RF3 could accelerate the dissociation of the class I RF from Initiation the ribosome, and structures of ribosomes bound to RF3 suggest that ribo- Kelvin Caban1, Michael Pavlov2, Sandip Kaledhonkar3, Ziao Fu3, somal intersubunit conformation plays a role in RF3 function, but the Joachim Frank3,4,Ma˚ns Ehrenberg2, Ruben L. Gonzalez, Jr.1. interplay of conformation, other RFs and the mechanism of termination 1Department of Chemistry, Columbia University, New York, NY, USA, remain unclear. Here, we used a single-molecule method and 2Department of Cell and Molecular Biology, Uppsala University, Uppsala, fluorescently-labeled ribosomes and release factors to track these confor- Sweden, 3Department of Biochemistry and Molecular Biophysics, Columbia mational and compositional states in translation in real time. Our results University, New York, NY, USA, 4Department of Biological Sciences, show that RF3 binds to the post-termination ribosome and induces intersu- Columbia University, New York, NY, USA. bunit rotation to destabilize the interactions between the ribosome and the Initiation factor 2 (IF2) is a translational GTPase that helps ensure the fidel- class I RF. These results highlight the coordination between factor binding ity of bacterial translation initiation by selectively accelerating the rate with and ribosome conformation as critical for efficient transitioning from which the large ribosomal subunit joins to a small, or 30S, ribosomal sub- termination to recycling. unit initiation complex (IC) carrying an initiator N-formyl-methionyl-tRNA (fMet-tRNAfMet) in the peptidyl-tRNA binding (P) site. Genetic and 2940-Pos Board B148 ensemble kinetic studies of subunit joining indicate that IF2 couples the How Mechanical Forces on the Ribosome Modulate the Speed of Protein rate of subunit joining to the identity of the 30S IC-bound, P-site tRNA Synthesis by undergoing a GTP- and fMet-tRNAfMet-dependent conformational Benjamin Fritch1, Sarah Leininger1, Phillip Hudson2, Lee Woodcock2, change on the 30S IC. This conformational change, termed IF2 activation, Carol Deutsch3, Edward P. O’Brien1. 1 switches IF2 from a conformation that is inactive for rapid subunit joining Department of Chemistry, Penn State University, University Park, PA, USA, to one that is active. To elucidate the structural basis for IF2 activation, we 2Department of Chemistry, University of Southern Florida, Tampa, FL, USA, 3 have developed and used IF2-tRNA and IF2-IF2 single-molecule fluores- Physiology, University of Pennsylvania, Philadelphia, PA, USA. cence resonance energy transfer signals to measure the kinetics of IF2 bind- Mechanical forces acting on the ribosome can alter the speed of protein syn- ing to the 30S IC and to characterize the dynamics of 30S IC-bound IF2. thesis. These forces can arise from co-translational protein folding, indi- These studies demonstrate that GTP and fMet-tRNAfMet cooperatively in- cating that co-translational processes can regulate translation through crease the affinity of IF2 for the 30S IC and stabilize a unique conformation mechanochemical mechanisms. The factors governing force generation due of 30S IC-bound IF2. Moreover, we show that a mutation in domain III of to co-translational folding and the contribution of unfolded nascent chain IF2 that enables the factor to catalyze rapid subunit joining in the absence segments to the magnitude of the pulling force are unknown. Furthermore, of GTP or fMet-tRNAfMet does so by stabilizing this same unique confor- the mechanism by which force is transduced 10 nm to the ribosome’s cata- mation of 30S IC-bound IF2 in a GTP- and fMet-tRNAfMet independent lytic core, and how it modulates peptide bond formation are also unknown. manner. Using time-resolved cryogenic electron microscopy, we are Here, we address these issues using all-atom and coarse-grained molecular currently investigating the mechanism through which this unique conforma- dynamics simulations as well as in situ experimental measurements of tion of 30S IC-bound IF2 accelerates subunit joining. Based on our collec- changes in nascent-chain extension in the exit tunnel. We first show that tive findings, we propose a structure-based model for how the GTP bound- domain topology and stability play a key role in determining the magnitude form of IF2 couples the rate of subunit joining to the identity of the 30S IC- of the pulling force generated during co-translational folding. Next, we bound, P-site tRNA, thereby helping to ensure the fidelity of translation demonstrate that when the number of residues composing a nascent chain in- initiation. creases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that is transmitted through the polypeptide backbone to 2943-Pos Board B151 the ribosome’s catalytic core. Utilizing quantum mechanical calculations, we Identifying the RNA Kinetic Traps in Ribosome Assembly find that these pulling forces decrease the free energy barrier height to pep- Riley C. Gentry, Eda Koculi. tide bond formation indicating that elongating chains can accelerate transla- University of Central Florida, Orlando, FL, USA. tion. Since nascent protein segments start out as unfolded structural The ribosome is the cellular machine responsible for protein production in ensembles, these results indicate a universal pulling force is present during every living cell. While the final structure of the mature ribosome is known protein synthesis that increases as a protein elongates and modulates the from x-ray crystallography studies, the assembly process remains largely un- speed of synthesis. known. To fulfill the cells’ demand for proteins, ribosome assembly in vivo

BPJ 8724_8726 594a Wednesday, February 21, 2018 occurs fast and efficiently. Consequently, intermediates of the assembly pro- somal stalling and co-translational folding. To understand the details of these cess are short lived and do not accumulate in large amounts for investigation. interactions, a more quantitative understanding of the biophysical properties To increase the accumulation of in vivo intermediates of large subunit ribo- of the tunnel across different species is needed. In the present work, we some assembly in Escherichia coli, we take advantage of the helicase inactive compile and compare more than 20 recently obtained ribosome structures DbpA construct R331A. When R331A DbpA is expressed in cells, three large from cryo-EM data, coming from bacteria, archaea, and eukarya. Upon ex- subunit particles, with sedimentation coefficients of 27S, 35S, and 45S, accu- tracting the geometric properties of the tunnel for each species and devel- mulate. The particles rearrange to form the native 50S large subunit via par- oping new metrics to compare them, we show that the evolutionary tree allel pathways. We interrogated the RNA structure of the 45S particle using reconstructed using structural features of the tunnel is in remarkable agree- dimethyl sulfate and SHAPE chemical probing reagents combined with ment with known phylogenetic information based on DNA sequence evolu- Next Generation Sequencing. The structural probing experiments revealed tion, suggesting the importance of specific features associated with the shape perturbed secondary structures of the P-loop and the helices of the peptidyl- and structure of the tunnel. In particular, our analysis reveals high conserva- transferase center. Additionally, it revealed an incorrect orientation of helix tion in the upper region located between the PTC and the constriction site, 38, the finger of the A-site, and several critical, broken tertiary RNA contacts while more variation is observed in the lower region, where significant between distant regions of the 23S rRNA. The data suggests the peptidyltrans- shrinkage of the tunnel is observed in eukaryotes, associated with more spe- ferase center’s maturation is one of the final events during assembly. This se- cific interactions. Further, by introducing a new metric of charge conserva- vere misfolding explains the slow rearrangement of the 45S particle to the tion and applying it to aligned sequences of homologous ribosomal proteins native 50S subunit. Next, we will characterize the RNA structure of the and RNAs, we show that the charge density is highly conserved close to the 27S and 35S particles. The structural probing of the 27S and 35S particles tunnel. Interestingly, this charge variation along the tunnel, combined with will provide insights into the early steps of ribosome assembly, which remain its geometric variation, suggests a role in facilitating the movement of a complete mystery. charged polypeptide chains through the tunnel during the early stages of translation. Finally, this result is in agreement with 1) the variation of ribo- 2944-Pos Board B152 some elongation rates along the mRNA transcript (inferred from ribosome Dynamics of EF-TU and its Effect on Growth in Live Escherichia coli profiling data) and 2) the variation of amino-acid residue charges along Mainak Mustafi, James C. Weisshaar. the translated protein sequence, suggesting co-evolution of the proteomes Chemistry, University of Wisconsin, Madison, WI, USA. and the ribosome exit tunnel. In bacteria, elongation factor Tu (EF-Tu) is a translational co-factor which initiates the elongation cycle of translation. EF-Tu forms a ternary complex 2947-Pos Board B155 E. coli with aminoacyl-tRNA and GTP, which binds to one of four flexible L7/L12 Growth Phase Dependent Effects on Spatial Distribution of Chro- units on the ribosome 70S complex. This interaction tethers a charged tRNA mosomes and Ribosomes in close proximity of the ribosomal A site. This is followed by two tests to Sonisilpa Mohapatra. check for the correct aa-tRNA anticodon. The elongation cycle is extremely University of Wisconsin Madison, Madison, WI, USA. efficient and can complete in as little as 50 ms. As most of the aa-tRNA The super resolution fluorescence method, ‘‘photoactivation - localization copies don’t match the current mRNA, the tests must occur very rapidly. microscopy’’ (PALM) gives us the ability to explore in remarkable details In this study we present super-resolution tracking and diffusion analysis of how the central dogma of cell biology is spatially organized in live fluorescently labeled EF-Tu in live E. coli. From our diffusion data and E. coli. Previous studies on rapidly growing E. coli, have shown that ribo- copy number estimates we infer that the four L7/L12 sites are essentially somes and nucleoids are spatially segregated from each other. These data saturated with ternary complexes in vivo. This simultaneously tethers 4 are consistent with a picture in which most transcription of protein genes ternary complexes near the A site, creating a high local concentration that is also co-translational. Intriguingly, the picture is different in slow growing may greatly enhance the rate of testing of aa-tRNAs. Our data and a combi- species C. crescentus where ribosomes and DNA appear to be thoroughly natorial argument both suggest that the initial test for a codon-anticodon mixed. We have therefore investigated the spatial segregation of ribosomes match occurs in less than 1-2 ms per aa-tRNA copy. Furthermore, we test fromnucleoidinslowgrowingE. coli for comparison with slow growing the hypothesis of the dependence of cell growth on the diffusion of ternary cells. We have performed two color superresolution fluorescence imaging complexes in a crowded cytoplasm. of ribosomes and DNA binding HU protein in E. coli under two different growth conditions. For studying slow growing E. coli, we have chosen a 2945-Pos Board B153 minimal growth media, MBM þ 0.2% glycerol in which E. coli grows Dynamics of Eukaryotic Translation Initiation with a doubling time of 147 min comparable to the slow growing C. cres- Jinfan Wang, Joseph D. Puglisi. centus species. The axial line scans of ribosomes and HU show that they Department of Structural Biology, Stanford University School of Medicine, are anti-correlated with each other. The peak to trough ratio of ribosomal Stanford, CA, USA. axial line scans in slow growth conditions are however lower than good Temporally and spatially accurate protein synthesis is the key to maintain growth conditions indicating lower segregation. We have developed a healthy cell physiology. The initiation phase of translation in eukaryotic or- method to quantitatively measure the degree of segregation in the two ganisms is the rate-limiting step in protein synthesis, where more than 20 different growth conditions. We find that the phenomenon of co- polypeptides coordinate the assembly of an elongation-competent ribo- transcription translation is present in E. coli irrespective of growth somal complex on the mRNA template. Such a complex process is at the conditions. heart of translational control, and mis-regulation at this stage is linked to numerous human diseases including cancer. Unfortunately, our understand- 2948-Pos Board B156 ing of the mechanisms of initiation and its regulation is hindered by the Translational Control of Cancer Stem Cells lack of a dynamic picture the process. Here, we have developed in vitro Yasunari Kanda, Naoya Hirata, Shigeru Yamada, Daiju Yamazaki. single-molecule fluorescence methods to determine key compositional Division of Pharmacology, National Institute of Health Sciences, events and conformational dynamics during yeast translation initiation. Setagaya-ku, Japan. We have fluorescently labeled mRNAs, tRNAs, protein factors and ribo- Cancers are originated from a small population which is called cancer stem somes to track initiation in real time. Our results provide a dynamic frame- cells. Cancer stem cells can be identified by aldehyde dehydrogenase work for better understanding translational control and regulation in (ALDH) assay from various cancer cell lines as well as clinical samples. eukaryotes. However, growth regulation of cancer stem cells is not fully understood. In the present study, we examined translational control of cancer stem cells. 2946-Pos Board B154 We isolated ALDH-positive cells from MCF-7 cells and examined protein The Evolution of the Ribosome Exit Tunnel and its Impact on Translation synthesis. Rate of incorporated amino acids in ALDH-positive cells were Dynamics higher than that in ALDH-negative cells. To explore the genes which trans- Khanh Dao Duc1, Sanjit Batra2, Yun Song3. lation is upregulated in ALDH-positive cells, we next performed ribosome 1Computer Science, University of Berkeley California, Berkeley, CA, USA, profiling that provides genome-wide maps of protein synthesis by quanti- 2University of Berkeley California, Berkeley, CA, USA, 3Statistics and fying ribosome-protected mRNA using the deep sequencing. We analyzed Computer Science, University of Berkeley California, Berkeley, CA, USA. translation efficiency that is calculated by normalizing the ribosome- Interactions between the nascent polypeptide chain and the ribosome exit protected mRNA frequency to total transcript abundance and determined tunnel have been shown to impact translation dynamics, notably via ribo- approximately 1000 translation products in ALDH-positive cells, compared

BPJ 8724_8726 Wednesday, February 21, 2018 595a to ALDH-negative cells. These proteins include Ras and Cyclin-dependent 2951-Pos Board B159 kinase, confirming that ribosome profiling works well in the ALDH assay. Single-Molecule Analysis of Ribosome and Factor Assembly Pathways on a We are now currently screening the target protein for drug development. Viral mRNA Our strategy would reveal a novel growth regulator of cancer stem cells Alex G. Johnson, Joseph D. Puglisi. for drug discovery. Structural Biology, Stanford University, Stanford, CA, USA. Eukaryotic cells contain thousands of unique mRNAs undergoing translation 2949-Pos Board B157 by the ribosome. The process of mRNA selection thus acts as regulatory stage Cryo-EM Reveals Molecular Transformers in Organelles in which mRNAs compete for the available pool of ribosomes, and is controlled Alexey Amunts. by the interplay of mRNA, ribosomal subunits, and numerous ribosome- Stockholm University / SciLifeLab, Stockholm, Sweden. associated eukaryotic initiation factors (eIFs). To bypass several of these mo- 30 years of X-ray crystallography around ribosomes, emphasized by the lecular controls and commandeer cellular ribosomes, the Hepatitis C virus Nobel prize, have taught us the importance of the finest details of the ribo- (HCV) utilizes an internal ribosome entry site (IRES) within the 5’UTR of some structure. However, the last 3 years of the cryo-EM revolution have its positive-sense RNA genome to directly recruit ribosome to the start codon. shifted the focus towards ribosome evolution. This is thanks to the cryo- Here we describe a simple reconstitution model of translation initiation on the EM structures of ribosomes from mitochondria and chloroplasts that re- HCV IRES RNA, and the tracking of select fluorescently-labeled factors using vealed bizarre architectures such as mt-tRNA-Val substituting 5S, trapped single-molecule real-time imaging in zero-mode waveguides (ZMWs). Our nascent polypeptide in the absence of P-site tRNA, divaricated exit tunnels, system allows us to monitor the kinetics and assembly pathways of labeled ri- dedicated membrane anchor, intrinsic ribosomal GTPase in the head of the bosomes and factors on an immobilized RNA, and provides insights into the small subunit and more. molecular mechanisms governing mRNA selection by the human ribosome. Are these adjustments imposed by organelles to gain new functions or is it an exploration of a structural space on the molecular level? Can rapidly evolved 2952-Pos Board B160 evolutionary trajectories of these ribosomes help to predict aspects concerning Studying the Nascent Peptide Chain in the Ribosomal Exit Tunnel the future of the cytoplasmic counterparts? Have ribosomes from organelles Nadin Haase1, Wolf Holtkamp2, Reinhard Lipowsky1, Marina Rodnina2, adopted unique mechanisms for development and why they differ so much be- Sophia Rudorf1. tween organisms? 1Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 2Max I will present unpublished high resolution structures of previously unseen and Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany. most bizarre organellar ribosomes, including functional complexes that shed During protein synthesis, the nascent peptide chain traverses the ribosomal exit new light on these questions. tunnel. We monitor this co-translational movement via fluorescent probes attached to the N-termini of the nascent chains. Due to fluorophore quenching, 2950-Pos Board B158 the time-dependent fluorescence signal emitted by an individual peptide is Nascent Proteins Interact with Key Regions of the Outer Surface of the determined by co-translational events, such as secondary structure formation Ribosome and peptide-tunnel interactions. To obtain information on these local events, Andrew M. Fuchs, Valeria Guzman-Luna, Rayna Addabbo, the measured total fluorescence signal has to be decomposed into position- Silvia Cavagnero. dependent intensities. To this end, we describe mRNA translation as a Markov Chemistry, UW-Madison, Madison, WI, USA. process and assign a specific fluorescence intensity to each position of the ribo- The initial stages of a protein’s life in the cell are crucial for the proper bal- some. Combining the stochastic time evolution of the ribosomal position with ance between folding, misfolding and aggregation. While there is a consider- the observed time course of the total fluorescence, we obtain a system of linear able amount of knowledge pertaining to protein folding in vitro, very little is equations. We solve this system for the unknown position-specific intensities. known about protein folding in the cellular environment. The earliest stage Our results indicate that the N-terminus of poly-phenylalanine experiences ma- of protein folding in the cell, when nascent chains emerge from the ribo- jor environmental changes during elongation of the first five amino acids. some, are still poorly understood. Ribosomal components located within the exit tunnel and ribosomal outer surface, for instance, may interact with 2953-Pos Board B161 nascent proteins and affect their folding. Previous studies in the Cavagnero Ribosomal Protein Dynamics on the Human Ribosome group showed that ribosome-bound nascent chains (RNCs) derived from the Christopher Lapointe, Joseph Puglisi. intrinsically disordered protein PIR and its highly negatively charged mutant Structural Biology, Stanford University School of Medicine, Palo Alto, CA, exhibit significant spatial bias, suggesting potential interactions with the ri- USA. bosomal surface. We now present direct evidence for the presence of well- The human ribosome is a macromolecular machine comprised of four ribo- defined interactions under physiologically relevant conditions between somal RNAs and more than 70 ribosomal proteins. In parallel to their role in RNCs of PIR, PIR 1.2.3, and the foldable cofactor-free form of the bacterial the ribosome, some ribosomal proteins have extra-ribosomal functions that flavohemoglobin apoHmpH with specific ribosomal proteins and with the co- are implicated in human health. Furthermore, inclusion or exclusion of spe- translationally active chaperone trigger factor (TF). RNC-ribosome contacts cific ribosomal proteins in the ribosome is implicated in the selective trans- were probed by a combination of chemical crosslinking, Western blotting lation of specific human messenger RNAs (mRNAs). Thus ribosomal protein and LC-ESI-MS/MS. RNCs derived from both PIR and apoHmpH crosslink composition is likely a dynamic feature of translation. Unfortunately, the dy- to select ribosomal proteins near the ribosomal exit tunnel. Sucrose density namics of human ribosomal proteins on the ribosome remain unclear. We gradients were instrumental to ascertain that nascent chain - ribosomal pro- therefore established single-molecule fluorescence microscopy, using both tein interactions take place within the context of a single ribosome. Interest- zero-mode waveguide and total internal reflection fluorescence (TIRF) in- ingly, interactions with ribosomal proteins are strongly Mg2þ concentration- struments, and single-molecule Fo¨rster resonance energy transfer (smFRET) dependent. Yet, the solubility profile of apoHmpH upon release from the strategies to examine interactions between select ribosomal proteins, ribo- ribosome is not Mg2þ concentration-dependent, suggesting that the above in- somal subunits, and mRNAs. We have analyzed the kinetics of their interac- teractions may play a yet-unidentified regulatory role in translation. An tion with the ribosome and their dynamics during translation initiation on important emerging theme of this work is that the ribosome plays a funda- viral mRNA. We hope to reveal the compositional and conformational dy- mental role in the cell that extends beyond the mere catalysis of peptide namics of human ribosomal proteins during translation of viral and cellular bond formation. mRNAs.

BPJ 8724_8726 596a Wednesday, February 21, 2018

Posters: DNA Structure and Dynamics II improvements of the magnetic tweezers technique that enable i) fast tracking (kHz), ii) the capability to directly measure torque, and iii) the 2954-Pos Board B162 ability to perform parallel measurements on many ( 100) molecules at Sequence Selectivity, Cooperativity and Competition in the Equilibrium the same time. Using these capabilities, we have carried out high- Binding of Psoralens to DNA precision measurements of DNA mechanics and dynamics. Our results indi- Stephen A. Winkle, Sigal Dahan, Doreen Patichi, Cinthya Susanibar Tinoco, cate that the intrinsic torsional stiffness does not change with mono- or Giselle Valdes. divalent ion concentration and is approximately independent of temperature, Chemistry and Biochemistry, Florida International University, Miami, FL, for temperatures well below the melting temperature. Quantitative compar- USA. ison of high-resolution single moleculesmeasurementstocoarse-grained Psoralens, such as 8-methoxypsoralen, show sequence preference in their pho- simulations of DNA mechanics shows that taking into account the anisot- tocrosslinking of DNA for TA steps. This uv-induced covalent binding to DNA ropy of DNA and introducing a non-zero twist-bend coupling significantly is preceded by intercalative equilibrium binding. Previously, we have examined improves agreement with torque measurements. Further, we demonstrate the equilibrium binding of intercalators and groove binders using restriction that all atom molecular dynamics simulations are in quantitative agreement enzyme activity assays. Here we report results for restriction enzyme activity with single-molecule measurements of DNA twist-stretch coupling and assays of the binding of 5-methoxypsoralen [5-mop] and 8-methoxypsoralen correctly predict the temperature-dependence of DNA twist and of DNA [8-mop] to phiX174 RF DNA [psoralen/DNA base pair from 5.6 - 23]. Eight torsional stiffness, if the most recent force fields are used. Finally, we report restriction enzymes possessing differing cleavage sites and differing flanking direct measurements of the nucleation of DNA plectonemes that occurs in a sequences were employed. The results indicated that binding to flanking se- force and salt-dependent fashion on a time scale of 10-50 ms. Going beyond quences affects enzyme activity. For example, both 5-mop and 8-mop cause in- bare DNA, we have developed a magnetic tweezers assay to follow retro- hibition of BSSH II [cleavage site GCGCGC ]. The structural difference viral integration in real time, revealing several critical steps along the inte- between 5-mop and 8-mop creates differences in binding behavior. 5-mop pro- gration free energy landscape. Our data suggest that integration is driven by duces enhanced cleavage by DRA I (TTTAAA), suggesting binding at a site a ‘‘downhill’’ path in the free energy landscape. In particular, we find an separate from the cleavage site and suggesting that 5-mop produces a DNA ultra stable strand transfer complex that suggests an important role of conformational change. 8-mop produces inhibition of cleavage, suggesting resolving factors in vivo. binding at the cleavage site. Previously we examined selectivity in the binding of DB75, a groove binder [provided by D.W Boykin and W.D. Wilson, Georgia 2957-Pos Board B165 State University]. The presence of DB75 alters the binding of 5-mop near the The Sequence-Dependent Effects of Branch Migration BSSH II site suggesting possible competition. The presence of DB75 enhanced D.W. Bo Broadwater, Jr., Harold D. Kim. 5-mop effects near the STU I site [AGGCCT] suggests that, at this locale, the Physics, Georgia Institute of Technology, Atlanta, GA, USA. binding sites differ. The studies with combinations of DB75 and either 5-mop DNA strand displacement is a swapping reaction whereby a single DNA or 8-mop provide insight into the activity of these agents separately. strand invades a partially bound complementary duplex and dislodges a nearly identical strand. Over the past two decades, bioengineers have 2955-Pos Board B163 embraced this reaction as a tool in nanotechnology to create complex struc- Using Minicircles to Test the Role of DNA Bending in Mismatch Recogni- tures, logic circuits, and even robots. The mechanism is thought to initiate tion by Rad4/XPC with the binding of the original single nucleic acid strand to the toehold, Sagnik Chakraborty1, Debamita Paul2, Jung-Hyun Min2, Phoebe A. Rice3, the unpaired region of the partial duplex. It has long been thought that the Anjum Ansari1. 2 complementary strands compete for base pairs at a junction or ‘‘branch’’ 1Physics, University of Illinois at Chicago, Chicago, IL, USA, 2Chemistry, which undergoes a one-dimensional random walk until one of the strands University of Illinois at Chicago, Chicago, IL, USA, 3Biochemistry & is completely displaced. Three-strand branch migration phenomena similarly Molecular Biophysics, The University of Chicago, Chicago, IL, USA. occur at the heart of central biological systems such as homologous recom- Special proteins involved in cellular processes like gene regulation, replication, bination (D-loop) and the CRIPSR/Cas system (R-loop). Despite its wide- and repair bind to specific sites on DNA. Structures of site-specific protein- ranging importance, the sequence dependent effects of the branch migration DNA complexes reveal bent, kinked, or otherwise deformed DNA at the target step of strand displacement are largely unknown. In this study, we investi- site, suggesting that proteins distinguish specific from nonspecific sites in part gate the kinetics of three-strand branch migration and its relation to the by sensing differences in local DNA deformability. This intrinsic deformability sequence of bases occurring early in the branch migration region. We is also influenced by bending and/or topological strain (whether DNA is pre- used an innovative single-molecule FRET scheme to allow for direct mea- bent and/or supercoiled). Most studies addressing protein-DNA recognition surement of branch migration as well as a cost-effective exploration of mechanisms use short, linear DNA that do not reflect the dynamical interac- sequence and length dependence which reveal novel insights into the under- tions in the presence of bending/supercoiling, which are expected to have a pro- lying features of branch migration. We further investigate the effects of salt found impact on target recognition.Here, we investigate the dynamics of DNA and temperature. We present a quantitative model to explain the measured damage recognition by nucleotide excision repair protein Rad4 (yeast XPC or- waiting time distributions. tholog) in the context of DNA minicircles (MCs). Rad4/XPC recognizes a range of helix-destabilizing lesions caused by environmental toxins or UV- 2958-Pos Board B166 radiation. How DNA deformability and the affinity of Rad4 for model lesions Simultaneous AFM and FLIM Imaging with a SiR-DNA Probe Reveals are altered when there is bending strain are addressed. We designed 120-bp Structural Changes during DNA Condensation in Live Cell Nuclei MCs containing a CCC/CCC mismatch, previously shown to bind specifically Chetan Poudel, Nathan Curry, Kevin A. Feeney, Gabriele S. Kaminski to Rad4. As DNA of these lengths do not bend readily, we employed an inno- Schierle, Clemens F. Kaminski. vative approach where two 60 bp DNA fragments that contained a cognate Chemical Engineering and Biotechnology, University of Cambridge, site for a DNA-bending protein Hbb were pre-bent and then ligated to form Cambridge, United Kingdom. MCs with or without mismatches. Competition binding assays revealed that We show that the use of fluorescence lifetime imaging (FLIM) on the cell- Rad4 binds to both matched and mismatched MCs with significantly higher af- permeable DNA reporter dye SiR-DNA reports on the condensation state of finity compared with 24-bp linear DNA with CCC/CCC mismatch. Fluores- DNA in the nucleus of live cells. As opposed to other probes, SiR-DNA offers cence lifetime studies with a cytosine analog FRET pair incorporated in MCs the advantages of minimal toxicity, photostability, and the use of gentler far-red are ongoing to examine how the bending strain introduced affects intrinsic dy- light illumination compared to alternative, UV sensitive probes. This permits namics at the mismatched site compared with a corresponding matched site, single photon excitation and compatibility with super-resolution techniques and how that correlates with the observed increased binding affinities. and makes for a powerful probe for live-cell imaging. Using a variety of bio- logical and chemical processes known to induce DNA condensation and struc- 2956-Pos Board B164 tural modifications, we demonstrate that SiR-DNA FLIM informs on state Parallelized Magnetic Torque Tweezers Probe DNA Mechanics and Viral changes in live nuclei without the conventional requirement for cell fixation Integration with alternative probes. We verify our FLIM findings with simultaneous atomic Jan Lipfert. force microscopy (AFM) measurements in the same field-of-view and monitor Department of Physics, LMU Munich, Munich, Germany. nuclear stiffness in response to treatments that affect DNA condensation. The Magnetic tweezers are a powerful tool to probe single DNA or RNA mol- acquisition of such multi-parametric biophysical data enables us to highlight ecules and their complexes with proteins under controlled forces and tor- and study differences between types of nuclear modifications and their second- ques at the single molecule level. We have recently demonstrated several ary effects.

BPJ 8727_8730 Wednesday, February 21, 2018 597a

2959-Pos Board B167 topoisomerases are not able to keep pace with fork progression. The influence Single-Molecule FRET Investigations of Tandem Human Telomeric of topological strain on the dynamics of replication under these conditions is G-Quadruplex Structures not well understood. To investigate this issue we have developed a multidi- Emil L. Kristoffersen, Mikayel Aznauryan, Victoria Birkedal. mensional DNA manipulation platform that combines flow stretching and iNANO, Aarhus University, Aarhus, Denmark. magnetic tweezing. DNA flow stretching has emerged as a powerful approach Consecutive DNA G-quadruplexes, which are non-canonical nucleic acids to visualize replication kinetics at the single molecule level with massive mul- structures, can form from long guanine-rich sequences in the genome. One tiplexing. Combining this approach with magnetic tweezers provides torsional such region is the single-stranded human telomeric overhang, which contains control with over two orders of magnitude improvement in throughput. This the 5’-TTAGGG-3’ motif in multiple repeats. G-quadruplex structures can platform provides a powerful new tool to study DNA topology manipulating serve as regulatory switches in a number of biological processes and are consid- enzymes and torsional strain during replication in a massively parallel ered potential drug targets for cancer treatment (Balasubramanian, S. et al. Nat. manner. Rev. Drug. Discov., 2011). Single telomeric G-quadruplex structures are highly polymorphic (Dai, J et al. Biochimie, 2008), which can influence its interaction 2962-Pos Board B170 with proteins and small molecule drugs. Here, we investigate folding and poly- Inferring Transverse Statistics from Longitudinal Observables for morphism of DNA sequences capable of forming two consecutive G- Confined Wormlike Chains quadruplexes. Greg Morrison. We investigate the structure and dynamics of fluorophore labeled telomeric Physics, University of Houston, Houston, TX, USA. DNA sequences containing more than 37 bases able to form two consecutive The confinement of stiff macromolecules to nanochannels and nanoslits has G-quadruplexes using single-molecule Fo¨ster Resonance Energy Transfer recently provided a new platform for the study and manipulation of single mol- (FRET), circular dichroism spectroscopy and UV-melting experiments. Our ecules such as DNA and F-Actin. Since the readily accessible experimental ob- data show that various structures form under different cation conditions servables are found in the longitudinal axes (in-plane for nanoslits or along the (Kþ,Naþ or Liþ). G-quadruplex folding occurs in the presence of both Naþ nanochannel’s axis), a theoretical understanding of the transverse behavior of and Kþ, where we observe high FRET peaks. Our data suggest formation strongly confined macromolecules is desirable to fully understand the behavior of multiple different G-quadruplex structures under cellular like salt of these systems. conditions. In this talk, I determine the bending correlation functions for channel and slit confinement in the transverse directions analytically using a mean field 2960-Pos Board B168 approximation. I show the predictions from this approach satisfy known Atomistic Insights into DNA Twist Deformability and Fine Structure scaling laws for confined wormlike chains and more accurately capture the Korbinian Liebl. quantitative details of the transverse bending correlations than the existing Technical University Munich, Garching, Germany. weakly bending rod predictions. The predicted transverse and longitudinal DNA twist deformability plays a central role in many important biological correlation functions are coupled by a microscopic parameter that is accu- processes such as replication, transcription and repair. Although global me- rately estimated from the mean square end to end distance or radius of gyra- chanical properties of DNA can be studied with single-molecule experi- tion observed in the longitudinal dimensions for slit-confined wormlike ments and classical Molecular Dynamics (MD) simulations, molecular chains. This provides an analytical expression for the transverse correlation phenomena such as localized DNA denaturation are still not understood functions solely in terms of accessible observables for slit confined chains in atomic details. By integrating a torque acting on DNA duplexes during that agrees well with simulations. all-atom MD simulations, we unwind DNA molecules locally to induce 2963-Pos Board B171 denaturation. These simulations allow us to calculate changes in free en- De Novo Prediction of Human Chromosome Structures: Epigenetic ergy and in DNA structure along the unwinding process. The onset of local Marking Patterns Encode Genome Architecture denaturation results in an abrupt flattening of the free energy landscape and Michele Di Pierro1, Ryan R. Cheng1, Erez Lieberman Aiden2, evokes significant helical transitions, characterized by a sudden increase in Peter G. Wolynes1, Jose N. Onuchic1. stretching and breakdown in twist-bend coupling. Furthermore, simulations 1Center for Theoretical Biological Physics, Rice University, Houston, TX, under different levels of bending suppression reveal that the degree of USA, 2Baylor College of Medicine, Houston, TX, USA. twist-bend coupling largely determines the transition towards DNA dena- Inside the cell nucleus, genomes fold into organized structures that are char- turation. We also address the role of sequence dependence and investigate acteristic of cell type. Here, we show that this chromatin architecture can be the degree of reversibility of DNA unwinding. The population of backbone predicted de novo using epigenetic data derived from ChIP-Seq. We exploit substates, essentially BI/BII states, is closely related to DNA deformability the idea that chromosomes encode a one-dimensional sequence of chromatin and our simulations indicate that the presence of methyl groups promotes structural types. Interactions between these chromatin types determine the conversion from BII to BI states. By switching off non-bonded interactions three-dimensional (3D) structural ensemble of chromosomes through a pro- between methyl groups and targeted moieties during MD simulations, we cess similar to phase separation. First, a recurrent neural network is used to found out that a clash between methyl and sugar groups are the infer the relation between the epigenetic marks present at a locus, as assayed sterical origin for this effect. We expect that these results extend our cur- by ChIP-Seq, and the genomic compartment in which those loci reside, as rent understanding of DNA’s structure and flexibility and hence have measured by DNA-DNA proximity ligation (Hi-C). Next, types inferred important implications for comprehending many of its associated biolog- from this neural network are used as an input to an energy landscape model ical functions. for chromatin organization (MiChroM) in order to generate an ensemble of 3D chromosome conformations. After training the model, dubbed MEGA- 2961-Pos Board B169 BASE (Maximum Entropy Genomic Annotation from Biomarkers Associated A Multidimensional DNA Manipulation Platform Enables Massive Paral- to Structural Ensembles), on odd numbered chromosomes, we predict the lel Imaging of Replication Fork Dynamics chromatin type sequences and the subsequent 3D conformational ensembles Karl Duderstadt1,2, Rohit Agarwal1. 1 for the even chromosomes. We validate these structural ensembles by using Structure and Dynamics of Molecular Machines, Max Planck Institute of ChIP-Seq tracks alone to predict Hi-C maps as well as distances measured us- Biochemistry, Martinsried, Germany, 2Physik-Department, Technische € € ing 3D FISH experiments. Both sets of experiments support the hypothesis of Universitat Munchen, Garching, Germany. phase separation being the driving process behind compartmentalization. All cellular organisms rely on large macromolecular complexes, known as re- These findings strongly suggest that epigenetic marking patterns encode suf- plisomes, to coordinate the faithful and efficient duplication of chromosomes. ficient information to determine the global architecture of chromosomes and While the core architectural characteristics of replisomes are becoming clear, that de novo structure prediction for whole genomes may be increasingly the dynamic events that occur during replication remain obscure. In particular, possible. it remains unclear how replisomes overcome structurally diverse obstacles encountered on chromosomes, such as DNA damage, tertiary structures, and 2964-Pos Board B172 stalled protein complexes. Among these, unfavourable topological forms Intrastrand Base Pair Formation in Repetitive DNA Sequences represent a fundamental and intrinsic challenge. Strand separation due to heli- Marisa Mitchell, Carolina Dunbar, Thao Tran, Brian Cannon. case action leads to overwinding and the formation of positive supercoils. If Physics, Loyola University Chicago, Chicago, IL, USA. left unresolved, this strain can exert extreme torques that disrupt critical enzy- Repetitive trinucleotide DNA motifs at distinct genetic loci are responsible for matic events on chromosomes. Typically, topoisomerases resolve these unfav- triggering a broad group of hereditary, neurodegenerative diseases. The occur- ourable topological forms. However, there are numerous circumstances when rence of non-helical DNA structure within these motif-harboring regions has

BPJ 8727_8730 598a Wednesday, February 21, 2018 long been identified as a structural precursor that interferes with normal DNA topologically associated domains (TADs) is conserved, 10% of genomic processing and ultimately leads to pathogenic states. The precise role of these regions switches between active (A) and repressive (B) compartments. structures in this interference remains unresolved; therefore, an improved un- Through direct measurements of single genomic loci dynamics in living derstanding of these structures is critical for developing targeted therapies. cells, we find that lamin B1 can confine genomic loci dynamics in nuclear As a first step, we characterized the formation and dynamics of CAG and periphery. The Ig-like domain of lamin B1 has been known to mediate the CTG repeat DNA hairpins, a specific non-helical structure that can form within interaction between Lamin B1 and chromatin. Disruption of the lamin B1- these trinucleotide sequences, which are associated with numerous forms of chromatin interaction by loss of the Ig-like domain resulted in increased spinocerebellar ataxia and Huntington’s disease. DNA constructs were de- chromatin mobility. These results suggest that lamin B1 tethers to chromatin signed that contain a specific number of either CAG or CTG repeats, and and organizes its 3D structure at the compartment level. We propose they were labeled at specific sites with the fluorescent dyes Cy3 and Cy5. Using that like yeast, mammalian cells also use tethering as a mechanism to orga- single-molecule fluorescence microscopy, we directly imaged nanoscale nize and compartment their genome to adapt complex intra-nuclear changes in distance between the fluorescence dyes for individual DNA mole- processes. cules in real time with sub-second resolution. The changes in interdye distance arise due to continuous conformational transitions between two hairpin states 2967-Pos Board B175 (open and closed). The time traces revealed that the CTG repeats form more Directly Resolving Activated and Barrierless Events in DNA Oligonucleo- stable hairpins than the CAG repeats. Strikingly, both sequences exhibit highly tide Dehybridization Across Many Decades in Time dynamic behavior with rapid open and closing rates. In addition, increases in Paul J. Sanstead, Andrei Tokmakoff. the monovalent salt concentration increased the likelihood of the hairpin in Chemistry, University of Chicago, Chicago, IL, USA. the closed conformation. Analysis of the open and closing behavior revealed Since the initial discovery of the double-helical structure of DNA our under- two reasons for this observation: 1) the closed form of the hairpin was longer standing of nucleic acids has progressed far beyond a static archive for the lived at higher salt concentrations and 2) the transition from the open to the storage of genetic information. Whether in biological roles or DNA-based closed state occurred faster. nanotechnology, many of the most important functions of DNA are highly dy- namic. Despite remarkable experimental progress, many of the most detailed 2965-Pos Board B173 descriptions of the critical motions are still relegated to simulation. Additional Trapping the Intermediate Conformations during Flexible DNA Cyclisa- experimental insight is necessary to fully understand and characterize nucleic tion by SMFRET Measurements acid folding in real time. To this end we are developing an infrared (IR) spec- Tapas Paul, Padmaja Prasad Mishra. troscopy based approach to characterize DNA folding. We explore a model Saha Institute of Nuclear Physics, Kolkata, India. set of DNA oligonucleotides in which the placement of GC pairs is varied Chromatin is a complex of macromolecules consisting of a combination of in an otherwise AT sequence. Equilibrium linear and two dimensional infrared DNA, RNA, protein etc. Its main function comprises in packaging of the (2D IR) spectroscopy were used to characterize the degree of heterogeneity in long length DNA ( 2 m) where the DNA bending, looping, twisting and the dimer ensemble, with some sequences demonstrating significant popula- stretching plays an important role. In vivo DNA bending is a protein driven tions of frayed helices. A simple statistical lattice model was developed to process, however, the intrinsic bendability of DNA dramatically influences aid interpretation of experimental results. Through temperature jump IR spec- cyclisation/looping dynamics that relies on DNA ligase to covalently join troscopy, the dehybridization of the oligonucleotides was triggered and short sticky ends of a dsDNA. The looping and the cyclization assessment tracked in real time from nanoseconds to milliseconds, allowing the direct are very important to quantify the bending properties of DNA. We have observation of pre-melting events, dissociation into monomer strands, and re- studied the DNA looping dynamics, in a protein-free assay to investigate hybridization all in a single measurement. Variable initial temperature, fixed the cyclization of single DNA molecules in real time. To monitor the final temperature transient experiments are employed to explore the essential dynamics during the loop formation with different loop composition, we features of the underlying free energy surface. We identify both activated have chosen the telomeric G-quadruplex along with other poly A and poly dissociation as well as seemingly barrierless unzippering of the termini, G DNA sequences at the looping region. The conformation dynamics revealing that both processes are necessary to describe the hybridization have been studied by a combination of ensemble and single-molecule mechanism. FRET techniques. It has been observed that the tendency of loop formation 2968-Pos Board B176 of G-quadruplex (telomeric sequence) as well as poly G is comparatively þ Cell Density Dependence of DNA Looping in E. coli Cultures favoured in presence of K ion. However, the process less favoured in Justin P. Peters, Vishwas N. Rao, Nicole A. Becker, L. James Maher. case of Poly G system due to its structural conformation. When Poly A, Mayo Clinic, Rochester, MN, USA. due to its flexible structure, is found to have a tendency to form loop. The Sequestering a gene promoter inside a tightly bent DNA loop is a common looping rates and looping probability density, known as the J factor (jM) mechanism of gene repression in bacteria. Many factors contribute to the are extracted. This observation influences reconcile of the looping probabil- strength of this type of gene repression, including the mechanical properties ity and to underscores the importance of determining the intrinsic shape of of the looped DNA and the concentration and binding strength of the protein dsDNA. that anchors the repression loop. Additionally, E. coli cells undergo numerous highly regulated and dynamic transitions during the well-known phases of a 2966-Pos Board B174 bacterial growth curve (e.g. lag, log, and stationary). We hypothesized that Lamin B1 Tethers to Chromatin and Organizes Its High-Order Structure the probability of DNA looping would similarly depend on the growth status 1 2 1 1 2 Lei Chang , Mengfan Li , Shipeng Shao , Boxin Xue , Yingping Hou , of the E. coli culture. To test this idea, we utilized a previously characterized 2 2 1 Ruifeng Li , Cheng Li , Yujie Sun . engineered episomal repression loop model containing elements of the well- 1Biodynamic Optical Imaging Center (BIOPIC), School of Life Sciences, 2 characterized lac operon with a b-galactosidase reporter gene. We tested mul- Peking University, Beijing, China, Peking-Tsinghua Center for Life tiple episomal constructs, each containing a different operator to operator Sciences, Academy for Advanced Interdisciplinary Studies, Peking spacing, grown to three different cell densities (as measured by absorbance University, Beijing, China. at 600 nm) and assessed repression loop formation. We discuss these findings Lamin B1 constitutes a key component of nuclear lamina and is implicated and relate them to DNA supercoiling, which may also influence repression loop in nuclear skeletal, gene regulation, differentiation, and senescence. Howev- formation. er, whether and how lamin B1 organizes the three-dimensional (3D) struc- ture of chromatin remain elusive. Here, we applied a combination of 2969-Pos Board B177 chromosome conformation capture (Hi-C), super-resolution imaging, and Visualization of DNA Holliday Junctions via a High throughput Microflui- single chromosomal loci tracking to characterize the chromosome architec- dic Assay ture in wild type and lamin B1 depleted human breast tumor cells. Globally, Harrison Khoo1, Sy Redding2. the Hi-C interaction matrices of lamin B1 depleted cells indicate a relative 1University of California, Berkeley, Berkeley, CA, USA, 2Department of loss of long-range and gain of short-range interactions within chromosomes. Biochemistry and Biophysics, University of California, San Francisco, The epigenetic marker H3K27me3 shows a dramatic redistribution from Berkeley, CA, USA. the periphery to a more homogeneous nuclear dispersion, as revealed DNA Holliday junctions are four-way DNA structures comprising of two sister by super-resolution imaging. At the chromosome territory (CT) level, chromosomes that naturally form during chromosome segregation and DNA trans-interaction between chromosomes increases significantly. At the repair and replication. Misregulation of Holliday junction dissolution can compartment level, although the overall organization of the chromatin into lead to mitotic failure, toxic gene conversion, and DNA lesions, all of which

BPJ 8727_8730 Wednesday, February 21, 2018 599a are associated with human disease. Despite extensive research on DNA Holli- factor suppressing viral transfection and gene delivery. Here, we use fluores- day junction interactions, the structure itself has never been visualized in detail cence microscopy and custom single-molecule conformational tracking algo- at the single-molecule level. Thus, we propose a high throughput single- rithms to measure center-of-mass transport and time-varying conformational molecule microfluidic assay to trap and study DNA Holliday junctions in sizes and shapes of single 115 kbp DNA molecules diffusing in networks real time. This assay is derived from an established microfluidic technique, of actin filaments and microtubules. We determine the role of cytoskeleton DNA curtains, that captures large linear strands of DNA on a lipid bilayer. Hol- protein concentration and rigidity (actin vs. microtubules) on dynamics of liday junctions are synthetically fabricated in vitro for observation. This ring and linear DNA. Corresponding measurements with monomeric actin research will offer novel insight into genetic recombination and its role in dis- and tubulin identify the roles that network rigidity versus excluded volume ease, allowing for further investigation of factors influencing Holliday junction play in transport. resolution. 2973-Pos Board B181 2970-Pos Board B178 Single Molecule FRET Observations of Folding for DNA Hairpins Con- Synthetic Chlorophyll-A Derivatives Stabilize DNA G-Quadruplex taining Trinucleotide Repeats Structures Pengning Xu, Keith Weninger. Yasunobu Nagano1, Tamaki Endoh2, Shin Ogasawara1, Naoki Sugimoto2,3, Physics, NCSU, Raleigh, NC, USA. Hitoshi Tamiaki1. Expansions of tracks of Trinucleotide Repeats (TR) in DNA is associated with 1Graduate School of Life Sciences, Ritsumeikan University, Kusatsu, Japan, disorders including Huntington disease, fragile X syndrome and myotonic dys- 2FIBER, Konan University, Kobe, Japan, 3Front. Innov. Res. Sci. Tech., trophy. Unusual TR conformations may cause mutagenic DNA replication and Konan University, Kobe, Japan. affect downstream protein translation changes. We have used single molecule A DNA G-quadruplex structure is one of the higher-order DNA structures FRET (smFRET) to study the dynamics of DNA containing repeated Cytosine- found in a telomere region of chromosomes and suppresses enzymatic Adenine-Guanine (CAG) triplet patterns that can self-assemble into loops and telomere-elongation reactions which frequently occur in cancer cells. haripins. Thermodynamic characteristics such as transitional entropy and Therefore, ligand compounds stabilizing such a DNA G-quadruplex struc- enthalpy acquired from studies of single molecules are compared with results ture is expected as a new-type of anti-cancer agents. To evaluate the inter- from DNA melting bulk analysis. We have found complex patterns of hairpin action of the ligand with DNA G-quadruplexes in an aqueous solution, the stability and cooperativity that depend sensitively on the number of triplet examined ligands must be highly water-soluble. Such ligands have been repeats. usually substituted with hydrophilic substituents in a molecule. Here we report synthesis of several chlorophyll-a derivatives, in which their inner Posters: Membrane Dynamics II nitrogen is methylated, to enable their complexation and stabilization with a DNA G-quadruplex in an aqueous solution. Their binding constants 2974-Pos Board B182 with the DNA G-quadruplex structure were determined by optical spectro- Molecular Coupling of the Lipid Membrane Elasticity and In-plane scopic techniques. The values using cationic N-methylated chlorophyll-a Dynamics 1 2 2 1 derivatives were larger than those using neutral unmethylated analogs. In Kuan-Yu Tsang , Lai Yei-Chen , Yun-Wei Chiang , Yi-Fan Chen . 1Chemical and Materials Engineering, National Central University, Taoyuan, addition, the thermodynamic stabilities of several G-quadruplexes with 2 the synthetic ligands were analyzed by CD spectroscopy and will be Taiwan, Chemistry, National Tsing Hua University, Hsinchu, Taiwan. discussed. Biomembranes exhibit liquid and solid features concomitantly, with their in- plane fluidity and elasticity tightly regulated by cells. Here, with electron 2971-Pos Board B179 spin resonance and analyses on the elasticity data from the literature, we pre- Stretch and Dynamics of Single Chromatin Molecules Confined in Nano- sent experimental evidence supporting the existence of the dynamics- fluidic Channels elasticity correlations for lipid membranes and propose a mechanism William M. Rosencrans1, Fan Liu2, Nikolay V. Berezhnoy3, involving molecular packing densities to explain the correlations. This paper Anatoly Zinchenko4, Lars Nordenskio¨ld5, Johan R.C. van der Maarel2. thereby unifies, at the molecular level, the two aspects of the continuum me- 1Physics, Colgate University, Hamilton, NY, USA, 2Physics, National chanics long used to model the two membrane features. We also raise the University of Singapore, Singapore, Singapore, 3Singapore Center for possibility that the known cellular regulations on membrane fluidity and Environmental Life Sciences Engineering, Nanyang Technological elasticity are merely two manifestations of the membrane homeostasis that University, Singapore, Singapore, 4Graduate School of Environmental regulates the molecular packing densities. This ultimately may elucidate Studies, Nagoya University, Nagoya, Japan, 5School of Biological Sciences, the universal physical principles governing the cellular phenomena Nanyang Technological University, Singapore, Singapore. involving biomembranes. Inside the nucleus of eukaryotic cells, chromatin is compacted into a condensed state without compromising its accessibility for the machinery of life. The high 2975-Pos Board B183 degree of compaction in tandem with accessibility are key factors in the com- Investigating the Transbilayer Distribution of Cholesterol in Asymmetric plex regulation of the genome. In this study, we report the static and dynamic Unilamellar Vesicles using Small-Angle Scattering 1 2 1 properties of bacteriophage T4 DNA (165.6kbp) reconstituted with recombi- Christopher T. Boughter , Milka Doktorova , Wen-hung Chou , 1 1 1 nant human histone octamers and confined inside nanochannels of various Jessica M. Morgan , Steven A. Redford , Vedant Sachdeva , 1 1 3 cross-sectional diameter (100 - 250 nm). The reconstituted chromatin complex Elizabeth D. White , Vilmos Zsolnay , Frederick A. Heberle , 1 is thought to be a good model for long segments of heterogeneous human chro- Adam T. Hammond . 1University of Chicago, Chicago, IL, USA, 2Weill Cornell Medical College, matin. We measured the mean stretch and its fluctuations for various environ- 3 mental conditions and histone loading fractions. Two relaxation modes are New York, NY, USA, Oak Ridge National Laboratory, Oak Ridge, TN, observed, with the slowest one pertaining to partial opening up of the complex. USA. The efficiency of chromatin compaction by various multivalent ions and Cholesterol is the most abundant component of eukaryotic plasma membranes condensing agents is explored and discussed in terms of nucleosome- (PMs), accounting for 30-50% of the total lipid. In addition to modulating the nucleosome interaction. structural and mechanical properties of lipid bilayers, cholesterol promotes the formation of orderedþdisordered fluid phase separation in model membranes, 2972-Pos Board B180 and may similarly contribute to microdomain or ‘‘raft’’ formation in the PM. Mobility and Conformational Dynamics of Large DNA Diffusing through A major unresolved question is how PM properties and functions are influ- Cytoskeletal Networks enced by the asymmetric transbilayer distribution of lipids, including choles- Kathryn Regan, Rachel Dotterweich, Shea Ricketts, terol. While the distribution of most phospholipids and sphingolipids is Rae Robertson-Anderson. known, determining the average transbilayer distribution of cholesterol has Physics & Biophysics, University of San Diego, San Diego, CA, USA. been challenging due to its rapid translocation between the PM leaflets. Pre- The high concentrations of proteins crowding cells greatly influence intracel- vious attempts to probe the cholesterol distribution in cell membranes using lular DNA dynamics. These crowders, ranging from small mobile proteins to fluorescence-based methods have yielded conflicting results. Moreover, large cytoskeletal filaments such as semiflexible actin and rigid microtubules, studies using model vesicles have been limited due to the difficulty of prepar- can hinder diffusion and induce conformational changes in DNA. Previous ing asymmetric liposomes. Making use of recent advancements enabling the studies have mainly focused on the effect of small mobile crowders on production of asymmetric large unilamellar vesicles (aLUVs) via DNA transport; however, the rigid cytoskeleton has been identified as a key cyclodextrin-mediated lipid exchange, we prepared cholesterol-containing

BPJ 8727_8730 600a Wednesday, February 21, 2018 aLUVs in which one leaflet was enriched in egg sphingomyelin (eSM), and been clearly established. Here we describe a recently discovered bioelectric the other in phosphatidylethanolamine (POPE). We then used small-angle puzzle that deepens this mystery — nanosecond bipolar pulse cancellation, neutron and x-ray scattering combined with isotopic labeling strategies to in which the physical and biological effects of a unipolar electric pulse are probe the average cholesterol location within these bilayers. An analysis of greatly attenuated or cancelled by a second pulse of opposite polarity. The scattering data, together with complementary Molecular Dynamics simula- pulse duration must be less than 1 ms. Bipolar pulses of longer duration are ad- tions, provides insight into the transbilayer distribution of cholesterol in asym- ditive, as if each phase were a separate unipolar pulse, with positive and nega- metric membranes. tive pulses equally effective. Cancellation occurs even when the second phase of the bipolar pulse is delayed by as much as 100 ms. Bipolar pulse cancellation 2976-Pos Board B184 is a part of an expanding body of experimental results that cannot be explained Lateral Distribution and Mobility of Transmembrane Proteins in Plasma by standard electropermeabilization models. Results of measurements of Membrane Vesicles electroporation-induced molecular transport across the cell membrane include Guillermo S. Moreno-Pescador1, Emilie L. Veje1, Henrik O¨ tsbye2, bipolar pulse cancellation with pulses as short as 2 ns, transport patterns that Szabolcs Semsey3, Robert Daniels2, Poul Martin Bendix1. vary with pulse duration, and clear evidence for the rapid recovery of the 1 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark, plasma membrane resting potential, even while the membrane has not been re- 2 Department of Biochemistry and Biophysics, Stockholm University, paired and is still permeable to ions and small molecules. These experimental 3 Stockholm, Sweden, Functional genomics, University of Copenhagen, observations support the view that a robust and predictive model of electropo- Copenhagen, Denmark. ration must go beyond simple dielectric shell representations to take into ac- Cellular morphology has the potential to affect cellular functions at different count all of the permeabilizing structures and processes that appear after a scales. Positive and negative membrane curvatures are found in structures porating electric pulse —the electropermeome. like filopodia and invapodia or during the release of progeny viruses from in- fected host cells and is essential for initiation of the budding process for envel- 2979-Pos Board B187 oped viruses. The affinity of membrane proteins for high curvature regions or The Imaging FCS Diffusion Law for Multiple Diffusive Modes lipid ordered phases, for example, play a key role in the proliferation of dis- Sapthaswaran Veerapathiran, Thorsten Wohland. eases. One such protein is the influenza neuraminidase (NA), which is respon- Biological Sciences, National University of Singapore, Singapore, Singapore. sible for enzymatically hydrolyzing sialic acid residues to facilitate release of The plasma membrane is a heterogeneous matrix, which regulates key cellular the progeny virus from the infected cell after completion of the viral assembly. functions. It is organized at different length scales by lipid and protein do- The curvature affinity of both transmembrane and peripheral membrane pro- mains and the cytoskeleton meshwork. To explain the function of the plasma teins have been studied in artificial membrane vesicles and also in living cells. membrane it is necessary to understand its organization. However, this orga- However, the environment in synthetic membranes differs substantially from nization is beyond optical resolution and therefore dynamic measurements are the plasma membrane. The use of living cells poses significant problems used to indirectly prove the existence of domains and barriers by analyzing the with quantification of sorting and quantification of tube diameters and addition- mode of diffusion of probe molecules. One of these tools is the Fluorescence ally the control over membrane curvature is difficult. Also, both synthetic mem- Correlation Spectroscopy (FCS) diffusion law, which plots the diffusion time brane vesicles with simple lipid compositions and living cells with an of a probe versus the observation area to differentiate between free diffusion, underlying actin skeleton might not adequately reflect the dynamics of the domain confined diffusion, and meshwork hindered hop diffusion by the plasma membrane. Here, we show an experimental assay to study membrane plot’s y-intercept (t0). However, a description of t0 has only been given for proteins in their native plasma membrane environment. Mechanical and optical pure diffusive modes, and in many experimental cases, it is not evident that manipulation of derived Giant Plasma Membrane Vesicles (GPMVs) from cells a protein will undergo only one kind of diffusion. Here, we investigate how expressing NA-gfp construct, allow us to have full control over membrane cur- the diffusion law’s t0 value changes in the case of mixed or convoluted diffu- vature by pulling out membrane tethers using an optical trap. This new assay, sive modes. Our experimental results supported by simulations, show that ab- that allows us to create a wide range of curvatures, can be used for studying the solute t0 values are weighted averages depending on the various diffusion curvature affinity of transmembrane proteins in their native lipid environment, modes contributing to the protein mobility. By contrast, the relative changes quantify curvature sensing and diffusion. of t0 values upon disruption of a diffusion mode, typically by targeted drug treatment, are a direct indicator of the probe’s diffusion modes. These findings 2977-Pos Board B185 support earlier results on epidermal growth factor receptor (EGFR) measure- Lipid Lateral Ordering of Raft Domains Defined by High-Field EPR ments (Bag et al. Biophys. J. (2015), 109(9) 1925-1936), a protein which Zahra Hayati, Pavanjeet Kaur, Likai Song. shows domain confinement and hop diffusion, but which shows under all cir- National High Magnetic Field Laboratory, Tallahassee, FL, USA. cumstances a positive t0 value. Finally we demonstrate that two probes, which EPR spectra at 94 GHz or higher are sensitive to lipid lateral ordering, which pro- show different diffusion modes, can be distinguished in a single autocorrela- vides key information on the structure and dynamics of biological membranes as tion function, and the diffusion law can be reconstructed separately for the well as protein-lipid interactions. High-frequency and high-field EPR improves two components. spectral resolution through increased g-factor resolution, enabling the determina- 2980-Pos Board B188 tion of the motionally averaged gxx-gyy anisotropy, which reflects lipid lateral ordering. Lipid rafts are domains of plasma membranes enriched with cholesterol The Role of Traction in Membrane Curvature Generation and sphingolipids, and are essential for cellular functions. Here, we investigated Haleh Alimohamadi1, Ritvik Vasan1, Julian Hassinger2, the lateral ordering of lipid rafts using high-field EPR. The analyses reveal that Jeanne Stachowiak3, Padmini Rangamani1. 1 2 lateral ordering is more sensitive to raft-domain formation than the conventional University of California San Diego, La Jolla, CA, USA, University of 3 order parameter. Lateral ordering is a sensitive parameter to both lipid phase tran- California, Berkeley, Berkeley, CA, USA, University of Texas at Austin, sition and cholesterol/sphingomyelin content. Our data also show that raft and Austin, TX, USA. non-raft domains exhibit distinct lateral order profile across the lipid bilayer. Addi- Membrane curvature can be generated by a variety of different molecular tionally, we characterized the membrane interaction and peptide-induced lipid- mechanisms such as protein scaffolding, heterogeneity, cytoskeletal forces lateral-ordering changes of an antibacterial g-AApeptide (AA1). The results illus- that act as inputs. These mechanisms have the net effect of generating stresses trate that AA1 interacts strongly with bacterial membranes, inducing significant on the bilayer that are translated into distinct final shapes of the membrane as lipid lateral-ordering and membrane thinning. Our findings suggest that AA1 in- outputs. We sought to reverse this input-output relationship by using the teracts and disrupts bacterial membranes through a carpet-like mechanism. Taken observed shape of a curved membrane to extract physical quantities such as together, these results highlight the sensitivity of high-field EPR for elucidating the magnitude of the applied forces acting on the bilayer. To do this, we first membrane order and dynamics as well as protein-lipid interactions. calculated the normal and tangential tractions along the membrane surface us- ing the shape and material properties of the bilayer. Then, externally applied 2978-Pos Board B186 forces were computed by taking a contour integral of tractions on the region 1 D 1 = 0? — Nanosecond Bipolar Pulse Cancellation and the Electroper- boundary. We applied this approach to three different examples to demonstrate meome its utility - membrane tube formation, emergence of line tension at the interface Esin B. Sozer, P. Thomas Vernier. of two spontaneous curvature fields, and the formation of podosome protrusions Frank Reidy Research Center for Bioelectrics, Old Dominion University, in human macrophage cells. These examples demonstrate that this analysis and Norfolk, VA, USA. accompanying workflow can be used to extract meaningful measurements of Even after many years of investigation, the mechanisms underlying cellular re- force and line tension from membrane shapes in a wide variety of biological sponses to nonlethal, membrane-permeabilizing, pulsed electric fields have not contexts.

BPJ 8727_8730 Wednesday, February 21, 2018 601a

2981-Pos Board B189 behavior. We use coarse-grained molecular dynamics simulations together Fine Tuning of Microscopic Properties in Two-Component Zwitterionic- with replica exchange umbrella sampling to calculate the transfer free energy Anionic Lipid Bilayers: Determinant Role of H-Bonding of one cholesterol molecule from the bilayer to bulk water, from which we infer Roman G. Efremov1,2, Darya V. Pyrkova1, Nikolay A. Krylov1,3. the chemical potential. Our results demonstrate the sensitivity of the chemical 1Lab. of Biomolecular Modeling, Shemyakin-Ovchinnikov Institute of potential to the degree of PC tail unsaturation and show that cholesterol has the Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian greatest affinity to saturated PC lipids. Contrary to recent experiments and the Federation, 2National Research University Higher School of Economics, conceptual models, our results indicate that the chemical potential increases Moscow, Russian Federation, 3Joint Supercomputer Center, Russian linearly with increasing cholesterol concentration for all lipid types, suggesting Academy of Sciences, Moscow, Russian Federation. the absence of critical cholesterol concentrations at which the bilayer organiza- Structure, dynamics, and functioning of hydrated lipid bilayers - model cell tion changes dramatically. The increase of the chemical potential with choles- membranes - are governed by a thin balance of intermolecular interactions be- terol content also suggests that cholesterol prefers bilayers with lower tween constituents of these systems. Besides the hydrophobic effect, which de- cholesterol concentration. termines the overall bilayer skeleton, important contribution is made by H- bonds between lipids, water, and ions. This determines crucial phenomena in 2984-Pos Board B192 cell membranes: dynamic clustering, hydration, fine tuning of microscopic Multicomponent Vesicle Membranes: Influence of Material Properties physico-chemical properties, which permit fast adaptation of membranes to David Salac, Prerna Gera. external agents (e.g., proteins). Characteristics of H-bonds (strength, spatial Mechanical and Aerospace Engineering, University at Buffalo, Buffalo, NY, location, etc.) dramatically depend on local polarity properties of water-lipid USA. environment. Here, we calculated free energies of H-bonded complexes be- It is well known that the material properties of multicomponent lipid mem- tween lipids and water in explicit solvents of different polarity (water, meth- branes influence of the dynamics of vesicles. In this work computational tools anol, chloroform) mimicking membrane environment at different depth. The are used to systematically explore the influence of variable membrane proper- strongest H-bonds were observed in nonpolar environment, although the over- ties, such as bending rigidity, spontaneous curvature, and domain line tension, all bilayer organization imposes serious limitations on the distribution of and fluid properties such as increased vesicle viscosity on the dynamics of various types of H-bonds over hydrophobic/hydrophilic regions (corresponding deflated three-dimensional vesicles. In addition to reproducing various experi- to dielectric media with low and high permeability). This creates a delicate bal- mental equilibrium results, the results demonstrate rich behaviour when ance, which determines a unique H-bonding pattern for each particular lipid exposed to externally driven fluid flow and external fields. bilayer. This was confirmed via atomistic molecular dynamics (MD) of several 2985-Pos Board B193 hydrated lipid bilayers. Understanding of the factors regulating H-bonding pro- Diffusive Modes of Archaea Bolalipid Membrane pensities in such systems is indispensable for rational design of new membrane- Sergei I. Mukhin, Daria Makitruk, Daniyar Gabdullin. like materials with predefined properties. One example - an artificial lipid with Theoretical Physics and Quantum Technologies, Moscow Institute for Steel engineered hydroxyl group - is studied via MD simulations. It is shown that and Alloys, Moscow, Russian Federation. such lipids can induce significant changes of key characteristics of model mem- One of the distinctions of archaea cell is bolalipid membrane. A feature of the branes. This opens new avenues in goal-oriented design of artificial membranes bolalipid membrane is the presence of intrinsically multicomponent content. with engineered properties. Bolalipid molecules can exist in two major configurations: integral shaped (O- Acknowledgements: Russian Science Foundation (14-50-00131), Russian forms) and hairpin shaped (U-forms). The U-forms surrounded by the O- Foundation for Basic Research (16-04-00578), RAS MCB Program, Supercom- forms cause local curvature of the membrane. Ability of the U-shapes to puter Center ‘‘Polytechnical’’ (St. Petersburg Polytechnic University), Joint move inside the bolalipid layer and dependence of their potential energy on Supercomputer Center of RAS (Moscow). the local curvature of the membrane cause lateral diffusive flaws of U-forms 2982-Pos Board B190 under the membrane’s bending fluctuations. For a theoretical calculation of Shape Transformation of Biomembrane Induced by Banana-Shaped Pro- the bending modes of bolalipid membrane with small concentration of U- tein Rods forms we take the energy functional of isotropic elastic thin plate with dy- Hiroshi Noguchi. namic nonzero local spontaneous curvature J0 proportional to the local con- Institute for Solid State Physics, University of Tokyo, Kashiwa, Japan. centration of U-forms. Motion of U-forms is described by Fick’s laws in In living cells, morphology of biomembranes is regulated by various proteins. the presence of dynamic potential field effectively created by the membrane’s Many of these proteins contain a banana-shaped binding module called BAR local bending fluctuations. Resulting system of self-consistent equations is (Bin-Amphiphysin-Rvs) domain. We have studied how anisotropic sponta- solved perturbatively in the long-wavelength limit. As a result, we have found neous curvatures of banana-shaped protein rod induce effective interaction be- two brunches of frequency dispersion: purely diffusive brunch corresponding tween the proteins and change membrane shapes by using implicit-solvent to the lateral motion of U-forms, and damped bending modes of the mem- meshless membrane simulations [1-6]. The self-assembly of the rods is divided brane. Prediction is made for the spectral intensity distribution of the mem- to two directional assemblies at the low rod density [1] and polyhedral and brane’s fluctuations, that can be measured by e.g. neutron scattering high-genus vesicles are formed at the high density [2,3]. A small spontaneous technique. curvature perpendicular to the rod can remarkably alter the tubulation dynamics at high rod density whereas minor effects are only obtained at low density [4]. 2986-Pos Board B194 Two types of the protein rods with opposite rod curvatures cooperatively Analytical Calculation of Diffusion Coefficient Drop at the Liquid-Gel induce straight bumps and stripe structures [5]. The addition of small mem- Phase Transition in Lipid Membrane 1,2 1 1 brane inclusions with isotropic spontaneous curvature accelerates or suppresses Timur Galimzyanov , Boris Kheyfets , Sergei Mukhin . 1Theoretical Physics and Quantum Technologies, National University of the tabulation depending on their curvatures [6]. [1] H. Noguchi, EPL 108, 2 48001 (2014). [2] H. Noguchi, J. Chem. Phys. 143, 243109 (2015). [3] H. No- Science and Technology MISIS, Moscow, Russian Federation, A.N. guchi, Phys. Rev. E 93, 052404 (2016). [4] H. Noguchi, Sci. Rep. 6, 20935 Frumkin Institute of Physical Chemistry and Electrochemistry RAS, (2016). [5] H. Noguchi and J.-B. Fournier, Soft Matter 13, 4099 (2017). [6] Moscow, Russian Federation. H. Noguchi, Soft Matter (2017) DOI: 10.1039/C7SM01375B. Diffusion coefficient drop by an order of magnitude at the liquid-gel phase tran- sition in the lipid membranes so far was missing theoretical description. Sub- 2983-Pos Board B191 diffusion regime, which takes place on 1ps-10ns timescale, is captured by Cholesterol Chemical Potential in Mixed Phosphatidylcholine/Cholesterol our microscopic model and shows a jump of the self-diffusion coefficient. Bilayer: Model Predictions and Computer Simulations We developed the analytical theory of the first order liquid-gel phase transition Nihit Pokhrel, Lutz Maibaum. of a lipid bilayer using the microscopic model of semi-flexible strings. We have Chemistry, University of Washington, Seattle, WA, USA. shown that the van der Waals attraction between the lipids tails is the essential Cholesterol plays a vital role in maintaining the structure of cellular membranes component of the free energy. In the framework of the free volume theory, we by inducing order and increasing packing. There are multiple competing calculated a diffusion coefficient in the subdiffusive regime drop by an order of models that aim to describe the structure of cholesterol-containing phospho- magnitude at the main phase transition. We found that the main contribution to lipid bilayers, and these models make specific predictions for the concentration the diffusion drop is due to compression factor as opposed to activation-like dependence of the cholesterol chemical potential. In this work, we systemati- factor. The calculated temperature dependencies of the major thermodynamic cally study four phosphatidylcholine (PC) lipids with different degrees of un- characteristics of the lipid membranes including diffusion coefficient, mem- saturation to investigate which of these models best describes the bilayer brane thickness, area and volume per lipid molecule are in a good quantitative

BPJ 8727_8730 602a Wednesday, February 21, 2018 agreement with experimental data. The work was supported by the Russian Sci- 2990-Pos Board B198 ence Foundation (project #17-79-20440). Membrane Mediated Forces on Proteins during Diffusion and Binding Ana-Suncana Smith. 2987-Pos Board B195 FAU Erlangen, Erlangen, Germany. Membrane Mediated Cooperative Behavior of Spherical Nanoparticles Cells connect to other cells with the help of highly specific membrane-anchored Eric J. Spangler1, P.B. Sunil Kumar2, Mohamed Laradji3. adhesion proteins. This adhesion process is typically regulated by the complex 1 Biomedical Engineering, University of Memphis, Memphis, TN, USA, biochemical network of the cell. However, on the molecular level, local defor- 2 3 Physics, Indian Institute of Technology, Palakkad, India, Physics, mations, thermal and active fluctuations1 dominate every motion of the pro- University of Memphis, Memphis, TN, USA. teins, the cell membrane and the surrounding water. Nanoparticle (NP) based technologies, which are becoming increasingly prev- Recent collaborative effort which lead to the understanding of the coupling be- alent component in industrial development, have many important potential tween the protein diffusivity, activity, and organization within the membrane medical applications including diagnosis, imaging, drug delivery, hypother- during adhesion2,3 will be presented. Using a variety of modelling techniques4, mia, and photothermal therapy. Since the plasma membrane is the point of en- we explain the origin and the impact of membrane-mediated long-range forces try of cells, biomedical applications of NPs require understanding of their between proteins, which affect macromolecular assemblies’ complexation interactions with lipid membranes (LMs). Mixing NPs with soft materials, rates5, and change the diffusive behaviour of its constituents6. such as polymers and liquid crystals, often leads to cooperative behavior of [1] C. Monzel, D. Schmidt, C. Kleusch, D. Kirchenbuchler,€ U. Seifert, A.-S. NPs manifested in their self-assembly. Recent experiments have shown that Smith, K. Sengupta, R. Merkel. Measuring Fast Stochastic Displacements of the adhesion of NPs onto LMs leads to their aggregation. In order to under- Bio-Membranes with Dynamic Optical Displacement Spectroscopy. Nature stand this cooperative behavior, we conducted large scale and systematic mo- Commun. 6,8162,2015. lecular dynamics simulations of spherical NPs self-assembly mediated by [2] T. Bihr, S. Fenz, E. Sackmann, R. Merkel, U. Seifert, K. Sengupta, A.-S. their adhesion onto LMs using a coarse-grained implicit solvent model. In Smith. Association Rates of Membrane-Coupled Cell Adhesion Molecules. addition to the linear chains and tubes, indicated earlier by other researchers Biophys.J. 107,L33,2014. using dynamic triangulation Monte Carlo method, we observed additional [3] D. Schmidt, T. Bihr, S. Fenz, R. Merkel, U. Seifert, K. Sengupta, A.-S. novel self-assemblies corresponding to bitubes and rings. The phase diagram Smith. Crowding of receptors induces ring-like adhesions in model mem- of the system is determined as a function of NPs size, adhesion strength, and branes. BBA: Mol. Cell Res. 1853,2984,2015. number density on the LM. The stability of these self-assemblies, particularly [4] T. Bihr, S. Fenz, D. Schmidt, R. Merkel, U. Seifert, K. Sengupta, A.-S. bitubes and rings, was investigated using simulated annealing as well as free Smith. Membrane fluctuations mediate lateral interactions between cadherin energy calculations. bonds. Nature Physics 13,906,2017. [5] T. Bihr, U. Seifert, and A.-S. Smith. Multiscale approaches to protein- 2988-Pos Board B196 mediated interactions between membranes-Relating microscopic and macro- The Binding and Aggregation of Anisotropic Nanoparticles on Cylindrical scopic dynamics in radially growing adhesions. NJP 18,083016,2015. Lipid Membranes [6] T. Bartossek, N. G. Jones, C. Sch€afer, M. Cvitkovic, M. Glogger, H. R. 1 1 2 Alexander D. Olinger , Eric J. Spangler , P.B. Sunil Kumar , Mott, J. Kuper, M. Brennich, M. Carrington, A.-S. Smith, S. Fenz, C. Kisker, Mohamed Laradji3. 1 M. Engstler. Structural basis for the shielding function of the dynamic trypano- Biomedical Engineering, University of Memphis, Memphis, TN, USA, some VSG coat. Nature Microbiology, 2017 doi:10.1038/s41564-017-0013-6. 2Physics, Indian Institute of Technology, Palakkad, India, 3Physics, University of Memphis, Memphis, TN, USA. 2991-Pos Board B199 Golgi and endoplasmic reticulum in eukaryotic cells owe their complex mem- Doping of Hopanoids in Bilayers Modulates Oligomerization of Proteorho- brane conformations to specialized curvature inducing proteins. Using coarse- dopsin grained molecular dynamics simulations, we investigated the aggregation and Eric Sefah, Blake Mertz. binding of anisotropically curved nanoparticles to cylindrical lipid membranes Chemistry, West Virginia Univiversity, Morgantown, WV, USA. that are reminiscent of the BAR-domain containing proteins. Here we consider Proteorhodopsin (PR) is a bacterial membrane protein that functions as a light- only the case where the nanoparticle–nanoparticle interaction is repulsive and activated proton pump. The efficiency of proton pumping in PR is intimately only the concave surface of the nanoparticle interacts attractively with the lipid linked to its organization into oligomers [1]. Furthermore, the degree of oligo- head groups. We found that the ability of a nanoparticle to bind to a cylindrical merization in PR can be tuned by the composition of the membrane environ- membrane depends on the nanoparticle-lipid interaction strength, mismatch in ment [2]. However, the specific relationship between membrane composition nanoparticle-membrane curvature, and the nanoparticles’s arclength. In partic- and oligomerization of PR is unclear. In particular, the effect of hopanoids, ular, we found that the minimum interaction strength required for a single nano- the prokaryotic equivalent to cholesterol, on the ability for PR to form oligo- particle binding increases with mismatch in nanoparticle-membrane curvature mers remains unknown. Our hypothesis is that hopanoids have specific binding or increasing the nanoparticle arclength. Additionally, nanoparticles were sites on PR that stabilize membrane–protein interactions as well as influence found to accommodate a tilt angle on cylindrical membranes having a radius the degree of oligomerization of the protein. In this work, we have used of curvature less that of the bound nanoparticles. This tilt angle is well main- coarse-grained molecular dynamics simulations to determine the effect of tained for nanoparticles with large arclength, while shorter nanoparticles are two hopanoids, diploptene and bacteriohopanetetrol, on the behavior of a able to rotationally diffuse more freely. These results are consistent for larger model 1-palmitoyl-2-oleoyl-sn-glycero-3-phophoethanolamine–1-palmitoyl- numbers of nanoparticles where they aggregate into various structures depend- 2-oleoyl-sn-3-phosphoglycerol (POPE–POPG) bilayer containing PR. Our re- ing on nanoparticle-lipid interaction strength, mismatch in nanoparticle- sults show that in the presence of diploptene or bacteriohopanetetrol, mem- membrane curvature, and the nanoparticle’s arclength. This aggregation by branes undergo a noticeable decrease in area per lipid, with diploptene many nanoparticle is reminiscent of protein aggregates formed by the BAR- having a greater effect. We also observe that the presence of hopanoids depress protein family, in spite of the simplicity of our nanoparticles. clustering of PR, with the rate further decreasing as hopanoid concentration is increased. For any hopanoid-containing system, diploptene forms higher order 2989-Pos Board B197 clusters compared to bacteriohopanetetrol. Our findings thus provide a useful Microscopic View on Non-viral Mediated Transfection connection between the presence of hopanoids in bacterial membranes and Bart M. Bruininks, Paulo C. Telles de Souza, Siewert Jan Marrink. how they modulate membrane protein clustering into oligomers. [1] Hussain Science and Engineering, University of Groningen, Groningen, Netherlands. et al. J. Mol. Biol. (2015) 427:1278. [2] Maciejko et al. JACS (2015) 137:9032. Complexes of dsDNA and cationic lipids (lipoplexi) can be used as an alterna- tive for viral-vector mediated gene transfer. Gene transfer by lipoplexi does not 2992-Pos Board B200 trigger the immune system, which is a major problem with viral vectors, and A New Computational Modeling Framework for the 3D Flow and Shape production of the lipoplexi is relatively cheap. However, the transfection effi- Dynamics of Cellular Membranes cacy of most non-viral vectors is much lower compared to viral-vectors. There- Roger A. Sauer1, Amaresh Sahu2, Yannick A.D. Omar1, fore optimisation of lipoplex mediated gene transfer is required for it to be a Kranthi K. Mandadapu2. valid alternative. In this research we show how to construct a lipolex at the 1Mechanical Engineering, RWTH Aachen University, Aachen, Germany, nanoscale using Molecular Dynamics and the CG Martini force field. We use 2Chemical & Biomolecular Engineering, University of California at this model to study lipoplex-membrane fusion and show how transfection of Berkeley, Berkeley, CA, USA. dsDNA occurs over a simplified representation of the endosomal membrane Cellular membranes are characterized by complex mechanical behavior that is and vesicle. very challenging to describe accurately at continuum length scales. They

BPJ 8727_8730 Wednesday, February 21, 2018 603a undergo large shape changes, they exhibit non-trivial surface flows, they un- verse hexagonal phases. We could show a partial reversion of these peptide- dergo phase transitions, and they interact with surrounding objects. Modeling induced phase changes by the application of high pressure, demonstrating those requires solving coupled partial differential equations on evolving that a less dense packing of the monoolein membranes achieved by the fusion surfaces. peptides promotes the negative curvature of the membrane. Interestingly, the This work presents a new, three-dimensional modeling framework to describe secondary structures of the fusion peptides appear unaffected by monoolein flu- and simulate the mechanical behavior of cellular membranes. It is based on idfluid phase transitions, suggesting that the fusion peptides are the structure advanced, recently developed modeling techniques in mathematics, physics dominant species in the fusion process of lipid membranes. and computational analysis. Its mathematical description is entirely formulated in the general theory of differential geometry, since it is capable of describing 2995-Pos Board B203 arbitrary surface flows and shape changes. Its physical description is based on a The HOPS/Class C Vps Complex Tethers Membranes via a Direct Protein- rigorous thermodynamical treatment, since it provides coupling relations for Membrane Interaction the various physical fields. Its computational description is based on so- Christopher Stroupe. called isogeometric surface finite elements, since they are capable of represent- Molecular Physiology and Biological Physics, University of Virginia, ing surface flow, shape and constitution to high accuracy. The capabilities of Charlottesville, VA, USA. the new framework are illustrated by several challenging numerical examples. Membrane tethering is a physical association of two membranes before their Those include endocytosis, adhesive contact and deformation-induced surface fusion. Many membrane tethering factors have been identified, but the interac- flows. tions that mediate inter-membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sort- Posters: Membrane Fusion and Non-Bilayer ing/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low- Structures curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly-curved liposomes to Ypt7p-bearing low-curvature lipo- 2993-Pos Board B201 somes even when the high-curvature liposomes are protein-free. Phosphoryla- Liposome-Membrane Fusion Rates Altered by Dose and Location of Short- tion of the curvature-sensing amphipathic lipid-packing sensor (ALPS) motif Chain Alcohols from the Vps41p HOPS subunit abrogates tethering of high-curvature lipo- Dixon J. Woodbury, Devin M. Fuller, Miguel A. Ibarra, somes. A HOPS complex without its Vps39p subunit, which contains one of Austin L. Zimmerman. the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds Physiology and Developmental Biology, Brigham Young University, Provo, high-curvature liposomes and Ypt7p-bearing low-curvature liposomes. Thus, UT, USA. HOPS tethers highly curved membranes via a direct protein-membrane interac- Many studies on the effects of ethanol in humans have focused on its interac- tion. Such high-curvature membranes are found at the sites of vacuole tethering tions with membrane protein receptors, however we recently showed that and fusion. There, vacuole membranes bend sharply, generating large areas of short-chain alcohols dramatically alter the rate of liposome fusion to a planar vacuole-vacuole contact. We propose that HOPS localizes via the Vps41p membrane (Biophy. J.; 2017; 112:121). To understand the molecular mecha- ALPS motif to these high-curvature regions. There, HOPS binds via Vps39p nism(s) whereby alcohols alter fusion, we utilized the nystatin/ergosterol to Ypt7p in an apposed vacuole membrane. fusion assay and recorded fusion rates in response to increasing doses of al- cohols on one or both sides of the membrane (BLM). Addition of ethanol typi- 2996-Pos Board B204 cally excited fusion when applied on the cis (liposome) side, and inhibited on MiD51 and Mff Co-assemble in Cardiolipin-Enriched Membrane Micro- the trans side. Other short-chain alcohols followed a similar pattern. Addition domains to Cooperatively Regulate Drp1-Mediated Mitochondrial Fission to both sides allows the alcohol to quickly equilibrate in all membranes. Since Patrick Macdonald1, Natalia Stepanyants1, Abeer Singh2, Ryan Clinton3, cis addition also allows the alcohol to interact with both membranes, we ex- Laura Osellame2, Michael Ryan2, Rajesh Ramachandran1. pected double-sided addition to increase fusion rates. However, a decrease 1Physiology and Biophysics, Case Western Reserve University, Cleveland, was generally observed. To isolate the effect of alcohol on just the vesicle OH, USA, 2Monash University, Melbourne, Australia, 3Pharmacology, Case membrane, we altered the osmotic gradient across the planar membrane Western Reserve University, Cleveland, OH, USA. such that the water flux might drive alcohol out of the membrane. Alcohol’s Mitochondrial outer membrane (MOM)-anchored transmembrane protein effect on fusion rates changed in response to the osmotic gradient. To analyze adaptors, mitochondrial dynamics proteins of 49/51 kDa (MiD49/51) and mito- further the effects of alcohol on just the vesicle membrane, we used differen- chondrial fission factor (Mff), function to recruit the cytosolic mechanochem- tial scanning calorimetry (DSC) to observe how short-chain alcohols shift the ical GTPase, dynamin-related protein 1 (Drp1) toward mitochondrial fission. melting temperature (Tm) of liposomes made of DPPC. As expected, at low Whether these adaptors function independently or cooperatively in regulating doses (0.4-4% v/v), alcohols decrease Tm by several degrees Celsius. Howev- Drp1 function remains controversial, as are their mechanisms in Drp1- er, at higher alcohol doses, Tm increases toward control, perhaps reflecting catalyzed mitochondrial fission. MiD49/51 and Mff can independently recruit interdigitation of the lipid tails. We are developing a kinetic model to fit these Drp1 to the mitochondrial surface, yet mitochondrial fission is impaired in data. By utilizing this characteristic of the fusion assay, we hope to determine each adaptor’s absence suggesting that their functions are cooperative and the effects of alcohol via simultaneous exposure to both vesicle and planar non-redundant. Using reconstitution approaches in model membranes and mul- membrane. tiple fluorescence spectroscopic and imaging techniques, we demonstrate that both MiD49/51 and Mff preferentially partition into cardiolipin (CL)-enriched 2994-Pos Board B202 membrane microdomains characteristic of MOM-MIM contact sites that regu- Viral Fusion Peptides Incorporated in Monoolein Membranes: Secondary late cristae remodeling and cytochrome c release during apoptotic mitochon- Structure and Lipid Phase Behavior drial fission. Surprisingly, MiD49 and MiD51 utilize their cytosolic Artem Levin, Claus Czeslik, Roland Winter. nucleotidyltransferase domains to specifically bind CL. In the presence of TU Dortmund University, Dortmund, Germany. CL, MiD49/51 co-assembles with Mff and Drp1 to constitute a ternary copol- The viral membrane fusion is mediated by fusion proteins located on the virion ymer that is morphologically distinct from Drp1 assembled in the presence of surface. The process involves major changes of the membrane curvatures lead- either adaptor. We propose that MiD51 specifically recruits Drp1 dimers, which ing to non-bilayer structures. For a better understanding of the way fusion pro- then associate with Mff to constitute a ternary polymeric complex that exhibits teins steer this process, we have studied the interaction of two different reduced Drp1 GTPase activity and helps retain the transition-state conforma- hydrophobic segments, HA2-FP and TBEV-FP, which are known as ‘‘fusion tion of the Drp1 polymer on the membrane to effect membrane constriction. peptides’’, with monoolein mesophases as a function of temperature and pres- Thus, we propose MiD49/51 and Mff cooperatively regulate Drp1 function dur- sure at limited hydration. The fusion peptides are derived from the influenza ing apoptotic mitochondrial fission. virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne enceph- alitis virus envelope glycoprotein E (TBEV-FP). The changes of the monoolein 2997-Pos Board B205 phase behavior upon binding the peptides have been determined by X-ray Broadly Neutralizing Anti-HIV-1 Antibodies Do Not Inhibit HIV-1-ENV- diffraction. FTIR spectroscopy has been used to analyze the concomitant sec- Mediated Cell-Cell Fusion ondary structures of the peptides. We have found that the interaction of the Nejat Duzgunes, Michael Yee, Deborah Chau. fusion peptides with monoolein as well as the change of the temperature and Department of Biomedical Sciences, University of the Pacific, San Francisco, pressure dependent lipid phase behavior differs markedly depending on the CA, USA. fusion peptide. However, they both destabilize the fluid lamellar phase and Objectives: An effective HIV-1 vaccine must be able to block infection by a favor phases with negative curvature, i.e. inverse bicontinuous cubic and in- wide range of viral isolates. PG9, PG16, PGT121, and PGT145 antibodies

BPJ 8727_8730 604a Wednesday, February 21, 2018 were identified from culture media of activated memory B-cells of an infected angle neutron and x-ray scattering data further reveals the structural conse- donor and shown to neutralize many HIV strains, recognizing conserved epi- quences of the asymmetric distribution of DMPC in a POPC bilayer. To inter- topes on the viral envelope protein, gp120. The PG9, PG16, and PGT145 anti- pret and better understand the experimental observations, we developed a new bodies recognize V1/V2 conformational epitopes, whereas PGT121 recognizes protocol for constructing tension-free asymmetric bilayers for MD simulations, a V3 epitope involving carbohydrates. Since HIV-1 spreads via both free vi- and used it to simulate the experimentally measured membranes and validate rions and cell-cell fusion, we examined the effect of the antibodies on HIV-1 the simulation conditions. An estimate of the elastic energy of mixing in the Env-mediated cell-cell fusion. asymmetric membranes was obtained from simulations of asymmetric and cor- Methods: We used the HIV fusion assay developed previously in our labora- responding symmetric bilayers by direct comparison of the resulting elastic tory. Clone69TRevEnv cells (NIH AIDS Reagent Program) that express Env properties (including bending rigidity and area compressibility) of the individ- in the absence of tetracycline (‘‘HIV-Env cells’’) were plated, and then labeled ual bilayer leaflets. with Calcein-AM Green (Invitrogen). Highly CD4þ SupT1 cells were labeled with CellTraceÔ Calcein Red-Orange (Invitrogen), and then incubated with 3000-Pos Board B208 the adherent HIV-Env cells, with or without antibodies. Antibodies were ob- Kinetic Models of Zika Virus Membrane Fusion 1 2 2 1 tained from the International AIDS Vaccine Initiative and Polymune Scientific. Robert Rawle , Elizabeth Webster , Steven Boxer , Peter Kasson . 1Mol Phys and Biol Phys, Univ of Virginia, Charlottesville, VA, USA, Lectins were purchased from EY Labs. Syncytia were observed under a Nikon 2 Diaphot inverted fluorescence microscope. Stanford University, Stanford, CA, USA. Results: Monoclonal antibodies PG9, PG16, 2G12, PGT121, and PGT145 (at Zika virus is an emerging pathogen which can cause mild febrile illness in up to 20 mg/ml) had little or no inhibitory effect on fusion between HIV-Env adults and congenital abnormalities in infants following infection. To study and SupT1 cells. By contrast, Hippeastrum hybrid agglutinin at 1mg/ml the entry process of Zika virus, we have recently reported the hemi-fusion completely inhibited fusion. kinetics of Zika using a single virus lipid mixing assay [Rawle, Webster, et Conclusion: Antibodies PG9, PG16, PGT121, and PGT145 are ineffective al., submitted; see also companion poster/presentation by E. Webster]. against cell-cell fusion, indicating that transmission of the virus or viral genetic We observed pH-dependent hemi-fusion efficiencies, with relatively pH- material would not be inhibited. Broadly neutralizing antibodies need to be insensitive kinetics. Here we present kinetic models to explain these findings. screened for their inhibitory effect on Env-mediated cell-cell fusion. We employ both a traditional chemical kinetics approach, as well as an adap- tation of a previous numerical model which accounts for geometrical arrange- 2998-Pos Board B206 ment of and interactions between viral proteins at the virus/host membrane Single Particle Content Transfer Assay for Surface-Tethered Virus Mem- interface. We evaluate the ability of each of these to reproduce the observed brane Fusion data and how sensitively they recover parameters of the molecular kinetics Katherine N. Liu1, Robert J. Rawle2, Elizabeth R. Webster1, involved. We also show, in contrast to previous analyses of other enveloped vi- Steven G. Boxer1. ruses, that off-pathway intermediates are required to explain our observed 1Department of Chemistry, Stanford University, Stanford, CA, USA, single-virus kinetics data. Together, these kinetic models offer hypotheses 2Department of Molecular Physiology and Biological Physics, University of for future mechanistic studies of Zika virus fusion, as well as providing a Virginia, Charlottesville, VA, USA. framework to examine the membrane fusion kinetics of other flaviviruses. Enveloped virus membrane fusion proceeds through a proposed hemifusion in- 3001-Pos Board B209 termediate, followed by the formation of a fusion pore through which the viral Ebola Virus Spike Glycoprotein Recruits Cholesterol for Efficient Fusion genome is transferred to a target cell. We have previously reported an assay to Jinwoo Lee, Alex J.B. Kreutzberger, David A. Nyenhuis, study viral fusion that uses synthetic DNA-lipids to tether virions to target ves- Elizabeth A. Nelson, Volker Kiessling, David S. Cafiso, Judith M. White, icles in the absence of a receptor (Biophysical Journal, 111, 123-131 (2016)). Lukas K. Tamm. This approach uses a self-quenched concentration of fluorescently labeled University of Virginia, Charlottesville, VA, USA. lipids added to the viral envelope and yields information that is limited to lipid Cholesterol serves critical roles in membrane fusion of enveloped viruses by mixing. Here, we expand this assay to study the mechanism of viral content modulating physical properties such as thickness, fluidity, and curvature of bio- transfer. We first tether influenza virions to a surface using DNA-lipid conju- logical membranes. The spike glycoprotein GP of Ebola virus (EBOV) is a gates, then bind content-labeled vesicles to virions using an orthogonal class 1 viral fusion protein sharing similar structural features with Influenza DNA-lipid sequence. The kinetics of individual, pH-triggered content mixing hemagglutinin and HIV gp120/gp41. Fusion of EBOV in the late endosome re- events is then detected by fluorescence microscopy. This approach avoids the quires Niemann-Pick C1 protein as a receptor, which is an endosomal choles- difficult step of content labeling viruses and should be applicable to any envel- terol transporter. Thus, cholesterol might be important in EBOV membrane oped virus. fusion and cell entry. Here we present evidence that the EBOV GP membrane proximal external region/transmembrane (MPER/TM) domains recruit choles- 2999-Pos Board B207 terol to a local environment that enhances fusion. Virus-like particle (VLP) cell Interleaflet Coupling in Asymmetric Membranes: Protocols and entry and lipid mixing fusion assays show that entry and fusion depend on the Revelations cholesterol concentration in the viral membrane. Cholesterol titration experi- 1,2 3 4 Milka Doktorova , Frederick A. Heberle , Boris Dzikovski , ments using NMR revealed that several glycines, most notably G660, on the 4 3 5 Siddarth Chandrasekaran , John Katsaras , Gerald Feigenson , GP2 TM domain are critical for its interaction with cholesterol. The interaction Harel Weinstein2. 1 in the G660L mutant is diminished and shows a more open angle between the Tri-Institutional PhD Program in Computational Biology and Medicine, MPER and TM domains in the mutant compared to WT, as measured by pulsed Weill Cornell Medical College, New York, NY, USA, 2Physiology and 3 EPR experiments. Single particle fusion experiments (by TIRF microscopy) Biophysics, Weill Cornell Medical College, New York, NY, USA, Biology show that cholesterol in the viral membrane enhances the probability and ki- and Soft Matter Division, Oak Ridge National Laboratory, Oak Ridge, netics of fusion. The G660L mutant shows a higher probability of hemifusion, TN, USA, 4Chemistry and Chemical Biology, Cornell University, Ithaca, 5 which correlates with a 4-fold lower cell entry efficiency of mutant compared to NY, USA, Molecular Biology and Genetics, Cornell University, Ithaca, NY, WT VLPs. Taken together, it appears that a quite strong interaction of choles- USA. terol with the TM domain of GP is important to facilitate cell entry of EBOV by In the life of cells, the many functional roles of the plasma membrane require membrane fusion. tight regulation of properties including structure and dynamics. These proper- ties depend on the lipid composition of the membrane, so that differences in the 3002-Pos Board B210 functions of the two membrane leaflets are best served by an asymmetric lipid Novel Labeling Strategy for Automated Detection of Single Virus Fusion distribution. In addition to exposing certain types of lipids to the extracellular and Assessment of HIV-1 Protease Activity in Single Virions or intracellular spaces, the transbilayer lipid distribution also affects the prop- Chetan Sood1, Ashwanth C. Francis1, Tanay M. Desai2, Gregory Melikyan1. erties of the membrane itself. How these effects contribute to normal cell func- 1Pediatrics, Infectious Diseases, Emory University, Atlanta, GA, USA, 2Carl tion remains a largely open question. To address this question we have Zeiss Microscopy, Thornwood, NY, USA. developed both in vitro and in silico protocols for building asymmetric model A common approach to visualizing single-virus fusion utilizes a fluorescently membranes and investigating the dynamics, energetics, and structural conse- labeled Gag that, upon processing by HIV-1 viral protease, yields fluid-phase quences of interleaflet coupling. For a set of compositionally asymmetric fluorescent proteins trapped within the virus particle. Upon loss of viral mem- extruded liposomes containing DPPC and different lower melting temperature brane integrity this content marker rapidly diffuses out of the particle, and, in lipids we have obtained ESR data on the acyl chain order and rotational diffu- combination with a viral core fiducial, such as a fluorescently tagged Vpr, can sion of the lipids in each bilayer leaflet. Joint analysis of a large set of small- be used to reliably report single-particle fusion. But, this labeling approach is

BPJ 8727_8730 Wednesday, February 21, 2018 605a limited by an uncontrolled incorporation of the two markers, resulting in an membrane. Ongoing experiments are examining the role of lipids in LASV- excess of single-labeled particles and lack of correlation between intensities mediated cell-cell fusion. of the content and core markers. Here, we introduce a novel bi-functional mCherry-2xCL-YFP-Vpr label that encodes both the content (fluid-phase 3005-Pos Board B213 mCherry) and fiducial (core-associated YFP-Vpr) markers on a single Target Membrane Spontaneous Curvature Modulates ITS Poration by construct separated by a 2xCL linker that contains a tandem of cleavage sites Influenza Virus targeted by the viral protease. We show that the bi-functional label is effi- Sourav Haldar, Elena Mekhedov, Paul S. Blank, Joshua Zimmerberg. ciently incorporated and processed in single-virus particles and can be used LCMB, NICHD/NIH, Bethesda, MD, USA. to reliably report single-virus fusion in imaging assays. Further, by taking Enveloped viruses merge with plasma or endosomal membranes to transfer advantage of the nearly perfect colocalization and correlated intensities of their genetic material into the host cells. Specialized proteins on the viral content and fiducial markers in single-particles we are able to automate fusion membrane catalyze the merger of viral and cellular membranes. For influ- event detection in both fixed and live cells, significantly increasing assay enza virus, fusion between viral and endosomal membranes is mediated by throughput. Finally, we show that the loss of Forster Resonance Energy the surface glycoprotein haemagglutinin, which is triggered by a low endo- Transfer (FRET) signal between YFP and mCherry is a good predictor of viral somal pH induced conformational change. Although influenza is a global protease activity, and that the maturation status of single virions can be in- health concern and one of the most widely studied viruses, molecular de- ferred by both FRET efficiency and YFP/mCherry signal ratio. Together, tails about its fusion process remain elusive. Based on a high resolution these results demonstrate that the new labeling strategy offers a number of ad- cryo-electron microscopy investigation of hemifusion structures, a new in- vantages compared to the previous approaches, including reading out single- termediate, lipidic-junction, has been identified. It was posited that, beside virus maturation status and increasing reliability and throughput of single- the canonical hemifusion-stalk pathway, there is rupture-insertion pathway virus fusion imaging assays. that leads to membrane edge formation, dependent on the target membrane lipid composition. Membrane edge formation would lead to loss of internal 3003-Pos Board B211 content, alternatively allow soluble contents to enter the volume defined by Simulations and Experiments Show a Mechanistic Role for Influenza the target membrane. We carried out membrane poration experiments, Fusion Peptides in Membrane Bending and Fusion Stoichiometry based on a giant unilamellar vesicle (GUV) influx assay in the presence Peter Kasson. of influenza virus over a wide range of target GUV lipid compositions. Uppsala University and University of Virginia, Uppsala, Sweden. Our results show that in the presence of influenza virus at low pH, lipid Membrane fusion by influenza virus is exquisitely sensitive to the N-terminal compositions producing edge formation, exhibit poration. The probability residues of the membrane-inserted fusion peptide. Just how single-residue sub- of poration is dependent on the monolayer spontaneous curvature of the stitutions at the N-terminus of this peptide can cause large changes in fusion target membrane. Our data is well described by a Poisson model and is phenotype and viral infectivity has remained a puzzle in the field. Here, we consistent with the idea that hemifusion pathways are dependent on the use a combination of atomic-resolution simulations of viral membrane fusion spontaneous curvature of the target membrane. We are testing our hypoth- and fluorescence microscopy to probe the role of the fusion peptide N-terminus esis that the influenza fusion peptide is responsible for edge formation and in controlling fusion behavior. Surprisingly, we find that the formation of high- recapitulates virus induced target membrane poration using the GUV influx ly curved fusion intermediates is highly sensitive to the N-terminus in simula- assay. tions, and the fusion behavior of both mutant virus and hemagglutinin- 3006-Pos Board B214 expressing cells also suggests a difference in protein stoichiometry required Should I Splay or Should I Stay - How Lipids and Transmembrane Helices for fusion between wild-type and mutant fusion peptides. Together, these Determine Membrane Fusion data suggest mechanistic roles for the fusion peptide N-terminus that can be Katja Kolocaj1, Holger A. Scheidt2, James A. Frank3,4, Dirk Trauner3,5, directly connected to fusion behavior and infectious outcomes, helping explain Daniel Huster2, Dieter Langosch1. how these seemingly subtle changes to a membrane-inserted peptide can alter 1Chair of Biopolymer Chemistry, Technical University of Munich, Freising, the behavior of the entire viral fusion machinery. Germany, 2Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig, Germany, 3Department of Chemistry and Center for 3004-Pos Board B212 Integrated Protein Science, Ludwig-Maximilians-University of Munich, Lassa Fever Virus Glycoprotein Mediates LAMP1- and Low pH- Munich, Germany, 4Department of Materials Science and Engineering, Dependent Cell-Cell Fusion through a Stalk-Pore Mechanism Massachusetts Institute of Technology, Boston, MA, USA, 5Department of Ruben M. Markosyan1, Mariana Marin2, Fredric S. Cohen1, Chemistry, New York University, New York, NY, USA. Gregory B. Melikyan2. The mechanism of two membranes fusing and the transition states involved 1Physiology, Rush University Medical Center, Chicago, IL, USA, 2 are of tremendous interest. Fusion is driven by a highly regulated protein Department of Pediatrics Infectious Diseases, Emory University, Atlanta, machinery that cooperates with the lipids of the membrane. The questions GA, USA. as to how exactly fusogenic proteins and lipids interact, and which fusion- Lassa fever virus (LASV) GP glycoprotein-mediated entry into cells proceeds regulating properties are needed to generate membrane fusion, have yet to a through multiple distinct steps. Initial binding to the surface -dystroglycan be elucidated. Importantly, understanding conditions that initiate fusion is allows virus uptake and transport to late endosomes/endolysosomes. Here essential for optimization of lipid-based drug delivery systems and thera- acidic pH mediates virus dissociation from a-dystroglycan and binding to peutic targeting to organs of interest. Lipid splay has been implicated in the LAMP1 receptor that is thought to trigger LASV fusion. The difficulties inducing fusion by several modeling studies. Here, we correlated lipid associated with mechanistic studies of virus-endosome fusion prompted us splay, as determined by NMR spectroscopy, and membrane fusion, using to examine LASV GP-mediated fusion using a cell-cell fusion model. We lipid-mixing assays. We established splay-stabilizing conditions, by using show that cells expressing LASV GP fuse to mammalian cells upon exposure peptide-containing and peptide-free lipid environment. Additionally, we to low pH and that this fusion is promoted by ectopic expression of LAMP1 asked to which extent fusion-inducing TMDs cooperate with lipids. By and, even more significantly, by the LAMP1 mutant that resides in the plasma varying membrane components and including a photoswitchable lipid, we membrane. These findings suggest endogenously expressed LAMP1 is pre- were able to establish a molecular milieu that favors splay and enhances sent on the cell surface. Using this new cell-cell fusion model, we show full membrane fusion. Our results provide new insights into the role of lipid that LASV GP does not require priming by a receptor to undergo irreversible splay in the mechanism of membrane fusion, and thus strengthen the notion acid-mediated refolding. Additional mechanistic experiments showed that, of acyl chain protrusion as a crucial transition state involved in the merging similar to other viral glycoproteins, LASV GP-mediated fusion proceeded of two membranes. We established a deeper understanding of how manip- through a hemifusion intermediate, which can be arrested by exposure to ulating the molecular composition of lipid membranes affects membrane low pH in the cold, and that this intermediate can be converted to full fusion fusogenicity. by chlorpromazine treatment. We further demonstrate that GP-induced hemifusion requires the formation of a highly curved lipid intermediate 3007-Pos Board B215 (stalk) disfavored by incorporation of a positive-curvature lipid lyso- Combining MD Simulations and 31P NMR Spectroscopy to Decipher phosphatidylcholine into the plasma membrane. Real-time measurements of Lamellar to Hexagonal Phase Transition Promoted by Diverse Lipid Types transfer of fluorescent cytoplasmic markers revealed that LASV GP- Matthieu Chavent, Evert Haanappell, Alain Milon. mediated fusion pores did not enlarge as readily as those formed by other viral IPBS - CNRS, Toulouse, France. glycoproteins. The less efficient dilation of these fusion pores might be due to Transition from lamellar to non-lamellar phase is an important process for the difference in lipid/protein composition of late endosomes and the plasma biological phenomena such as vesicle fusion or lipid nano-domains

BPJ 8727_8730 606a Wednesday, February 21, 2018 organization. Finely understanding such biophysical state of lipids, espe- hydrophilic pore formation and consequent formation of dead-end intermedi- cially stalk formation, will help to develop more accurate force field and bet- ate increases with increasing of the insertion diameter. Deep insertion of ter understand experimental data from NMR spectroscopy. Here, we present fusion peptides promotes the fusion of membranes via classical stalk mecha- extensive work comparing Molecular Dynamic simulations (based on the nism, as it leads to the incurvation of the membrane and decrease of the hy- MARTINI force field) with 31P NMR spectroscopy. This allows bench- dration repulsion between the membranes. Increasing the diameter of deep marking the simulations on a very detail dataset of temperatures and lipid insertion leads to decrease of the energy barrier towards fusion, which indi- compositions (based on a variation of PE/PC lipids). We then performed cal- cates increase in fusion activity. Our results are in a good agreement with culations to emulate NMR spectra based on simulations data to directly experimental data and MD simulations. Thus, we show that the action of compare with experimental results. This shed new lights on NMR results fusion proteins can result in either leaky or non-leaky fusion or formation and help to explain the formation of stalks and the transition to inverse hex- of dead-end leaky intermediate. agonal phase. We then assessed both experimentally and computationally how insertion of diverse biologically important lipids - from cholesterol to 3010-Pos Board B218 bacterial virulence factors - may influence the phase transition. These results Study of the Fusion Mechanism of Fusogenic Cationic Liposomes with can then be replaced in a more biological context to understand the influence Anionic Model Membranes of lipids on cell membrane structuration. Rafaela R.M. Cavalcanti, Rafael B. Lira, Karin A. Riske. 3008-Pos Board B216 Unifesp, Sao Paulo, Brazil. Computational and Experimental Study of Dope and Pope Lipids in the Liposomes have been used as a drug delivery system in biological media. Inverted Hexagonal Phase: Effect of Water per Lipid, Temperature, However, they typically enter the cell via endocytic routes, which lower the delivery efficiency of the encapsulated material to the intracellular me- Salt Concentration, and Simulation Setup dium. A fusogenic liposomal system composed of DOTAP (cationic lipid) Mohsen Ramezanpour1, Bashe Y.M. Bashe2, Miranda L. Schmidt2, and the phospholipid DOPE in equal molar fractions showed high fusion Jenifer L. Thewalt2,3, D. Peter Tieleman1. 1Centre for Molecular Simulation, Department of Biological and delivery efficiencies with both biomimetic lipid vesicles and cells. Sciences, University of Calgary, Calgary, AB, Canada, 2Department To understand the role of the lipid composition in the fusion mechanism of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, and efficiency, DOTAP:DOPE liposomes were titrated with large unilamel- BC, Canada, 3Department of Physics, Simon Fraser University, Burnaby, BC, lar vesicles (LUVs) of different lipid composition, containing POPC (zwit- terionic lipid), POPG (anionic lipid in increasing concentrations), and Canada. Non-lamellar lipid phases such as the inverted hexagonal (HII) phase are of cholesterol (up to 30 mol%). Isothermal Titration Calorimetry (ITC), zeta special interest in nanomedicine, e.g. in the drug/gene release step of intra- potential and size measurements using Dynamic Light Scattering (DLS) cellular therapeutic delivery to the target cell. In this study, the structural were used to evaluate fusion efficiency along the titration. At the end of properties, including the deuterium order parameters and lattice distance, the experiments, the final mixture was observed with optical microscopy. of DOPE lipids in the HII phase were studied as a function of temperature ITC data showed that fusion is an exothermic process with a stoichiometry that depends on the POPG:DOTAP ratio. However, with the addition of using molecular dynamics (MD) simulation, NMR, and SAXS experiments. cholesterol in the acceptor system, an endothermic component appeared MD simulation was also used to provide deeper insights into the effect of water per lipid (WPL) and salt concentration on both structural and dynam- and eventually dominated the titration at the highest cholesterol fraction. ical properties of DOPE and POPE systems in HII phase. The preliminary The combined results of zeta potential and size of the liposomes showed results suggest that the WPL has a high impact on both deuterium order that when the zeta potential was close to zero, vesicle fusion/aggregation parameter and lattice distance. In principle, to be consistent and comparable was maximal. As the molar fraction of POPG in the system increased, with experimental data, the WPL in the simulation should be the same to the the size of the vesicles also increased, indicating high fusion efficiency. On the other hand, addition of cholesterol to the acceptor LUVs suppressed one in the corresponding experimental setup. However, the accurate mea- surement of the WPL inside the water channels of the HII phase is chal- the fusion efficiency. We can conclude that the efficiency of the DOTAP:- lenging experimentally since it requires the experiments to be conducted DOPE fusogenic system is modulated by the lipid composition of the in the low WPL regime. Given well-established force field parameters for acceptor membrane. the lipids, which is the case for both DOPE and POPE lipids, computer simu- lation can assist experimentalists in estimating the WPL. In addition, this 3011-Pos Board B219 will provide a methodology for lipid force field parameter development in Nanomechanics of Membrane Fission: Elasticity of the Precursor State 1 1 2 2 situations where only experimental data in the HII phase are available for Pavel Bashkirov , Ksenia Chekashkina , Anna Shnyrova , Pedro Arrasate , 3 4,5 that specific lipid. Finally, since experimental data for DOPE and POPE Peter Kuzmin , Vadim Frolov . 1Federal Scientific Clinical Center of Physical Chemical Medicine of FMBA lipids in the HII phase is available, these systems can form frameworks to 2 validate the simulation setup for HII systems required for the study of other of Russia, Moscow, Russian Federation, University of the Basque Country, lipids or lipid mixtures in this phase. Biofisika Institute (CSIC, UPV/EHU) and Department of Biochemistry and Molecular Biology, Leioa, Spain, 3A.N. Frumkin Institute of Physical 3009-Pos Board B217 Chemistry and Electrochemistry, Russian Academy of Sciences, Moscow, Leaky Intermediates and Possible Dead-End Configurations in Membrane Russian Federation, 4Biofisika Institute (CSIC, UPV/EHU) and Department Fusion of Biochemistry and Molecular Biology, Leioa, Spain, 5IKERBASQUE, Rodion Yu Molotkovskiy1, Timur R. Galimzyanov1,2, Piotr I. Kuzmin1, Basque Foundation for Science, Bilbao, Spain. Sergey A. Akimov1,2. Fusion and fission of cellular membranes are generally associated with cre- 1Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry of RAS, ation of extremely high membrane curvatures. Mechanical properties of lipid Moscow, Russian Federation, 2Theoretical Physics and Quantum bilayer bent to such curvatures remain poorly characterized. Here we report Technologies, National University of Science and Technology, Moscow, real time measurements of the mean curvature elastic modulus in lipid nano- Russian Federation. tubes (NT) constricted to curvatures characteristic for spontaneous mem- Planar lipid membranes fusion demands overcoming of energy barrier associ- brane fission. We used nanoscale electro-actuation to perturb the NT ated with deformations and hydration repulsion between the membranes. This shape and measured the resulting changes in the I/V characteristic of its barrier is surmounted due to the action of a complex of specific proteins called luminal conductance. We found that the NT deformations were accurately fusion proteins. According to the classical stalk mechanism, membrane fusion described by linear curvature elasticity with the appropriate macroscopic proceeds through a sequence of non-leaky intermediates. However, there is modulus, function of the NT lipid composition. Fission was not associated experimental evidence that during the fusion a pore can form in one of the with measurable changes in the linear elastic behavior. Importantly, membranes. Depending on mutual organization of the pore and fusion rosette, increasing of the curvature stress accelerated fission but also promoted for- either complete membrane fusion or dead-end intermediate can form. We use mation of pores and could even lead to structural destabilization of the NT the continuum elasticity theory to analyze possible trajectories of the system membrane. Such destabilization was associated with extreme protein crowd- evolution. We show that the main protein parameters effecting the fusion are ing on the NT surface while similar phenomenology was also observed in the depth of insertion of the fusion peptide into the membrane of the target fission driven by lyso-lipids. Our results indicate that physiologically rele- cell, the diameter of the fusion peptide, and the radius of the fusion rosette. vant leakage-free fission should be discriminated from that associated with According to our analysis, shallow insertion of fusion peptides promotes for- membrane destabilization. Dedicated fission proteins can utilize specific mation of a hydrophilic transversal pore in the membrane. The probability of ‘‘catalytic’’ strategies to minimize destabilization effects while achieving

BPJ 8727_8730 Wednesday, February 21, 2018 607a required rates of the membrane remodeling. The work was financially sup- complexin and synaptotagmin are closely coupled to SNAREs during synaptic ported by the Russian Science Fund No 17-75-30064. vesicle fusion and lead to synchronous fusion. Synaptotagmin-1 (Syt1) is a transmembrane protein found in synaptic vesicles and is the Ca2þ sensor for 3012-Pos Board B220 synaptic transmission. Syt1 has two Ca2þ binding domains, C2A and C2B, Molecular Interactions of Lipids and Mineral Surfaces along with a flexible linker from the membrane to C2A, and a linker between 1 2 1 Brenda L. Kessenich , John S. Loring , Sarah L. Keller , C2A and C2B. A polybasic patch in C2B has been shown to bind to anionic James J. De Yoreo2,3. 1 2 lipids such as phosphatidylserine (PS) and phosphatidylinositol (PIP2) in the Chemistry, University of Washington, Seattle, WA, USA, Pacific 2þ 2þ 3 absence of Ca .Ca binding to Syt1 allows non-polar residues nearby the Northwest National Laboratory, Richland, WA, USA, Materials Science and Ca2þ binding sites to insert into the membrane. While these mechanisms are Engineering, University of Washington, Seattle, WA, USA. relatively well-accepted, the precise biochemical and biophysical mechanisms Lipid assembly at mineral surfaces is a poorly understood phenomenon that for the Syt1 Ca2þ trigger remain unclear. In this work, we directly measure the reflects underlying lipid-lipid and lipid-substrate interactions, both of which interactions of Syt1-coated surfaces with anionic membranes including PS and are expected to be sensitive to hydration/dehydration cycles and have the po- PIP2 lipids by the surface forces apparatus technique, in order to mimic a Syt1- tential for significant environmental impacts. Organic matter derived from coated synaptic vesicle membrane interacting with the anionic plasma mem- plants and microbes represents a large and poorly constrained terrestrial car- brane. We perform site directed mutagenesis of the Ca2þ binding sites of bon pool that is stabilized through the interaction with mineral surfaces via an C2A and C2B, along with the polybasic patch in C2B, to fully map the site- unknown mechanism. The lipid fraction is particularly interesting as it im- binding energetics of Syt1 with membranes, both in the absence and presence pacts parameters such as soil water repellency and its assembly on mineral of divalent ions. The addition of Ca2þ leads to a dramatic increase in the bind- surface may inhibit digestion by microbial attack and consequent mobiliza- 2þ ing energy, around 10 kBT, due to the insertion of the hydrophobic C2B Ca tion of carbon. Furthermore, determination of molecular-scale interaction loop into the membrane. The results illuminate a passive role for C2A and a mechanisms of lipids and mineral surfaces may help elucidate the role of min- strong-binding, active role for C2B. erals in formation of pre-biotic membranes. To understand lipid-mineral interaction and assembly, as well as the impact of hydration/dehydration cy- 3015-Pos Board B223 cles, we are using infrared spectroscopy and atomic force microscopy (AFM) Investigation of Synaptic Vesicle Fusion Mechanisms with Novel Vesicular to observe humidity-dependent conformation and morphology for model sys- Force Microscopy tems of DOPC, DSPG, and DSPE on mica, montmorillonite, and kaolinite. Ines Luchtefeld€ , Tomaso Zambelli, Janos Vo¨ro¨s. Lipid morphology observed via both fluorescence and AFM is found to be het- Institue for Biomedical Engineering, ETH Zurich, Zurich,€ Switzerland. erogeneous in both liquid and air, with some dependence on the type of lipid The fusion of the lipid membranes of cells with those of vesicles plays an headgroup. AFM data also suggest that bilayers are the dominant unit of dry important role in many essential physiological processes such as the neuro- lipid structures on mineral surfaces, contrasting prior hypotheses in the soil transmitter release at neural synapses. However, the mechanisms behind these science field for transformation to monolayer structures. Preliminary mea- membrane fusion events are poorly understood due to a lack of appropriate surements of contact angle vs lipid coverage suggest both a sharp increase methods to facilitate their investigation. All available methods to date use in hydrophobicity at low coverage and oscillatory variations as coverage is supported or floating artificial lipid bilayers instead of free vesicles, and increased beyond that of a full bilayer. Changes are seen in both photo- thus do not provide a physiological model of the fusion processes. Here, we induced force microscopy and attenuated total reflectance infrared spectros- propose a novel experimental system for the investigation of the forces acting copy with varying humidity that may reflect changes in conformation. The during the membrane fusion of vesicles with lipid bilayers or cell membranes. relationship between these changes and both contact angle and soil water The system consists of an atomic force microscopy (AFM) cantilever with an repellency are the subject of current research. integrated microfluidic channel, namely the FluidFM. By applying negative 3013-Pos Board B221 pressure to the channel, objects like polystyrene beads can be attached revers- Membrane Fusion as a Function of Normal Forces and In-plane Tension ibly to the cantilever opening. Additionally, the described system provides the Andreas Janshoff. possibility to reversibly attach a membrane vesicle to the tip of the cantilever. Chemistry, Institute of Physical Chemistry, Goettingen, Germany. These vesicles can be either composed of artificial lipid bilayers with inte- Membrane fusion of lipid bilayers is prevented by substantial energy barriers grated membrane proteins or of native vesicles extracted from cell solutions. arising from removal of the hydration shell, formation of highly curved struc- By approaching the cantilever with the attached vesicle to the substrate, the tures and eventually fusion pore widening. We measured the impact of normal interaction between vesicle and substrate can be investigated. The force feed- and tensile forces on the fusion efficiency of two bilayers in the presence of back of the AFM system thus facilitates precise measurements of the forces SNAREs. Force-dependent lifetime of fusion intermediates were measured acting during the observed fusion events. One application for the experi- by means of membrane coated silica spheres serving as force probes in contact mental system described above is the investigation of the SNARE-mediated with supported lipid bilayers. Analysis of time traces obtained from force vesicle docking and fusion at the presynaptic button. Once established, a sys- clamp experiments allowed us to assign different intermediate states during tem for force controlled approximation of (native) vesicles to lipid mem- fusion. Force-dependent lifetime distributions of the various fusion states branes would thus enable the investigation of a multitude of interesting pave the way to determine the free energy barriers related to the passing of biological and biophysical questions and might bring new insights in many the hydration barrier, hemifusion and full fusion, the area of the transition areas including neuroscience. state and the zero-force lifetime of intermediates in SNARE-mediated mem- brane fusion. We also investigated the tension-dependency of fusion using 3016-Pos Board B224 two different strategies. One is based on dilatable supported bilayers and Molecular Conformation and Topography of a Synaptic Lipo-Peptide in the other one employing sessile giant liposomes. In both approaches fusion Simulated Synaptosomal Membrane Lipids 1 1 2 3 efficiency increases considerably with lateral tension and we identified a Julian Whitelegge , Piotr Ruchala , Alan Waring , Cameron Gundersen . 1NPI-Semel Institute, UCLA, Los Angeles, CA, USA, 2Medicine, UCLA, threshold tension at which the number of fusion events is increased 3 substantially. Los Angeles, CA, USA, Pharmacology, UCLA, Los Angeles, CA, USA. Synaptosomal proteins have a number of vicinal cysteine and lysine residue 3014-Pos Board B222 side chains that may be covalently palmitoylated via ester or acyl linkages. Direct Measurement of Site-Specific Binding Energetics of Synaptotagmin-1 Synthesis of a twenty-one residue peptide derived from the synaptosomal pro- with Anionic Lipid Membranes tein Synaptotagmin (sequence residues 72 - 92) using Fmoc chemistry with Clemence Gruget1, Jeff Coleman2, Oscar Bello2,3, Shyam Krishnakumar2,3, Cysteine carrying side-chain thioether linked palmitate was used to charac- Frederic Pincet1, James E. Rothman2,3, Stephen H. Donaldson, Jr.4. terize the structure and activity of the lipo-peptide with multilayer phospholipid 1Laboratoire de Physique Statistique, Ecole Normale Superieure, Paris, vesicles having POPC and POPS composition representative of synaptosomal France, 2Department of Cell Biology, Yale University School of Medicine, membranes. The peptide dissolved freely in chloroform, methanol, 1% formic New Haven, CT, USA, 3Institute of Neurology, University College London, acid in water (4/4/1; v/v) and its primary structure was analyzed by high- London, United Kingdom, 4Departement de Physique, Ecole Normale resolution electrospray-ionization mass spectrometry confirming accurate syn- Superieure, Paris, France. thesis. Both collisionally activated and electron-capture dissociation (CAD/ Upon arrival of an action potential at the neuronal synapse, calcium ions (Ca2þ) ECD) techniques provided complementary information on fatty acylation sites. are released, allowing soluble N-ethylmaleimide-sensitive factor activating Secondary structure analysis of the peptide after reconstitution in multilayer protein receptor (SNARE) proteins to fully zipper, leading to fusion of pre- vesicles using FTIR - ATR spectroscopy indicated that the dominant conforma- docked vesicles containing neurotransmitters. Regulatory proteins such as tion of the peptide in this environment was extended beta sheet. We

BPJ 8727_8730 608a Wednesday, February 21, 2018 hypothesize that the membrane-associated fatty beta motif modulates vesicle- ant unilamellar vesicles using the abovementioned (i) hybridizing DNA membrane fusion interactions. and (ii) coiled-coil peptide pairs as SNARE mimetics. We quantified the (hemi-)fusion process using two different FRET-based approaches which 3017-Pos Board B225 yielded similar results. Our system also offers the possibility to determine Complexin Binding to Membranes and Acceptor t-SNARE Complex LUV docking and fusion efficiencies. Content mixing assays indicate that Explains its Clamping and Stimulatory Effects on Fusion lipid mixing in the DNA-based system is a consequence of hemi- and not Binyong Liang, Alex JB Kreutzberger, Rafal Zdanowicz, Volker Kiessling, full fusion. For the peptide system, indirect labelling of lipidated peptides David S. Cafiso, Lukas K. Tamm. indicated their phase-specific partitioning. The incubation of peptide- University of Virginia, Charlottesville, VA, USA. decorated GUVs and LUVs resulted in a FRET efficiency decrease suggesting Complexin-1 is a SNARE effector protein that decreases spontaneous neuro- lipid mixing. In contrast to the DNA-based system, here low amounts of con- transmitter release and enhances evoked release. Complexin binds to the tent mixing implied full fusion events. Complementary experiments are fully or partially assembled four helical neuronal SNARE core complex currently performed using reconstituted SNARE proteins (SNARE proteins as revealed in competing molecular models derived from x-ray crystallog- were kindly provided by Claudia Steinem, Department of Biomolecular raphy. It is unclear how complexin binding to the post-fusion complex ac- Chemistry at Go¨ttingen University). This work is performed within the Max- counts for its effects upon spontaneous and evoked release in vivo.Using SynBio consortium which is jointly funded by the Federal Ministry of Educa- a combination of spectroscopic and functional imaging methods, we charac- tionandResearchofGermanyandtheMaxPlanckSociety. terize in molecular detail how complexin-1 binds to the 1:1 plasma mem- brane t-SNARE complex composed of syntaxin-1a and SNAP-25 while 3020-Pos Board B228 simultaneously binding the lipid bilayer at both its N- and C-terminal Interaction of Snare Mimetic Peptides with Lipid Bilayers ends. This complexin interaction reduces the affinity of synaptobrevin-2 Andrea Grafmueller, Swapnil Wagle, Reinhard Lipowsky. for the 1:1 complex thereby retarding SNARE assembly and vesicle docking Theory and Biosystems, Max Planck Institute of Colloids and Interfaces, in vitro. The results provide the basis for molecular models that account for Potsdam, Germany. the observed clamping effect of complexin beginning with its binding to the The coiled-coil forming peptides, ‘K’ (KIAALKE)3 and ‘E’ (EIAALEK)3 acceptor t-SNARE complex in the absence of the vesicle SNARE represent a minimal model for the action of SNARE proteins. Their action synaptobrevin-2 and the subsequent stimulated activation of the clamped 2þ on the membrane is typically pictured as a zipper-like closing of the coiled- complex by Ca and synaptotagmin. coil, bringing the membranes into close proximity. However, the individual 3018-Pos Board B226 peptides are significantly less ordered in solution and peptide K has been shown The Fusion Pore Lifetime during SNARE Mediated Fusion of Dense Core to interact with the membrane. Thus, the real coiled-coil assembly process is Vesicles with t-SNARE Containing Supported Membranes can be Modu- likely to be less straightforward and will possess different free energy contribu- lated by Asymmetric Lipid Distributions tions related to helix folding, coiled-coil assembly and competing interactions Volker Kiessling1, Alex J.B. Kreutzberger1, Binyong Liang1, with the membrane. Sung-Tae Yang1, J. David Castle2, Lukas K. Tamm1. To investigate these different factors, we perform molecular simulations and 1Physiology, University of Virginia, Charlottesville, VA, USA, 2Cell free energy calculations. We use all atom simulations to characterize the inter- Biology, University of Virginia, Charlottesville, VA, USA. actions of the peptides with lipid bilayers and to analyze the effect of bilayer The two leaflets of the plasma membrane contain different lipid composi- composition, peptide secondary structure, and the presence of additional resi- tions. Phosphatidylethanolamine (PE) is predominantly located in the inner dues often found at the peptide terminals. The potentials of mean force cytosolic leaflet and has been proposed to promote membrane deformation (PMF) for bringing the peptides to the bilayer surface are calculated for two and stabilize fusion pores during exocytotic events. Docking and fusion of bilayer compositions used experimentally: A neutral bilayer containing bulky neuroendocrine dense core vesicles (DCVs) purified from PC12 cells with PC head-groups, and a charged bilayer containing PG lipids. The PMF profiles supported membranes that contain the neuronal SNARE proteins syntaxin show minima for the interactions with the PG bilayers, whereas the bulkier PC 1a and SNAP-25A can be observed by recording the fluorescence of head groups present a considerable barrier for embedding the peptide in the mRuby-tagged neuropeptide Y in a total internal reflection microscope membrane, and the results are strongly dependent on the peptide secondary (TIRFM). Single DCV fusion events can be identified by distinct character- structure. istic time-dependent fluorescence line shapes. These line shapes can be To clarify the different free energy contributions to coiled-coil formation, a simulated by a mathematical model that includes the opening of a stable series of Metadynamics simulations are conducted which analyze the stability fusion pore followed by the collapse of the vesicle into the plane of the sup- of the alpha helical structure in solution, close to the bilayer and in the folded ported membrane. coiled coil. The energy contributions obtained from these all atom simulations We prepared supported t-SNARE membranes with different asymmetric PE can be used to obtain an optimized coarse grained-model for membrane distributions between the two leaflets and analyzed the life-time of the fusion fusion. pore and the fusion probabilities. With PE in the leaflet facing the DCVs, over- all fusion was most efficient and the extended fusion pore lifetime enabled 3021-Pos Board B229 notable detection of content release preceding vesicle collapse. All other PE SNARE Copy Number Determines the Size and Kinetic Properties of distributions decreased fusion efficiency, altered pore lifetime and reduced con- Nascent Fusion Pores tent release. With PE exclusively in the opposite leaflet, resolution of pore Huan Bao. opening and content release was lost. UW Madison, UW Madison, Madison, WI, USA. The first step in neurotransmitter and hormone release is the formation of a 3019-Pos Board B227 fusion pore. Capacitance measurements revealed that pore size can range Spatially Confined Membrane Fusion with SNARE Mimetics from 0.5 to 2 nm in neuroendocrine cells. However, the mechanisms that Tom Robinson1, Bastian Kubsch1, Torben Kliesch2, Andreas Janshoff2, determine pore size and dynamics are largely unknown due to the low abun- Reinhard Lipowsky1, Rumiana Dimova1. dance and ephemeral nature of these crucial structures. Here, we reduced the 1Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, number of available SNAREs in neurons and observed changes in transmitter Golm-Potsdam, Germany, 2University of Goettingen, Goettingen, Germany. release suggestive of alterations in fusion pore properties. To address this pos- Biological membrane fusion is involved in a number of essential processes sibility, we first employed reconstituted nanodisc/liposome-based fusion as- such as neurotransmission, exocytosis, and viral infection. It is crucial for says, using optical sensors to detect cargo flux through nascent pores. cells to spatially confine this process to specific organelles or sites in the Ensemble and single vesicle measurements revealed that increasing the num- plasma membrane. In nature, the SNARE complex induces fusion in eukary- ber of SNARE complexes enhances the rate of release of a given cargo, and otic cells. Its different constituents integrated into opposing membranes con- enables the escape of larger cargos. To determine whether this was due to nect the two bilayers by interacting in a zipper-like fashion. Different studies changes in pore size versus stability, we developed a novel reconstitution examined membrane fusion in artificial systems using SNARE mimetics. approach, based on nanodiscs and planar lipid bilayer electrophysiology, Fusion between pairs of small unilamellar vesicles have been shown using that affords msec time resolution at the single event level. Remarkably, both lipidated DNA [Chan et al., Biointerphases, 3:FA17, 2008] and peptide parameters were affected by SNARE copy number. Increasing the total num- [P€ahler et al., Biophysical Journal, 103:2295, 2012] ligand-receptor pairs. ber of v-SNAREs per nanodisc from three to five caused a two-fold increase in Here, we show strong evidence of the fusion of large unilamellar vesicles pore size and decreased the rate of pore closure by two orders of magnitude. (LUVs) to a spatially confined region within liquid-liquid phase-separated gi- Moreover, trans-SNARE pairing was dynamic as perturbation of v-/t-SNARE

BPJ 8727_8730 Wednesday, February 21, 2018 609a zippering disrupted the stability of fusion pores, particularly at the base of the time directly depends on the lipid membrane in which it is embedded. Howev- SNARE complex. In summary, trans-SNARE complexes are metastable and er, less is known about how a membrane-spanning channel affects the lipid the number of SNAREs recruited to drive fusion dictates fundamental prop- bilayer properties, particularly the elasticity. Here we use neutron spin echo erties of individual pores. spectroscopy (NSE) to measure both the collective bending and thickness fluc- tuation dynamics in dimyristoylphosphatidylcholine (DMPC) and dipalmitoyl- Posters: Protein-Lipid Interactions: Channels phosphatidylcholine (DPPC) bilayers containing gramicidin at low peptide/ lipid ratios. At these low concentrations, gramicidin incorporation did not 3022-Pos Board B230 have a measurable effect on the average bilayer structure but significantly Modulation of Membrane Proteins by Lipids impacted the collective membrane dynamics. The bending modulus of Carmen Domene. DMPC membranes increased with increasing gramicidin concentration, while Chemistry, University of Bath, Bath, United Kingdom. the trend in DPPC was non-monotonic and first decreased at low gramicidin Interactions of proteins with lipids play a key role in stabilizing the struc- concentrations before increasing at higher concentrations. In contrast, the thick- ture of membrane proteins within their lipid bilayer environment, and also ness fluctuation amplitude increased with increasing gramicidin concentration influence the function of membrane proteins. By selected examples from in both the DMPC and DPPC lipid bilayers. Notably, combining the bending the work we have carried out, I will provide an overview of the current and thickness fluctuation results revealed that the seemingly disparate concen- knowledge we have about the modulation by lipids of a Kþ-channel and tration trends in the DMPC and DPPC lipid bilayers were both consistent with a transporter. In particular, we adopt a computational approach to unmask an increase in coupling between the bilayer leaflets with increasing gramicidin details of the specific interactions of phospholipids with KcsA, revealing concentration. Together these results demonstrate that channel formation can how anionic lipids modulate its activity and how the lipid phase affects have considerable effects on the surrounding lipid membrane elasticity and Glut1 transport, in agreement with experimental evidence that has shown highlight the interplay between lipids and peptides in determining the mem- a close correlation between the composition and physical state of the mem- brane dynamics. brane bilayer and glucose transport activity via the glucose transporter GLUT1. 3026-Pos Board B234 Scaling Laws for Ionic Transport in Nanochannels: Bulk, Surface and 3023-Pos Board B231 Interfacial Effects Mathematical Models of Protein Induced Membrane Deformation Antonio Alcaraz1, Marı´a L. Lo´pez1, Marı´a Queralt-Martin2, Michael Grabe1, Neville Bethel2. Vicente M. Aguilella1. 1 1Laboratory of Molecular Biophysics. Department of Physics, University University of California, San Francisco, San Francisco, CA, USA, 2 2Biophysics Graduate Program, University of California, San Francisco, San Jaume I, Castellon, Spain, Program in Physical Biology, Eunice Kennedy Francisco, CA, USA. Shriver NICHD, National Institutes of Health, Bethesda, MD, USA. I will describe recent advances we have made in using fast, continuum elastic- The usual description of ion transport in membrane channels is based on dual ity theory to describe membrane deformations around proteins. I will show that model describing the channel conductance as the addition of bulk and sur- our calculations match the deformations predicted from all atom molecular dy- face contributions. This vision constitutes an idealization that it is extremely namics simulations for two proteins: gramicidin (a small antibiotic ion channel) useful for modelling purposes. However, there are no surface- and bulk- and nhTMEM16 (a member of the calcium activated chloride channel family). labelled counterions in real solutions, but only ions that due to thermal agita- Our calculations reveal that nhTMEM16 produces large distortions in the mem- tion continuously interchange their role. Furthermore, ion transport in brane potentially related to its ability to scramble lipids from one leaflet to the confined geometries may differ significantly from that in bulk conditions. other. This hypothesis is supported by atomistic simulations in which we Besides direct electrostatic interactions between the permeating ions and observe lipids flipping from one leaflet to the other. Experiments to test the lipid pore charges, other phenomena like interfacial access resistance or entropic flipping mechanism in a mammalian TMEM16 family member will also be effects due to obstacles and irregularities of the boundaries may play a role. discussed. We investigate here the limitations of the abovementioned two-state model by assessing experimentally the scaling behavior of channel conductance (G) 3024-Pos Board B232 with salt concentration (c) in structurally different protein and proteolipidic Determining the Specificity of Designed Peptide that Inhibits Antibiotic pores. Previous studies in both nanochannels have suggested a power law Resistance dependence G ca,wherea is an exponent that has been reported to attain Virangika K. Wimalasena, Jimmy Budiardjo, Cyril B.R. Cook, a variety of values depending of the system and the concentration regime. Joanna S.G. Slusky. We hypothesize here that scaling exponents found in a specific system arise The University of Kansas, Lawrence, KS, USA. from a particular interplay between bulk and surface effects, being the Antibiotic resistant bacteria is a looming public health threat. By 2050 antimi- distinction between them so subtle that the two-state model faints. In the crobial infection is predicted to outpace cancer as a cause of death. The broad case of biological pores, we show also that the presence of interfacial effects spectrum method of antibiotic resistance is caused by the over-expression of could give rise to an apparent universal scaling that does not reflect the chan- protein pumps that are responsible for the efflux of small molecules, like anti- nel actual characteristics. biotics. Each pump has an outer membrane component with structurally homol- ogous, multi-chain, beta barrel topology. However, among related bacteria the 3027-Pos Board B235 sequence similarity of these proteins is only around 25%. Herein, we have Membrane Permeabilizing Electric Fields Disrupt Water Channel Func- begun determining if the topologically similar but structurally dissimilar outer tion and Selectivity membrane pump proteins of P. aeruginosa, V. cholera, N. meningitidis, and Zachary A. Levine1,2. 1Department of Molecular Biophysics & Biochemistry, Yale University, New C. jejuni can substitute for their E. coli homolog. In addition, we have designed 2 a peptide that has proved successful in the inhibition of the efflux machinery in Haven, CT, USA, Department of Pathology, Yale School of Medicine, New E. coli. If the substitution is successful, we will determine if our designed pep- Haven, CT, USA. tide is effective in inhibiting the outer membrane efflux pump proteins of these External MV/m electric fields can permeabilize cellular membranes, allowing diverse organisms. influx and efflux of normally-impermeant molecules across the protective lipid bilayer. Utilization of short-duration permeabilizing electric fields (PEFs) have 3025-Pos Board B233 become widespread in the food industry, in biotechnology, and in medicine, Effects of Channel Forming Peptides on Lipid Bilayer Dynamics and where targeted high-voltage electric fields are used to transiently enhance the Leaflet Coupling transport of water and other large molecules into and out of cells. However, Elizabeth G. Kelley1, Michihiro Nagao1,2, Paul D. Butler1,3. despite the increasing use of PEFs in clinical and biological systems, their 1Center for Neutron Research, NIST, Gaithersburg, MD, USA, 2Center for interactions with transmembrane proteins remain largely unknown. These lim- Exploration of Energy and Matter, Department of Physics, Indiana Unversity, itations yield an incomplete picture of membrane electropermeabilization, Bloomington, IN, USA, 3Department of Chemical and Biomolecular where multiple components of the cell are likely perturbed (and possibly dena- Engineering, University of Delaware, Newark, DE, USA. tured) under PEFs. In order to test if PEFs affect transmembrane proteins, espe- Altering the material properties of lipid bilayers has been shown to affect the cially those that natively transport water, molecular dynamics simulations biological activity of a number of channel-forming peptides and proteins. A were carried out on human aquaporin 1 (hAQP1) water channels under prototypical example is gramicidin, in which the channel formation and life- cell-permeabilizing voltages. Simulations revealed significant changes to water

BPJ 8727_8730 610a Wednesday, February 21, 2018 orientation and flux through hAQP1 under PEFs, with voltage-dependent trans- TMEM16F-mediated lipid scrambling activity are also deficient in generation port rates that diverge from diffusive and osmotic experiments. Surprisingly, of a subtype of extracellular vesicle (EV) called ‘‘microvesicles’’ or ‘‘ecto- hAQP1 was able to transport Cl anions (but not Na cations) when 1-2 volts somes’’ though the relationship between scrambling and vesiculation is not was applied, despite its inability to do so under physiological conditions. The well understood. Microvesicles, which range between 100 nm to 1 mmin protein did not affect the construction of discrete lipid electropores around diameter depending on the cell type of origin, shed distinctively from the the periphery of the channel, or the timescales of lipid bilayer permeabilization, plasma membrane in a Ca2þ-dependent manner and are thought to carry however a small reduction in the protein’s alpha-helicity was detected. hAQP1 bioactive cargo in the form of RNAs, proteins, and lipids which can elicit bio- Cl conduction was also sensitive to the protonation state of the histidine residue logical activity in neighboring cells. Quantification of EVs using conventional located in the two-stage filter, suggesting that pH may also affect anion conduc- techniques is challenging due to the inherent difficulties of resolving particles tion. Taken together, these results suggest that PEFs have a direct effect on on such a diminutive scale. We have adapted the use of chemically-induced transmembrane water channels, even before membrane electropores interact giant plasma membrane vesicles (GPMVs) generation which can be moni- with them. Further studies of additional membrane components under PEFs tored in real time by conventional light microscopy to investigate the role are necessary if optimization of therapeutic electric fields is to be significantly of TMEM16F phospholipid scrambling activity in extracellular vesiculation. improved. Using the GPMV assay, we identify and characterize both inactivating and activating mutants that emphasize residues critical for TMEM16F function 3028-Pos Board B236 and that allow us to further examine the mechanism of lipid translocation Optimal Design of an Aquaporin Lipid Membrane System using Molecular through TMEM16F. Dynamics Simulation Hyunki Kim1, Moon-ki Choi1, Byungho Lee1, Soojin Jo1, Daejoong Kim2, 3031-Pos Board B239 1 2D Moon Ki Kim . PIP2 and CA are Both Required to Open TMEM16a Channels in 1School of Mechanical Engineering, Sungkyunkwan University, Suwon, Xenopus Laevis Oocytes Republic of Korea, 2Department of Mechanical Engineering, Sogang Maiwase Tembo, Anne E. Carlson. University, Seoul, Republic of Korea. Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA. Aquaporin is a water permeation channel protein found in many living cell Transmembrane member 16A (TMEM16a) is a widely expressed Ca2þ- acti- membranes. Aquaporin-lipid membrane system has been studied for desali- vated Cl- channel with various physiological functions ranging from mucosal nation application because of its inherent property that only permeates wa- secretion to regulating smooth muscle contraction. Despite its importance, we ter molecules and rejects all other ions and molecules. However, the are just beginning to understand the mechanisms that regulate TMEM16a permeability of aquaporin-lipid membrane system is not proportional to gating. Here we recorded Ca2þ-evoked Cl- currents passed by the endogenous the density of aquaporin. That is, high density of aquaporin rather lowers TMEM16a channels expressed in oocytes from the African clawed frog, Xen- the water permeability. Therefore, it is necessary to find an optimal aqua- opus laevis. Using the inside-out configuration of the patch clamp technique, porin density to develop the highest permeability in designing aquaporin- we have found that the TMEM16a-conducting currents run down within a few lipid membrane system. In this work, changes in the water permeability seconds of patch excision, despite the continued presence of Ca2þ. Current with respect to aquaporin density were calculated by molecular dynamics rundownis common amongst channels regulated by phosphatidylinositol simulation. Furthermore, the correlation between permeability and other pa- 4,5-bisphosphate (PIP2). Thus, we tested the hypothesis that TMEM16a is rameters including hydrophobic thickness and hydrophobic mismatch was potentiated by PIP2 using inside-out patches exposed to PIP2 sequestering investigated in order to propose an optimal design method without direct and recovering agents. First, we found that application of the PIP2 seques- permeability calculation which requires quite long time simulation. Conse- tering agents, neomycin and anti-PIP2, to intracellular surface of the patch quently, the relationship between hydrophobic mismatch and permeability sped TMEM16a current rundown by two-fold. Second, we applied Mg-ATP leads to the optimal density of aquaporin. It is also expected that this study to the intracellular surface of the patch to enable rephosphorylation of PI de- will be an important basis for the future design of aquaporin-lipid mem- rivatives into PIP2, thereby slowing PIP2 depletion. We found that these cur- brane system. rents ran down more slowly than the controls. In another series of experiments, we sought to recover TMEM16a current after rundown with 3029-Pos Board B237 PIP2 application. We applied the soluble dioctanoyl-PIP2 analog (diC8- 2þ The Functional Relationship between a Kir Channel and the Lipid PIP2) with Ca and observed greater than 40% TMEM16a current recovery. Membrane Conversely, application of soluble dioctanoyl-phosphatidyl inositol (diC8-PI), Benjamin Wylie, Collin Borcik, Emily Hardy. the backbone of PIP2 without the two phosphate groups, was not able to 2þ Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA. recover current, nor was diC8-PIP2 applied with no Ca . Taken together, 2þ The functional interplay between membrane proteins and lipids in biological our data demonstrate that TMEM16a requires both Ca and PIP2 to pass membranes promote and regulate essential biological functions. Inward- current. rectifier Kþ (Kir) channels are ion channels that regulate the resting membrane potential. Anionic lipids activate and regulate Kir channels, but structural de- 3032-Pos Board B240 tails are limited. Our lab uses modern solid-state NMR (SSNMR) methodology Interactions of Nicotinic Acetylcholine Receptors with Cholesterol and functional assays to show how different lipid environments modulate the and Polyunsaturated Fatty Acids in Model, Native-Like, and Oocyte activity and conformation of the prokaryotic Kir channel KirBac1.1. SSNMR Membranes reveals how functional lipids activate and regulate KirBac1.1. It is also found Liam Sharp, Grace Brannigan. that KirBac1.1, in turn, orders the surrounding membrane and nucleates the for- CCIB, Rutgers University, Camden, NJ, USA. mation of ordered and disordered lipid compartments. Nicotinic acetylcholine receptors (nAChR) are pentameric ligand gated ion channels, critical to signaling across synapses and the neuro-muscular junc- 3030-Pos Board B238 tion. While sensitive to boundary lipids, nAChR have been shown to be func- TMEM16F is a Calcium-Activated Phospholipid Scramblase Required for tionally dependent on cholesterol. This dependence on cholesterol has led to Chemically-Induced Giant Plasma Membrane Vesicles the hypothesis that nAChR resides within the cholesterol rich liquid ordered Tina W. Han1, Wenlei Ye1, Neville P. Bethel2, Mario Zubia1, domains. Using the MARTINI force field, coarse-grained molecular dynamic Michael Grabe2, Yuh Nung Jan1, Lily Y. Jan1. simulations were preformed, with nAChRs in quasi-native ternary mem- 1 2 Physiology, UCSF, San Francisco, CA, USA, Cardiovascular Research branes. Native nAChR membrane composition has an abundance of polyun- Institute, UCSF, San Francisco, CA, USA. saturated fatty acids (PUFAs), saturated fatty acids, and cholesterol. The Cellular membranes are composed of heterogeneously partitioned phospho- two PUFAs chosen for these simulations were Docosahexaenoic acid and Li- lipids such that certain lipid species are sequestered to the inner leaflet noleic acid. while others are exposed on the cell surface. Lipid asymmetry is established These simulations display nAChR consistently residing in the PUFA enriched and maintained by ATP-dependent unidirectional phospholipid translocases disordered domain, remaining nearby the liquid ordered domain. Analysis of which are colloquially referred to as ‘‘flippases’’ and ‘‘floppases’’. TMEM16F boundary lipid composition confirms nAChR boundary lipids are enriched in belongs to a third class of proteins aptly named ‘‘scramblases’’ which PUFAs. Further analysis of nAChR subunit-domain interaction show alpha 2þ mediate Ca -activated, energy-independent bidirectional translocation of subunits preference for cholesterol rich domains, while beta subunits show lipids across the bilayer, leading to transient or, in the case of apoptotic preference for PUFAs. Lastly, analysis shows PUFAs and cholesterol binding scrambling, sustained collapse of membrane asymmetry. Cells lacking non-annularly nAChR.

BPJ 8727_8730 Wednesday, February 21, 2018 611a

This study is being expanded to compare complex quasi-native synaptic and scrambling rate constants. The largest reduction in transport (>100-fold) oocyte membranes. The oocyte membrane, in particular, is an optimal model was observed for residues that localize to three, structurally-defined hot- for studying lipid-protein interactions, because it has a lower abundance of spots: a constriction of the groove near the mid-point of the membrane, n-3 PUFAs compared to the neuron. From our simulations, we find that differ- the extracellular entrance of the cavity formed by E313 and R432 and three ences in membrane composition are especially noticeable around nAChRs. residues on TM5 and 6 (A385, Q436, and Y439) which connect these con- Given that nAChRs no longer exhibit partitioning preferences in oocyte mem- strictions. We conducted molecular dynamics simulation of nhTMEM16 to branes, our initial simulations suggest that oocytes do not provide a sufficiently gain insights into the dynamic characteristics of the groove, and how these native-like environment for nAChR. enable the opening of a functional pathway for lipid translocation. Our computational experiments suggested that access of lipids to the pathway 3033-Pos Board B241 from the extracellular leaflet is controlled by the dynamic rearrangements Interactions of Plasma Membrane Criticality and GABAA Receptor of an interacting triad of polar residues (E313, E318 and R432), which Gating form the extracellular gate of the groove. Further, we find that two other res- Thomas R. Shaw1, Benjamin B. Machta2, Sarah L. Veatch3. idues, Y439 and Q436, are essential for lipid translocation by coordinating 1 Applied Physics, University of Michigan, Ann Arbor, MI, USA, the headgroups as they traverse the membrane. Mutagenesis experiments 2 Department of Physics and Systems Biology Institute, Yale University, New on these residues support the dynamic nature of the interactions between 3 Haven, CT, USA, Biophysics, University of Michigan, Ann Arbor, MI, these residues and the translocating lipids. We propose that the TMEM16 USA. grooves serves as a lipid permeation track and that dynamic rearrangements Mammalian plasma membranes exhibit lateral heterogeneity on spatial of its extracellular portion control lipid access and translocation through this scales between 10 and 100 nm. Past work in the Veatch lab has demonstrated pathway. that many treatments that potentiate gamma-aminobutyric acid type A recep- tor (GABAAR) currents also lower miscibility critical temperature Tc in plasma membrane derived vesicles, a key measure of membrane heterogene- 3035-Pos Board B243 ity. A recent mechanistic theory, developed with collaborators Ofer Kimchi Accessing the Desensitized State of pLGICs: Why is the Connectivity Branched for Inhibitory Receptors, but Linear for Excitatory Receptors? and Ben Machta, could explain the connection between Delta Tc and ion channel gating. The theory uses an Ising model of membrane heterogeneity, Robert Cantor. and additionally posits that the channel has differing preferences for its Chemistry, Dartmouth College, Hanover, NH, USA. neighboring lipids in the open vs the closed state. As a result, a change in The family of pentameric ligand-gated ion channels (pLGICs) includes both the critical temperature of the membrane changes the free energy cost of inhibitory and excitatory receptors. Fast-perfusion electrophysiology has satisfying the boundary conditions in each state, and thus the free energy dif- been used extensively to study the time-dependent ion currents induced by ference between the open and closed states. Here we present our ongoing their neurotransmitter agonists. Kinetic modeling of these current traces re- experimental efforts to test this theory in the context of the GABA R, by quires a minimum of three conformational states: resting, active, and desen- A sitized. Traces from inhibitory and excitatory pLGICs are so qualitatively measuring spatial cross-correlations between GABAARs and lipid probes of membrane heterogeneity, calculated from single molecule localization different that reproducing even their basic features requires different state microscopy data. In particular, we will test whether and how cross- connectivity: ‘‘branched’’ (the desensitized state is accessed from the resting correlations differ between the open and closed channel states, and then state) for inhibitory receptors such as GABAAR, but ‘‘linear’’ (the desensi- tized state is accessed from the active state) for excitatory receptors such as how this difference changes under membrane perturbations that alter Tc. We will supplement these measurements with whole cell patch clamp elec- AChR. rophysiology data for each set of conditions, and compare the results to the It is surprising that a property as fundamental as the connectivity among theoretical predictions of our model. these three states would differ for receptors within the same family. So, we explore the possibility that the connectivity is actually the same, but cor- 3034-Pos Board B242 responding states differ in function: Analogous states on the conformational Identification of the Extracellular Gate of a TMEM16 Scramblase free energy landscape have similar structure, but differ in their ion selec- Byoung-Cheol Lee, George Kelashvili, Maria Falzone, Harel Weinstein, tivity and conductivity (open or closed), relative free energies and agonist Alessio Accardi. binding affinities. Weill Cornell Medicine, New York, NY, USA. The plausibility of this hypothesis is tested using a recently developed ki- The TMEM16 family comprises Ca2þ-activated Cl- channels and phospho- netic model, in which agonist can modulate the receptor free energy land- lipid scramblases. The crystal structure of nhTMEM16, revealed an scape by adsorbing to the membrane in which the receptor is embedded. It important architectural feature of this protein family in the form of a was previously shown that even with only three conformational states, the bilayer-spanning hydrophilic groove (TM3-7) that is directly exposed to full range of complex behavior observed for GABAARisreproduced, the membrane’s hydrophobic core and provides a possible pathway for lipid including its response to anesthetics. In the present work, it is shown that translocation. To identify residues that regulate lipid translocation we probed this hypothesis can account for the differences between inhibitory and the putative lipid pathway of nhTMEM16 by replacing its pore-lining side excitatory receptors, such as the desensitization and deactivation in chains with the hydrophobic and bulky residue tryptophan. We used a 3-state agonist-induced traces, and particularly their opposite responses to Markov model to quantify the impact of these substitutions on the lipid anesthetics.

BPJ 8727_8730 612a Wednesday, February 21, 2018

Posters: Protein-Lipid Interactions: Structures 3038-Pos Board B246 Supported Tubulated Bilayers: A Novel System for Evaluating Protein- 3036-Pos Board B244 Mediated Membrane Remodeling Analyzing the Effects of Placing Central Arginine Residues within a Highly Peter J. Dahl1, Noah A. Schenk1, Alexandra H. Ranski1, Michael G. Hanna2, Anjon Audhya2, Gregory G. Tall1, Jefferson D. Knight3, Arun Anantharam1. Dynamic Transmembrane Alpha-Helix 1 Matthew J. McKay, Denise V. Greathouse, Roger E. Koeppe II. Pharmacology, University of Michigan, Ann Arbor, MI, USA, 2Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI, Chemistry & Biochemistry, University of Arkansas, Fayetteville, AR, USA. 3 Transmembrane domains will sometimes contain conserved ionizable resi- USA, Chemistry, University of Colorado Denver, Denver, CO, USA. dues which are essential for protein function and regulation. This work Fusion and fission of cellular membranes involve dramatic, precisely regulated aims to examine the effects of single Arg(R) residues within a highly dy- changes in membrane curvature mediated by a number of proteins whose mech- namic transmembrane peptide helix. We have modified the dynamic trans- anisms are not well understood. Despite several recent advances, current 4,20 4 20 methods for investigating curvature sensing and generation in real time using membrane GW ALP23 (acetyl-GGAW (AL)7AW AGA-[ethanol] amide) peptide to incorporate an Arg residue near the center of the peptide well-controlled systems remain limited. We have developed a novel system at position 12 or 14. Peptide orientation and dynamics were analyzed by based on supported lipid bilayers (SLBs) in which high ionic strength during means of solid-state NMR spectroscopy to monitor specific 2H-labeled lipid bilayer deposition results in incorporation of excess lipids in the bilayer, Ala residues. GW4,20ALP23 adopts a tilted orientation within lipid bilayers which results in the spontaneous formation of lipid tubules after sequentially and exhibits moderate to high dynamic averaging of NMR observables, washing with water and physiological ionic strength buffer solutions. We such as the 2H quadrupolar splittings or 15N-1H dipolar couplings, due to find that the process of tubule formation is the result of an ion-dependent competition between the interfacial Trp(W) residues on opposing helical spreading of the SLB; addition of a physiological ionic strength buffer solution faces. Here we examine how such extensive peptide dynamics are impacted free of divalent ions leads to expansion of the bilayer and formation of tubules, by the introduction of central Arg residues. R14 holds significant influence likely due to increased membrane tension. Conversely, the addition of divalent over the peptide’s behavior and restricts the peptide to low dynamics and ions results in contraction of the membrane and a proportional loss of tubules. a tilt that decreases as lipid thickness increases, all at the cost of partial These ionic conditions can be tuned for each experiment, allowing investigators C-terminal helix unwinding. Alternatively, R12 causes the host peptide to control the extent of tubulation. We show the utility of these supported tubu- to adopt an interfacial surface-bound orientation in DOPC bilayers. lated bilayers, which we term ‘‘STuBs,’’ with an investigation of Sar1, a small Interestingly, multistate behavior is exhibited by a single residue, Ala-9. Ras family G-protein known to influence membrane curvature. The addition of Furthermore, numerous N-terminal quadrupolar splittings generated by Sar1 to tubulated bilayers results in both further tubulation and tubule fission, of 2H-labeled residues do not fit to the same quadrupolar wave plot as the re- which fission is shown to be more dominant based on quantification of wide- maining C-terminal residues. Therefore, both results potentially implicate field fluorescence microscopy images. Individual tubule formation events are distortion of the R12 helix. The partial helix unwinding/distortion observed with polarized total internal reflection fluorescence microscopy behavior observed in both the R12 and R14 peptides could be due to the (pTIRFM), an imaging method that allows for semi-quantitative measurements competition of the terminal Trp residues attempting to compensate for of membrane deformations. Overall, STuBs is a simple experimental system, the Arg’s dominance over the peptide’s orientation. We thus surmise that useful for monitoring solute- and protein-mediated effects on membrane topol- while the arginine residues are prominent factors controlling the helix dy- ogy in aqueous media and in real-time, using widely available instrumentation. namics, the influence of the competing tryptophan residues cannot be 3039-Pos Board B247 ignored. Sphingomyelin-Cholesterol Complexes in Plasma Membranes Shreya Endapally1, Donna Frias1, Diana Tomchick2, Arun Radhakrishnan1. 3037-Pos Board B245 1Molecular Genetics, UT Southwestern Medical Center, Dallas, TX, USA, Neutron Spin Echo Detects Effects of the pH-Low Insertion Peptide on 2Biophysics, UT Southwestern Medical Center, Dallas, TX, USA. Membrane Thickness Fluctuations Sphingomyelin(SM) and cholesterol are lipids that are critical for the function Haden L. Scott1, Rana Ashkar2,3, Fred A. Heberle2, Robert F. Standaert2,4, of animal cells. Interplay between these two lipids is important for maintaining John Katsaras2,5, Francisco N. Barrera1. integrity of the myelin sheath that encases axonal cells and for maintaining 1Department of Biochemistry & Cellular and Molecular Biology, University 2 functionally distinct pools of cholesterol in the plasma membranes of fibroblast of Tennessee, Knoxville, TN, USA, Biology and Soft Matter Division, cells. The nature of the interaction between SM and cholesterol has been long Neutron Sciences Directorate, Oak Ridge National Lab, Oak Ridge, TN, 3 debated, with proposals ranging from complexes of the two lipids to no specific USA, Department of Physics, Virginia Polytechnic Institute and State interaction between these two lipids. University, Blacksburg, VA, USA, 4Biosciences Division, Oak Ridge 5 Here, we have developed a sensor to examine the interaction between SM and National Lab, Oak Ridge, TN, USA, Department of Physics and Astronomy, cholesterol in animal cell membranes. Our sensor is derived from a fungal toxin, University of Tennessee, Knoxville, TN, USA. ostreolysin A (OlyA). We find that OlyA binds to membranes of CHO-K1 ham- Lipid membranes are highly dynamic assemblies, exhibiting a hierarchy ster cells when they contain both SM and cholesterol, but not when levels of of motions ranging from rotation of an individual lipid molecule to collec- either lipid are diminished by treatment with sphingomyelinase (which degrades tive membrane fluctuations. The latter, particularly thickness fluctuations, SM) or cyclodextrin (which depletes cholesterol). In model membranes, OlyA have recently become experimentally accessible with the development of shows no binding to dioleoyl-phosphatidylcholine – cholesterol membranes neutron spin echo (NSE) techniques. Biological membranes contain even when the cholesterol concentration exceeds 50 mole%. Also, OlyA shows surface-associated and transmembrane proteins, both of which are key to no binding to SM-containing membranes when epicholesterol (a diasteromer of a cell’s well-being. However, how these proteins affect the membrane’s dy- cholesterol) is present at concentration exceeding 50 mole%. Using X-ray crys- namics, including thickness fluctuations, remains an open question. Here, tallography, we studied the interaction of OlyA with SM and cholesterol at the we use pH-low insertion peptide (pHLIP) to address this question. pHLIP atomic level. This structural analysis combined with detailed mutagenesis led us is highly sensitive to changes in pH, transitioning from a surface- to a single point mutation in OlyA that abolishes its cholesterol specificity while associated to a transmembrane configuration below a critical pH value, retaining SM specificity. Comparing the X-ray structures of these two versions and thereby allowing both classes of membrane proteins to be studied in a of lipid-bound OlyA combined with ligand docking simulations revealed two single system. In this study, we measured membrane thickness fluctuations distinct binding modes of OlyA to SM. We propose that one of these SM confor- with NSE, and the average membrane thickness with small-angle neutron mations reflects a complex with cholesterol. Studies in live cells show that this and x-ray scattering (SANS and SAXS, respectively). Both SANS and pool of SM/cholesterol complexes in plasma membrane is maintained at a con- SAXS showed that neither of pHLIP’s conformations affected the average stant level across a large range of cholesterol concentrations. bilayer thickness of a phosphatidylcholine/phosphatidylserine/cholesterol (PC/PS/Chol) membrane. On the other hand, NSE detected differences be- 3040-Pos Board B248 tween the two protein states. Specifically, pHLIP in the surface-associated Biophysical Studies of Model Lipid Membranes to Determine a Novel state decreased the amplitude of the membrane’s fluctuations while Mechanism of Daptomycin Inhibition by Lung Surfactant increasing their frequency. Strikingly, as a transmembrane helix, pHLIP Brenda Y. Lee1, Jeff H.Y. Lam2, Maureen M.W. Li2, Zoya Leonenko1. dampened the membrane’s fluctuations. We hypothesize that the suppres- 1Physics & Astronomy, University of Waterloo, Waterloo, ON, Canada, sion of thickness fluctuations may result from hydrophobic matching be- 2Biology, University of Waterloo, Waterloo, ON, Canada. tween the peptide and membrane, effectively pinning the membrane in Daptomycin is an antimicrobial peptide that is clinically used to treat severe in- place. fections caused by highly-resistant Gram-positive bacteria. However, in cases

BPJ 8731_8734 Wednesday, February 21, 2018 613a of pneumonia, daptomycin is curiously inhibited by lung surfactant and is the area of the binding surface, the monomers may not bind concurrently. This rendered ineffective against Gram-positive Streptococcus pneumoniae, the pri- in turn suggests that an allosteric mechanism for stabilisation of the productive mary cause of this illness. The reason for this is currently not understood. binding conformation is more likely. We have also used simulations to inves- We developed lipid model systems to mimic S. pneumoniae membranes, tissue tigate the relationship between conformational dynamics of the activation loop cell membranes, and both synthetic and natural (BLES) lung surfactant. and interactions with lipids in the cell membrane. Furthermore, large-scale CG Using the Langmuir-Blodgett trough, constant-area insertion assays were per- simulations (1 million particles) reveal how lipid composition and membrane formed. It was discovered that daptomycin inserts into lung surfactant to a curvature relate to the interactions of an ensemble of 25 kinases with a model greater extent than it does with bacterial membrane at physiologically- membrane. relevant calcium concentrations. Fluorescence spectroscopy data provided us PIP kinases are implicated in many cancers as a result of their influence on cell with results corroborating the findings from the insertion assays. Meanwhile, processes. Studies in mice and human tissue samples suggest that inhibition of compression isotherms provided data on monolayer compressibility. We found PIP5K1A is a potential target for treatment. Simulations of the interactions of that daptomycin and calcium can improve surfactant activity and performance, PIP5K1A with realistic models of cell membranes will aid design of drugs tar- suggesting the formation of multilayers at lower pressures. geted at these and other related lipid kinases. Atomic force microscopy and Kelvin probe force microscopy imaging pro- vided visual evidence of multilayer formation induced by the presence of dap- 3043-Pos Board B251 tomycin and calcium, confirming this mechanism of inhibition. The Flow of Proteins and Idealised Pores within the Membranes of Gram- Based on these results, we propose a novel model of daptomycin sequestration Negative Bacteria by lung surfactant (see figure), where (1) daptomycin inserts into surfactant, (2) Jonathan Shearer, Syma Khalid. lowers its compressibility, (3) induces multilayer formation, and (4) reinforces School of Chemistry, University of Southampton, Southampton, United its sequestration, rendering it unable to exert its bactericidal activity against Kingdom. S. pneumoniae. Gram-negative bacteria are protected by a complex cell envelope. Understand- ing the structure-dynamics-function relationships within the Gram-negative 3041-Pos Board B249 cell envelope is key to the ongoing fight against the emergence of bacterial Membrane Binding Properties of Bacillomycin-D Derivatives with Model resistance to antibiotics. The Gram–negative cell envelope consists of two Membranes Composed of Different Sterols membranes, separated by a cell wall. The membranes contain a variety of pro- Carlos Munoz-Garay1, Sathishkumar Munusamy1, teins, which make up to 25% of the bilayer surface area. Lipid-protein interac- Agustin Luna Bulbarela2, Romina Vazquez3, Vanesa Herlax4, Sabina Mate4, tions are significant in determining the function and organisation of membrane Leobardo Serrano Carreon2. proteins, through macroscopic bilayer properties or specific protein-lipid inter- 1Biophysics, Institute of Physical Sciences, Cuernavaca, Mexico, actions. Computational and experimental studies have revealed that proteins 2Bioengineering, Biotechnology Institute, Cuernavaca, Mexico, 3Instituto de and lipids diffuse together as complexes, which have dynamical properties Investigaciones Biquı´micas, La Plata, Argentina, 4Instituto de that differ from the properties of unbound lipids. However it is unclear how Investigaciones Biquı´micas, La Plata, Argentina. much the size, shape and biochemistry of the macromolecules contribute to Exploration of new antimicrobial agents and study of their mechanism of action these altered dynamics. are urgently required to address multidrug-resistant pathogens. Among the anti- Here we present the results of coarse–grained simulations that uncover the microbial agents, lipopeptides are promising antibiotic agents which exhibit ac- structural and dynamic effects of a series of outer membrane proteins tions against a broad spectrum of pathogenic bacteria and fungi with surfactant (OMPs) and functionlised carbon nanotubes (CNTs) on biologically relevant or pore activity. The primary action of these lipopeptides is the permeabiliza- models of the inner and outer membranes of E. coli. Investigating such a range tion of cellular membranes. In the present work, we have examined the binding of nanopores provides an insight into the effects the chemistry and topology of and permeabilizing ability of the Bacillomycin-D homologs such as an embedded protein have on the local membrane environment. We show that Bacillomycin-C14 and C16 on model membranes composed by different con- the flow of molecules in the outer membrane is asymmetrical, reflecting the centration of sterols. Bacillomycin-D homologs are cyclic lipopeptides with lipid composition of this membrane. Flow of proteins and lipids are highly seven a-amino acids and a b-amino fatty acid linked by an amide bond to correlated in the outer leaflet and much less so in the inner leaflet of the the constituent amino acid residue. Here, we explored the capacity of Bacillo- membrane. mycin- C14, and C16 to form itself monolayers and the ability of them to insert into a monolayer of PC-cholesterol and PC-ergosterol through Langmuir- 3044-Pos Board B252 monolayer studies. On the other hand, the binding efficiency of Bacillomycin Two-Color Sted Microscopy to Visualize S-Layer Biogenesis in Caulobacter Crescentus analogs was studied by measuring the intrinsic fluorescence of the lipopeptides 1 2 3 2 bound to liposomes of different composition. Further binding information was Colin J. Comerci , Jonathan Herrmann , Lucy Shapiro , Soichi Wakatsuki , W.E. Moerner4. obtained by quenching experiments with acrylamide. The Stern-Volmer con- 1 2 stants (Ksv) results suggest that lipopeptides have more membrane penetration Biophysics, Stanford University, Stanford, CA, USA, Structural Biology, Stanford University, Stanford, CA, USA, 3Developmental Biology, Stanford in POPC LUVs than POPC: Cholesterol LUVs. As ergosterol itself is acting as 4 a quencher for tyrosine, then binding affinity of LUVs of POPC: ergosterol University, Stanford, CA, USA, Chemistry, Stanford University, Stanford, could mislead the results. Results obtained from calcein release from liposomes CA, USA. treated with both lipopeptides indicate that lipopeptides are more active in li- Many bacteria and archaea are coated by a proteinaceous surface layer (S- posomes composed by PC: ergosterol than in those composed by PC and PC: layer) that can aid in pathogenicity, protect them from their surrounding envi- Cholesterol. ronment, and/or provide structural stability. The Gram-negative bacterium, Caulobacter crescentus, is enclosed by an S-layer composed of the protein 3042-Pos Board B250 RsaA. RsaA is the most abundant protein in Caulobacter, representing Multiscale Simulations of Membrane Recognition by Lipid Kinases 10% of the bacteria’s total protein production. RsaA forms a hexagonally Sarah-Beth Amos1, Antreas C. Kalli2, Jiye Shi3, Mark S.P. Sansom1. packed 2D pseudocrystal and is attached to the outer cell membrane via a lipo- 1University of Oxford, Oxford, United Kingdom, 2University of Leeds, polysaccharide. Because of the small size of bacteria (1 mm), visualizing the Leeds, United Kingdom, 3UCB Pharma, Brussels, Belgium. S-layer necessitates the use of high-resolution imaging techniques. Due to this, Peripheral membrane proteins associate with lipids in the plasma membrane in most previous work on RsaA has used electron microscopy, imaging the S- many important cell signalling processes. Type I phosphatidylinositol phos- layer on frozen or negative stained cells. We utilize two-color STimulated phate kinase A (PIP5K1A) is one such example, which phosphorylates the Emission Depletion (STED) microscopy, a form of super-resolution micro- head group of the lipid phosphatidylinositol 4-phosphate (PI4P) to generate scopy that breaks the diffraction limit (250 nm), to image RsaA with molec- phosphatidylinositol (4,5)-bisphosphate (PIP2). ular specificity in living Caulobacter cells at 60 nm resolution (FWHM). On The dynamic interactions of peripheral proteins with their target membranes is cells with a full S-layer, new RsaA is added as discrete patches that are local- difficult to capture on experimentally. A recent crystal structure at 3.3 A˚ reso- ized mostly at the cell poles and division plane. On cells that lack a complete S- lution enables the further investigation of the structure and dynamics of layer due to culturing in low calcium media, however, new RsaA grows as PIP5K1A and its interactions with lipids in the plasma membrane. patches localized randomly along the cell length. This suggests normal S-layer Here we use coarse-grained (CG) and atomistic (AT) MD simulations of the growth is localized by properties inherent to the complete S-layer structure or PIP5K1A protein kinase to investigate the nature of monomer and dimer bind- other cellular factors. Our work provides insight into the mechanism of S-layer ing to the plasma membrane and their effects on lipid clustering. The AT sim- growth, potentially informing antibiotic research and the biophysics of other ulations suggest that whilst dimerisation of the kinase results in an increase in membrane-associated protein assemblies.

BPJ 8731_8734 614a Wednesday, February 21, 2018

3045-Pos Board B253 3047-Pos Board B255 Mechanisms Governing Protein Clustering and Shape Changes in Biolog- Structural Lipids Stabilise Functional Oligomers of the Eukaryotic Purine ical Membranes Symporter UapA Sunil Kumar Palakurissi Balagopal1, Sreeja K.K.2. Euan Pyle1, Antreas Kalli2, Zoe Hall3, Bernadette Byrne4, Argyris Politis5. 1IIT Palakkad, Palakkad, India, 2University of Pennsylvania, Philadelphia, 1Chemistry, King’s College London and Imperial College London, London, PA, USA. United Kingdom, 2Leeds Institute of Cancer and Pathology, University of A characteristic feature of eukaryotic cells is the variety of membrane bound Leeds, Leeds, United Kingdom, 3Chemistry, University of Cambridge, organelles, distinguished by their unique morphology and chemical composi- Cambridge, United Kingdom, 4Chemistry, Imperial College London, tion. Despite the differences in membrane composition across organelles, rami- London, United Kingdom, 5Chemistry, King’s College London, London, fied, tubular or sheet-like shapes are generic large scale morphologies observed United Kingdom. in internal membranes, which suggest involvement of common underlying UapA from Aspergillus nidulans is a eukaryotic membrane transporter that me- principles. While there is detailed knowledge of the molecular processes diates proton-dependent uptake of xanthine and uric acid. To date, the role of involved in membrane remodelling at short scales, our understanding of the un- lipids in stabilising eukaryotic transporter oligomers remains unclear. We use derlying physical principles governing large scale morphogenesis is still native mass spectrometry (MS) in conjunction with molecular dynamics rudimentary. (MD) simulations and in vivo studies to investigate the role of interfacial lipids One common aspect is that membranes are subject to the action of curvature in stabilizing UapA oligomers. We show that UapA exists primarily as a dimer sensing and curvature generating proteins which modulate local membrane and that it binds two lipid molecules. Using lipidomics we identified three clas- shape and lipid composition. Even at relatively small values of surface ses of phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine coverage these proteins can cluster through to a variety or processes and trigger (PE) and phosphatidylinositol (PI) co-purified with UapA. Next we delipidated morphological changes which are important for singling. the UapA and subjected it to native MS analysis. Interestingly, delipidation re- Another important feature, specific to organelle membranes, especially those in sulted in dissociation of the UapA dimer into individual protomers. Subsequent the trafficking pathways, is that they are subject to and driven by a continuous addition of PI or PE reformed the UapA dimer and enabled recovery of its flux of membrane bound materials, on time scales comparable to membrane bound lipids, suggesting a key role of PI and PE lipids in the stabilisation of relaxation times. the dimeric interface. MD simulations allowed predicting a putative lipid- Here we present Monte Carlo Simulation models that tests the effect of binding site near the UapA dimer interface. Mutational analyses further the above processes on the morphology of the membranes. We find that the confirmed that this lipid-binding site is essential for the quaternary structure steady state shapes obtained as a result of such active processes, bear a and function of UapA. striking resemblance to the ramified morphologies of organelles in vivo, Concluding, we performed the first in-depth investigation of the role of lipids in pointing to the relevance of nonequilibrium fission-fusion in organelle the oligomerisation and function of a eukaryotic transporter. Importantly, we morphogenesis. identified specific lipids crucial for maintaining UapA transporter in a func- We show that, due to membrane curvature and composition interactions, the tional dimeric state. Overall, the hybrid strategy used here, provide the enticing curvature-inducing membrane-nematogens can aggregate spontaneously, prospect to allow investigation of the function of lipid binding to a wider range even at low concentrations, leading to a variety of membrane morphologies of membrane-bound proteins. such as tubular and the sheet conformations. Strong lipid-protein interaction can result in fast protein clustering indicating a route to a lipid mediated signal 3048-Pos Board B256 Pseudomonas Aeruginosa amplification. Investigating Structural Properties of Exou Toxin Upon Interaction with Liposome and Nanodisc Bilayers by EPR Spectroscopy 3046-Pos Board B254 Tzvia I. Springer, Samantha Kohn, Jimmy Feix. Interplay of Curvature Sensing and Generation Mediated by Peripheral Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA. Membrane Proteins Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative opportunistic Sachin Krishnan Thekke Veettil1, Sovan Lal Das2,3, pathogen that commonly infects the lungs of cystic fibrosis (CF) patients and Sunil Kumar Palakurissi Balagopal1,4. severe burn victims. One of the four type III secreted effector proteins of P. aer- 1 2 Department of Physics, IIT Madras, Chennai, India, Department of uginosa is ExoU, a bacterial phospholipase A2 (PLA2) enzyme evolved to uti- 3 Mechanical Engineering, IIT Kharagpur, Kharagpur, India, Department of lize host ubiquitin (Ub) and ubiquitinated proteins for enzymatic activation, 4 Mechanical Engineering, IIT Kanpur, Kanpur, India, Department of Physics, leading to the destruction of host membranes. ExoU has three major domains: IIT Palakkad, Palakkad, India. a catalytic domain, a linker domain, and a four-helix bundle located in the C- Curvature sensing refers to the ability of proteins to bind onto membranes terminus. Sequence and structural homology of the four-helix bundle are weak depending on their local curvature. As it offers a method for protein localiza- across various genus and species, suggesting unique structural and functional tion independent of the chemical composition, curvature sensing plays an features that may play a role in the molecular mechanism of activation. Previ- essential role in many of the fundamental cell biological processes. Several ous work identified regions within helix IV of the four-helix bundle that are classes of proteins such as the BAR-domain family and dynamin have been necessary for both activation and localization of ExoU to the lipid bilayer, identified as capable of sensing curvature. Once bound, these proteins can and suggested a synergistic mechanism of activation involving ubiquitin and alter the local shape of the membrane through curvature generation mecha- membrane binding. We are interested in understanding conformational changes nisms like scaffolding, hydrophobic insertion, steric repulsion etc. Such mech- in ExoU upon membrane binding in the presence and absence of ubiquitin. We anisms are believed to be responsible for stabilizing highly curved regions of hypothesize that the C-terminal four-helix bundle maintains essential intramo- intracellular membrane compartments. These two processes - curvature lecular interactions with the catalytic domain that are necessary for enzymatic sensing and curvature generation - occur simultaneously and influence one activation. In this study, site-directed mutagenesis, continuous wave (CW) EPR another. While various mechanisms have been proposed for curvature sensing spectroscopy, CW power saturation, and double electron-electron resonance as well as curvature generation, the interplay between them is not well (DEER) were used to investigate the structure and membrane localization of understood. helix IV in the presence of LUVs and nanodiscs. Results show that ubiquitin In this work, we employ grand canonical Monte Carlo simulations and binding facilitates membrane penetration by helix IV, and demonstrate the mechanical-thermodynamical mean field models to address the above problem. utility of nanodisc bilayers for the study of protein-membrane interactions. In the simulations, membrane is modeled as dynamically triangulated surface Supported by NIH grant GM114234. and the local protein concentration using a scalar field. Using these techniques, we quantify the interdependence of curvature sorting and curvature generation. 3049-Pos Board B257 Specifically, we look at how the physical parameters such as the protein Investigating the Conformational Dynamics and Membrane Interaction intrinsic curvature, the membrane tension, and protein-protein interaction influ- Near the Catalytic Serine of Exou Upon Interaction with Diubiquitin ences the binding affinity and morphology of the membrane. As a limitting and Membranes by EPR Spectroscopy case, we obtain adsortion isotherms and curvature sorting curves for a non- Samantha Kohn, Tzvia Springer, Jimmy Feix. deformable vesicle. The curves obtained using MC simulations matches well Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA. with analytical predictions from the mean field model for protein adsorption Pseudomonas aeruginosa (P. aeruginosa) is a Gram negative, opportunistic on vesicles. Our results conforms with observations in experiments using Sin- pathogen that is a rising health concern as an ESKAPE pathogen resistant to gle Liposome Curvature (SLiC) assays and membrane nanotubes pulled from multiple antimicrobial agents. P. aeruginosa manipulates the host response Giant Unilamellar Vesicles (GUVs). by translocating effector proteins into the host cell via a type III secretion

BPJ 8731_8734 Wednesday, February 21, 2018 615a system (T3SS). P. aeruginosa’s most cytotoxic T3SS effector, ExoU, is a 3052-Pos Board B260 potent patatin-like phospholipase with a catalytic serine-aspartate dyad that de- Mechanisms of Membrane Remodelling Mediated by Short Form of the stroys host cell membranes once activated by non-covalent interaction with Mitochondrial Inner Membrane Fusion Protein OPA1 ubiquitin. Two crystal structures of ExoU in complex with its chaperone Danyang Zhang1, Yan Zhang1,2, Tongxin Niu1, Edward H. Egelman2, SpcU have been reported, however the activated conformational state of Fei Sun1. ExoU in the presence of ubiquitin remains unknown. Consequently, ExoU’s 1Institute of BiophysicsChinese Academy of Sciences, Beijing, China, mechanism of action remains poorly understood. This study focuses on eluci- 2University of Virginia, Charlottesville, VA, USA. dating the conformational dynamics, membrane interaction, and structure of In mammals, mitochondrial inner membrane fusion is mediated by OPA1. In sites near the catalytic serine upon interaction with a ubiquitin cofactor and the physiological state, OPA1 undergoes proteolytic processing to form a membrane substrate. Site-directed spin labeling (SDSL) in conjunction with long isoform (L-OPA1) that is membrane anchored, and a short isoform (S- electron paramagnetic (EPR) spectroscopy was used to examine the motional OPA1) that lacks the membrane anchor but can still interact with membranes. dynamics of sites near the catalytic serine upon interaction with diubiquitin A combination of both long and short isoforms is required for membrane (diUb) and membranes. Membrane penetration of the same sites upon interac- fusion. However, neither the mechanism of OPA1 mediated inner membrane tion with diUb and membranes was investigated using power saturation EPR fusion is understood, nor are the functions of the long and short form spectroscopy in the presence of various paramagnetic relaxation agents. OPA1s. Since S-OPA1 has been reported to deform the membrane surface Changes in spin label motion and membrane penetration were observed for and cause the formation of lipid tubules, we focus on the structure of the S- sites near the catalytic serine in the presence of diUb and membranes com- OPA1-membrane complex. We present a tube structure of helical assemblies bined, but not in the presence of either diUb or membranes alone, suggesting of a truncated S-OPA1 in the apo state; an extended structure with GTPgS that a synergistic interaction with both ubiquitin and membranes is necessary bound was also solved through tomography and sub-volume averaging. The to form an active catalytic site. Supported by NIH grant GM114234. structure shows that S-OPA1 oligomerizes into a helical polymer through a stalk region similar to dynamin proteins, and the building block is a dimer of 3050-Pos Board B258 the stalk region. The binding of non-hydrolyzable GTP analogs causes a The Minimum Conditions for Bax to Induce Apoptotic Membrane Pores conformational change between the GTPase domain and the stalk region, re- Yei-Chen Lai, Yun-Wei Chiang. sulting in the rearrangement of the dimers and an expansion of the tube. This Chemistry, National Tsing Hua University, Hsinchu, Taiwan. is very different from dynamin and Drp1, which produce restricted tubes due BAX acts as a gatekeeper in the regulation of mitochondria-dependent to GTP hydrolysis. These results help explain the molecular mechanism of apoptosis. Under cellular stress, BAX is activated to transform into a lethal OPA1-mediated membrane remodeling during mitochondrial fusion. oligomer that cause mitochondrial outer membrane permeabilization (MOMP). Four events in the activation process were previously identified 3053-Pos Board B261 to be pivotal, but it remains unclear which is the minimum requirement for Human Domain Swapped Cytochrome C: The Evolutionary Governor of the activation. Here, we explore the issue using spin-labeled ESR and fluores- Apoptosis? cence spectroscopic tools. We show that none of the four is a determinant that Harmen B. Steele, James T. Rogan, J.B. Alexander Ross, Bruce E. Bowler. decisively results in the BAX-induced MOMP. The minimum requirement for Biochemistry and Biophysics, University of Montana, Missoula, MT, USA. isolated BAX to cause MOMP is the part of outer mitochondrial membrane The oxidation of the lipid cardiolipin (CL) by Cytochrome c (cytc) has been containing cardiolipin (CL). Without the presence of CL, isolated BAX inter- proposed to be involved in the initiation of the intrinsic pathway of apoptosis. acts with membrane lipids to form an off-pathway oligomer that lacks ability Reports and studies of domain-swapped dimer (DSD) conformations of cytc, to cause MOMP. An important implication of our study is that BAX can be both from our lab and others, have been appearing in the literature. We hypoth- activated by two alternatives, one being the apoptotic activator proteins esize the DSD has evolved as an evolutionary switch providing tighter regula- requiring no presence of CL-containing membrane, and the other being tion of the intrinsic pathway of apoptosis. Data presented here will show the only the presence of CL-containing membrane. The two activation pathways human DSD is kinetically more stable than horse or yeast DSDs. Evidence can happen in parallel and both lead to the formation of active membrane- will be presented suggesting that while yeast required the unfolding of both associated BAX. subunits, the human DSD does not require complete unfolding of each subunit for dissociation to happen. Peroxidase data acquired from pH 6.0 to pH 8.0 will 3051-Pos Board B259 be presented showing the human DSD has a seven-fold increase in activity Structural Characterization of Membrane-Associated BCL-2 Family compared to the monomer. Similar data from the Hirota lab indicate that the Proteins Equine dimer only shows a four-fold increase of activity over the monomer. Yong Yao, Vindana Ekanayake, Pavel Ryzhov, Francesca M. Marassi. Additionally, a comparison of monomer versus DSD cytc-CL binding, moni- Sanford Burnham Prebys Research Institute, La Jolla, CA, USA. tored by fluorescence correlation spectroscopy (FCS), will be presented. Initial The BCL-2 family proteins are central regulators of programmed cell death. results indicate a micromolar range Kd for cytc binding to CL nanodiscs, Their functions are regulated by their interactions with a wide range of proteins, whereas no significant binding is observed for DOPC nanodiscs. both within and outside the BCL-2 family as well as with intracellular mem- branes, most notably, the mitochondrial outer membrane, where cell fate is ul- 3054-Pos Board B262 timately decided. The family members share sequence similarity across four Correlating Structure and Function of Non-Native Cytochrome C: The BCL-2 homology (BH1-BH4) motifs, of which BH3 is highly conserved and Relationship Between Iron Spin State and Peroxidase Activity essential for cell death. Several also contain a hydrophobic C-terminus that an- Gabrielle Lewis, Bridget Milorey, Reinhard Schweitzer-Stenner. chors the proteins to intracellular membranes. The family includes both death Drexel University, Philadelphia, PA, USA. antagonists that protect the cell against apoptotic stimuli, death agonists that The hemeprotein cytochrome c, widely known for its role as an electron carrier, kill the cell. Interactions with membranes and lipids are critical for BCL-2 pro- also plays an important role in apoptosis or programed cell death when binding tein function, but structural studies have focused primarily on forms of the pro- to cardiolipin. To this end its function has to switch from electron carrier to teins made soluble by deleting the C-terminal tail or adding detergent. As a peroxidase. While it is understood that this requires a structural transition result, the molecular mechanisms controlling BCL-2 function are incompletely into a non-native state, an exact structure - function relationship has still to understood. Here, we present NMR structural data for the anti-apoptotic protein be identified. Our group recently obtained different conformational ensembles BCL-XL, and the pro-apoptotic protein BID, associated with detergent-free of partially unfolded cytochrome c bound to cardiolipin-containing liposomes, lipid particles. We show that BCL-XL can be incorporated in lipid bilayer one with a hexacoordinate low-spin iron populated at neutral pH and another nanodiscs. BCL-XL is anchored to the nanodisc lipid bilayer membrane by one with a mixture of penta-and hexacoordinate high-spin iron adopted at tight association of its C-terminal tail, while the N-terminal head retains the ca- slightly acidic pH. The switch between these two ensembles involves the pro- nonical BCL-2 protein fold, with the surface groove solvent-exposed and avail- tonation of a non-native histidine ligand. We used optical spectroscopy to mea- able for BH3 ligand binding. The transmembrane C-terminus of BCL-XL sure and quantify the peroxidase activity of these conformations as a function forms an a-helix with a significant degree of conformational dynamics. We of substrate concentration. It is generally expected that significant peroxidase further show that tBID, the truncated active form of pro-apoptotic BID, can activity requires a penta- or hexacoordinate high-spin state of the heme iron. form soluble lipoparticles that are fully capable of engaging the canonical This would facilitate the reduction of water and the binding of an oxygen to groove of its partner BCL-XL. The results indicate that the BCL-2 family pro- a ferryl heme iron. Our data indicate that the Michaelis-Menten constant, teins exist as lipid-associated entities, in both their cytosolic and membrane- Km, of the reaction is larger at pH 6.5 (high spin heme) than at pH 7.4 (low bound states, and help reconcile a number of recent results involving spin heme). While these results establish a clear correlation between heme membrane-associated processes. spin/ligation state and peroxidase activity, they also corroborate earlier findings

BPJ 8731_8734 616a Wednesday, February 21, 2018 indicating that even the non-native hexacoordinate low-spin state exhibits sig- The misfolding and aggregation of the intrinsically disordered microtubule bind- nificant peroxidase activity which is likely to involve a proton transfer from ing tau protein into b-sheet enriched, highly ordered fibrils are linked to the path- H2O2 to the second histidine ligand which would lead to its dissociation ogenesis of a wide range of neurodegenerative disorders. The mechanism and from the heme iron. driving forces of tau fibrillogenesis in vivo is still unknown. Specifically, struc- tural details of the early events during the aggregation process that triggers the 3055-Pos Board B263 misfolding and assembly of the otherwise soluble and stable tau remain poorly Implementing a Statistical Thermodynamic Model to Describe Function- defined. We investigated the role interfaces play in modulating the structure C ally Relevant Cytochrome - Cardiolipin L-Site Binding and aggregation propensity of four tau proteins. The largest human isoform Bridget Milorey, Reinhard Schweitzer-Stenner. hTau40 and two truncated constructs K32 and K18 were used. Additionally, a Drexel University, Philadelphia, PA, USA. mutant hTau40/3Epi was used to mimic hyper-phosphorylation that is unknown The L-site binding of ferricytochrome c to cardiolipin (CL) - containing lipo- to occur early during disease process. All tau proteins were found to be highly sur- somes at slightly acidic pH has attracted interest owing to its potential role in con- face active, and favorably interacted with anionic 1,2-dimyristoyl-sn-glycero-3- verting this classical electron transfer protein into a peroxidase. Various lines of phospho-(1’-rac-glycerol) (DMPG)lipid monolayers at the air/water interface. evidence presented by Nantes and coworkers suggest that this mode of binding is When intercalated into the membrane, tau underwent structural compaction to electrostatic, active below pH 7, and involves residues K22, K25, and K27 as well adopt a conformation with a density close to that of folded proteins. Strikingly, as H26 and H33. We combined several spectroscopic techniques to characterize tau at the membrane surface gave rise to in-plane diffraction peaks, as detected L-site binding thermodynamically and to identify concomitant structural by grazing-incidence x-ray diffraction (GIXD), with a d-spacing of 4.74 A˚ . changes. To this end we employed and extended a recently presented thermody- This d-spacing exactly matches the b-sheet spacing in amyloid fibrils, indicating namic model that describes cytochrome c binding to CL- containing liposomes as that these 2D crystallites formed in membrane are tau proteins misfolded in a b- a two-step process, where native-like liposome-bound conformers convert to sheet conformation. Moreover, the misfolded tau also self-assembled into large more unfolded conformers with an increase in CL concentration. These partially oligomeric aggregates, as the size of these tau crystalline domains is around unfolded species are characterized by a loss of the M80 ligand, which is likely 200 A˚ . Our study supports a general tau aggregation mechanism where tau’s replaced by H26 and/or H33. At acidic pH, the partially unfolded low-spin spe- inherent surface activity drives tau-membrane interactions, inducing the misfold- cies converts into a mixture of penta- and hexacoordinate high-spin species at ing and self-assembly of tau into b-sheet enriched oligomers that could subse- moderate-to- high CL concentration. This change of the heme iron’s spin state quently seed tau fibril growth and deposition into diseased tissues. is most likely caused by the protonation of the non-native histidine ligand, which is facilitated by low effective pH at the liposome surface. Results of our analysis 3058-Pos Board B266 of binding isotherms obtained in the absence and presence of NaCl confirmed the Effect of Cholesterol on Membrane Pore Formation by Amyloid b25-35 electrostatic character of L-site binding. The inhibiting effect of sodium ions is Nabin Kandel1, Jason O. Matos1,2, Suren A. Tatulian1. attributed to the accumulation of cations in the liposome’s double layer, which 1Physics, University of Central Florida, Orlando, FL, USA, 2Biochemistry reduces its surface potential. Thus, L-site binding is clearly distinct from cyto- and Biophysics Grad. Program, Brandeis University, Waltham, MA, USA. chrome c - CL interactions at neutral pH where only the conformational change One of the cytotoxicity mechanisms of amyloid b (Ab) peptide in Alzheimer’s on the membrane surface is affected by the presence of NaCl. disease is membrane permeabilization, yet the mechanism of pore formation 2þ 3056-Pos Board B264 remains unclear. Here, Ca -permeable membrane pore formation by a cyto- Isothermal Titration Calorimetry and Vesicle Leakage Assays Highlight toxic fragment of Ab (Ab25-35) has been analyzed by biophysical methods. the Differential Behaviors of Tau Repeat Segments Upon Interaction Quin-2-loaded lipid vesicles composed of 0.3 mole fraction of anionic lipid with Anionic Lipid Membranes 1-palmitoyl-2-oleoyl-phosphatidylglycerol, xchol mole fraction of cholesterol, Lexus Tatge, Sidney Dicke, Paige Engen, Samantha Ealy, Megan Culp, and (0.7 - xchol) mole fraction of zwitterionic lipid 1-palmitoyl-2-oleoyl-phos- phatidylcholine were prepared at xchol = 0, 0.05, 0.1, 0.2 and 0.4. CaCl2 was Larry R. Masterson. 2þ Chemistry, Hamline University, St.Paul, MN, USA. added externally so that Quin-2 was sequestered from Ca ions. Quin-2 fluo- Alzheimer’s Disease (AD) is the sixth-leading cause of death in the United rescence increased exponentially upon addition of Ab25-35, which was inter- States and is due to protein misfolding. One of the proteins implicated in this preted as membrane pore formation by the peptide, calcium influx and misfolding is Tau, which is a protein that stabilizes the cytoskeletal structure binding to intravesicular Quin-2. The kinetics and the magnitude of fluores- in neurons. Tau contains four repeat segments (R1-R4) that are crucial to its cence enhancement were used to evaluate the second order rate constant of function, but are also the site of the misfolding that forms neurofibrillary tan- pore formation (ka), the affinity constant between the peptide molecules gles (NFTs). These NFTs inhibit signals between neurons and invokes pro- (Kp), and the number of peptide units in the pore (n). Circular dichroism sug- cesses which cause degradation of the neuronal cell. A number of previous gested mostly b-sheet structure for the peptide in the presence of lipid vesicles. studies reveal that anionic membranes interact with tau and induce misfolding Attenuated total reflection Fourier transform infrared experiments were per- of the protein. Here, we have investigated how each repeat segment of tau in- formed on Ab25-35 embedded in lipid multilayers deposited on a germanium teracts with anionic and neutral membranes using isothermal titration calorim- plate. These data suggested a-helical and b-sheet structures for the peptide eter (ITC) and leakage assays. We have determined the equilibrium binding in a lipid environment. The structure of the peptide, i.e., the a-helix/b-sheet constants for peptide: membrane interactions, along with the thermodynamic ratio, varied as a function of cholesterol content in the membranes. These parameters DH, DS, and DCp. We found that R2 and R3 repeat segments conformational transitions correlated with changes in pore formation parame- interact more favorably with anionic membranes, and none of the segments in- ters (Kp, ka, n). The data suggest that cholesterol affects Ab membrane pore < teracted strongly with neutral membranes. The differential behaviors of these formation by multiple mechanisms. At moderate concentration (xchol 0.1), sequences was also evident in their driving forces to membrane binding - cholesterol supports pore formation possibly through cholesterol-peptide in- both classical and nonclassical hydrophobic effects were observed among these teractions. Further increase in xchol causes pore inhibition by a membrane so- sequences. Leakage assays showed that all sequences display lytic activity to lidification mechanism. At xchol = 0.4, cholesterol causes lipid disorder and anionic membranes but not neutral membranes. Taken together, these results membrane destabilization. align with models of NFT formation that invoke misfolding events at the mem- brane surface. Further exploration will include experiments using NMR to see 3059-Pos Board B267 how the phosphates on the side chains of the amino acids play a role in the Characterization of Membrane-Bound Alpha-Synuclein with the Thiocya- structural composition of the repeat segments. nate Vibrational Probe Group Franklin A. Kostas, Kavita Shroff, Kristen E. Fiore, 3057-Pos Board B265 Daniel M. Konstantinovsky, Casey H. Londergan. Lipid Membrane Templated Misfolding and Self-Assembly of Intrinsically Haverford College, Haverford, PA, USA. Disordered Tau Protein Alpha-synuclein (aS) is a membrane-associated human protein found in the Jaroslaw P. Majewski1, Emmalee M. Jones2, Jacek Biernat3, pre-synaptic terminals of neurons. Its function is unclear but it is known to Eckhard Mandelkow4, Eva Y. Chi2. aggregate to form Lewy bodies, characteristic of Parkinson’s disease and 1Division of Molecular and Cellular Biosciences, National Science related disorders. aS adopts multiple stable conformations: at least two Foundation, Alexandria, VA, USA, 2Department of Chemical and Biological different amphipathic helix-based conformations have been reported at inter- Engineering, Center for Biomedical Engineering, University of New Mexico, faces with lipid membranes, and many experiments suggest a range of different Albuquerque, NM, USA, 3Max Planck Unit for Structural Molecular Biology, membrane-bound structures that depends on the membrane composition and Max Planck Institute, Hamburg, Germany, 4Center for Neurodegenerative curvature. The thiocyanate probe group, which reports on its local environment Diseases (DZNE) and CAESAR Research Center, Bonn, Germany. in IR spectroscopy, was used to characterize the lipid-binding dynamics at 11

BPJ 8731_8734 Wednesday, February 21, 2018 617a sites of interest in aS. Prior work in the Londergan lab involved unacetylated may be an important mechanism. In this study, we use atomistic Molecular aS, although in physiological settings nearly all aS is found to be N-terminally Dynamics simulations to capture the molecular detail of the SEVI- acetylated. Site-directed mutagenesis was used to create new single-cysteine membrane interaction in an attempt to confirm the binding mechanism. mutated aS variants, which were then coexpressed in Escherichia coli with We show that binding between SEVI and the membrane depends on the N-terminal acetylase B (NatB) and purified to yield N-terminally acetylated membrane composition of charged lipid species via electrostatic interac- aS. These proteins were cyanylated chemically at cysteine, and the resulting tions. We also show that the helical conformation of SEVI is dramatically SCN probe groups are characterized via IR in the presence of POPA/POPC changed upon binding to the membrane. However, large variations are large unilamellar vesicles, SDS micelles, and DOPE/DOPS/DOPC small unila- observed when different molecular mechanics force fields are used, and mellar vesicles to reveal differences in the membrane-bound structural we comment on the performance of these force field and their consistency distributions. with experimental results. 3060-Pos Board B268 3063-Pos Board B271 Structure of E. coli SecA Bound to Lipid Vesicles and Nano-Discs Cyclooxygenase 1 Lipid Interactions Revealed by All-Atom and Coarse- Guillaume Roussel, Stephen H. White. Grained Molecular Dynamics Simulations Physiology and Biophysics, University of California, Irvine, Irvine, CA, Besian I. Sejdiu, D. Peter Tieleman. USA. Biological Sciences, University of Calgary, Calgary, AB, Canada. Bacterial SecA ATPase binds to the SecYEG translocon to secrete soluble pro- Cyclooxygenase 1 (COX1) is an integral protein that is monotopically attached teins across the inner membrane into the periplasm. SecA (monomer MW about to the endoplasmic reticulum. It is an important enzyme in the biosynthesis of 100 kDa) exists in three-states in Escherichia coli: free in solution, bound to in- prostaglandins, where it is responsible for converting arachidonic acid into ner membrane, and bound to SecYEG. Much effort has been devoted to char- prostaglandin H2. COX1, along with COX2, are targeted by nonsteroidal acterizing the quaternary structure of cytosolic SecA and SecYEG-bound anti-inflammatory drugs (NSAIDs) and as such, they are of vital importance SecA, but there is no general consensus as to the oligomeric state (monomer in the development of new drugs that selectively target COX2, without or dimer) or the topology of SecA bound directly to membranes. The goal of affecting the activity of COX1. Current research has shown that the lipid envi- this project is to determine the energetics and orientation of SecA bound to lipid ronment can be directly and functionally involved in membrane protein struc- vesicles and nano-discs. Using intrinsic Trp fluorescence, we show that SecA ture and activity, and as such, it has to be taken into account. Here, we partitions very strongly into both vesicles and nano-discs with water-to- performed extensive all-atom and coarse-grained simulations to study how bilayer free energies of transfer of about 7 kcal/mol. To determine the topol- the membrane environment affects the structural activity of COX1. We ogy of the protein bound to the membrane, we started with a Cys-less SecA embedded the protein in a variety of compositionally-different lipid bilayers construct and then introduced single Cys residues at strategic locations within and performed microsecond long molecular dynamics simulations. The resul- SecA in order to covalently link the NBD fluorophore (7-nitro-benzen-2-oxa- tant trajectories revealed unique insights into how bulk properties of the bilayer 1,3-diazol-4-yl), whose fluorescence is sensitive to the polarity of the environ- influence the structure of COX1 and vice versa. We also observed a unique ment. By monitoring the change in fluorescence emission of the NBD-labeled interaction between COX1 and cholesterol, which we believe could play an Cys residues upon the addition of lipid vesicles, we have determined, from a important role in the activity of COX1. The latter result is surprising when large number of mutants, the disposition of the SecA dimer on lipid vesicles. considering that cholesterol is only minimally present in the endoplasmic Supported by NIH grant RO1 GM74637. reticulum. 3061-Pos Board B269 Investigating the Membrane Association of the Human N-Terminal Posters: Excitation-Contraction Coupling II Acetyltransferase 60 (hNaa60) 3064-Pos Board B272 Qaiser Waheed, Nathalie Reuter. 2D D Department of Molecular Biology, Computational Biology Unit, University The Effect of Oestrogen on Ca and Na Regulation in Heart Failure 1 1,2 1 of Bergen, Bergen, Norway. Jahn M. Firth , Hsiang-Yu Yang , Alice J. Francis , 1 1 N-terminal acetyltransferases (NATs) are members of GCN5-related N- ace- Anita Alvarez-Laviada , Kenneth T. MacLeod . 1Myocardial Function, Imperial College London, London, United Kingdom, tyltransferase (GNAT) superfamily, known for playing catalytic role in N- ter- 2 minal acetylation of the proteins. Recent studies revealed that Naa60 (NatF) is Tri-Service General Hospital, National Defense Medical Center, Taipei, the only candidate from the NAT family that localizes to the Golgi apparatus, Taiwan. In this work, we show that long-term absence of oestrogen impairs cardiac and that only the C-terminal tail (Naa60182-242) is important and sufficient for 2þ þ this association. Since the existing X-ray structure is missing the C-terminal function and produces detrimental changes to Ca and Na regulation in car- diomyocytes following pressure-overload heart failure (HF). Oestrogen supple- tail (Naa60185-242), we modeled this part containing two amphipathic helices. The modeled structure was then used to investigate the membrane association mentation reverses the negative effects of ovariectomy following HF. by molecular dynamics simulations. Results from implicit membrane model Pressure-overload HF was induced by aortic constriction (AC) in female guinea pigs. To examine the effects of long-term absence of oestrogen on HF progres- (IMM1) simulations show that Naa60185-242 associates with the membrane interface with its helices lying horizontally. In order to further characterize sion, selected ovariectomy (OV) animals underwent AC (ACOV). Pellets con- the interactions involved we performed atomistic simulations using the taining 17b-oestradiol (1mg, 60-day release) were placed subcutaneously in selected ACOV animals (ACOVþE). Electrophysiological and fluorescence CHARMM36 force field. Naa60185-242 was docked on three different phos- 2þ þ pholipid bilayers; two anionic (POPC:PI4P = 92:8, POPC:POPS = 75:25) techniques were used to assess Ca and Na regulation 150 days post- and one neutral POPC:PSM:CHOL = 70:20:10. Analysis of the interactions operatively. ACOV animals heart weight/body weight ratios increased by 11% and in vivo between Naa60182-242 and the lipids revealed a favorable association to anionic membranes. In vitro assays and cellular localization experiments fractional shortening decreased by 14% compared with the AC group, suggest- also verified that Naa60 is a peripheral membrane protein and favor mem- ing greater impairment of cardiac function following pressure-overload in the branes containing PI4P (Aksnes et al. 2017). Furthermore we built a full absence of oestrogen. Action potential duration increased in all three interven- tions but the changes were oestrogen-independent. While ICa and fractional SR model of Naa60 (Naa601-242), which we subsequently subjected to MD sim- 2þ ulations at a phospholipid bilayer, revealing how the globular part of the pro- Ca release were unaltered, myocytes from ACOV animals typically had reduced Ca2þ transient amplitudes, slower transient decay kinetics, decreased tein is oriented with respect to the membrane. Our work is relevant to the 2þ 2þ mechanism of N-terminal acetylation of transmembrane proteins at the Golgi SR Ca contents and increased Ca spark frequencies and spark mediated 2þ þ þ þ apparatus. SR Ca leak compared with the AC and ACOV E groups. The Na /K ATPase current densities and Naþ extrusion rates were reduced by 13% and 3062-Pos Board B270 19%, respectively, in parallel with a 17% increased INa,L current densities Conformational Changes of Sevi Precursor Peptide Pap248286 Upon following ACOV compared with the AC group. Interestingly, myocytes iso- Membrane Binding lated from ACOV animals supplemented with 17b-oestradiol (ACOVþE) typi- þ þ Shushan He, Lutz Maibaum. cally had similar Ca2 and Na regulation compared with the gonad-intact AC Department of Chemistry, University of Washington, Seattle, WA, USA. group. Amyloid fibers formed from the semen-derived enhancer of viral infection Here we present that long-term deprivation of oestrogen, in an animal model (SEVI) peptide can drastically enhance the infectivity of the HI virus. Mul- whose electrophysiological and hormonal status is akin to human, exacerbates tiple recent experimental studies have suggested that nonspecific binding of the detrimental effects of pressure-overload HF. Oestrogen supplementation re- monomeric forms of SEVI in a helical conformation to lipid membranes verses the negative effects of ovariectomy following AC.

BPJ 8731_8734 618a Wednesday, February 21, 2018

3065-Pos Board B273 Calcium release required for a proper cardiac contraction is performed by a D TNF-Alpha Mediates Gender Specific Ca2 Signalling Dysfunction in macromolecular protein complex, the calcium release complex composed of Type 2 Diabetes the ryanodine receptor (the sarcoplasmic reticulum calcium channel RyR2), Gema Ruiz-Hurtado1, Carmen Delgado2, Ana-Maria Gomez3, the calsequestrin CSQ2 (a calcium binding protein), and a number of asso- Laetitia Pereira3. ciated proteins among which triadin, calmodulin, and junctin. Recessive 1Unidad de Hipertensio´n, Instituto de Investigacio´n iþ12 Hospital forms of CPVT are genetically induced by mutation in genes encoding tria- Universitario 12 de Octubre, Madrid, Spain, 2Instituto de Investigaciones din or calsequestrin. We used a triadin knock out mice model presenting with Biomedicas Alberto Sols, Consejo Superior de Investigaciones Cientı´fica, CPVT-like phenotype in which we observed an important decrease of calse- Madrid, Spain, 3UMRS 1180, INSERM, Chaˆtenay-Malabry, France. questrin protein level as well as Ca2þ homeostasis impairment and suscepti- Cardiac inflammation, with secretion of pro-inflammatory cytokine, such as the bility to ventricular tachycardia resulting in sudden cardiac death. We Tumor Necrosis Factor alpha (TNF-a), participates in the development of heart assessed different approaches to rescue protein expression in cardiomyocytes failure, notably during diabetic cardiomyopathy, with gender-dependent effects. and to correct intracellular calcium releases and cardiac function: pharmaco- Yet, the underlying mechanism is still unclear. Here, we studied the role of logical treatment or viral gene transfer based-approach (using AAV2/9 en- þ TNF-a on Ca2 signalling alteration observed in male and female type 2 diabetic coding the cardiac triadin or calsequestrin).We showed that a fine tune of mouse model, the db/db mice. We measured plasmatic level of TNF-a as well as the relative re-expression of both the triadin and calsequestrin is required expression level of TNF-a receptors (TNF-R1 and TNF-R2) and TNF-a to correct cardiomyocytes Ca2þ signaling and rescue the cardiac function. þ converting enzyme (TACE). We assessed Ca2 signalling in isolated cardio- Our results also demonstrate the possibility of using AAV mediated gene de- þ þ myocytes loaded with Fluo3-AM by recording Ca2 transient, Ca2 spark fre- livery as a possible treatment for recessive forms of CPVT due to triadin þ quency and SR Ca2 load by confocal microscopy. While plasmatic level of mutations. TNF-a was unchanged in heart from male and female db/db, we observed gender specific differences in expression level of TNF-R1, TNF-R2 and 3068-Pos Board B276 TACE. Male db/db presented both higher TNF-R2 (relative density in %: T-Tubule Loss is a Prominent Feature of HFrEF but not HFpEF 1 2,3 1 4 147512.57 for db/db vs. 10059.2 for WT, p<0.05) and TACE expression level Michael Frisk , Michael Frisk , Christopher Le , Christen P. Dahl , 2 5 5 5 (relative density in %: 141511.3 for db/db vs. 99.9857.41, for WT p<0.05) Ida G. Lunde , Vibeke M. Almaas , Lars Gullestad , Svend Aakhus , Ole M. Sejersted2, Theis Tønnessen2, William E. Louch2. compared to WT. Those differences were associated with distinct alterations 1 2 2þ 2þ IEMR, University of Oslo, Oslo, Norway, IEMR, Oslo University Hospital of the Ca transient amplitude and decay, Ca spark frequency and SR 3 Ca2þ load from male and female mice. In conclusion, in db/db mice, the alter- Ulleval, Oslo, Norway, KG Jebsen Center for Cardiac Research, Oslo, Norway, 4Research Institute for Internal Medicine, Oslo University Hospital, ation of TNF-a signalling is gender specific and associated with a dysregulation 5 of Ca2þ signalling. Rikshospitalet, Oslo, Norway, Department of Cardiology, Oslo University Hospital, Rikshospitalet, Oslo, Norway. 3066-Pos Board B274 T-tubules are invaginations of the sarcolemmal membrane which are important D Role of Epac2 in High Glucose-Induced SR Ca2 Leak and Arrhythmia for effective excitation-contraction coupling. Accumulating data indicate that Magali Samia el Hayek1, Donald Bers2, Ana-Maria Gomez1, cardiomyocyte t-tubular structure is disrupted during heart failure with reduced Laetitia Pereira1. ejection fraction (HFrEF), resulting in impaired Ca2þ release and contractility. 1UMRS 1180, Inserm, Chaˆtenay-Malabry, France, 2Department of Much less is known about t-tubule structure during heart failure with preserved Pharmacology, UC Davis, Davis, CA, USA. ejection fraction (HFpEF). Based on our recent data, indicating that increased Epac2 (Exchange Protein directly Activated by cAMP) has recently emerged as wall stress promotes t-tubule degradation during HFrEF (Frisk et al. Cardiovasc a critical player in cardiomyopathies, such as heart failure and arrhythmia, also Res, 2016), we hypothesized that t-tubule structure would be safeguarded by seen in diabetes. Recently, we found that diabetic hyperglycemia leads to maintained wall stress during HFpEF. In HFpEF patients with mild diastolic þ CaMKII-dependent SR Ca2 leak, a downstream effector of Epac2. However, dysfunction (E/e’ = 10.952.7 and deceleration time < 235ms), we observed þ the role of Epac2 in hyperglycemia-mediated SR Ca2 leak is still unknown. that t-tubule density was maintained compared to healthy patient hearts. Inter- þ Here, we tested the significance of Epac2 in high glucose-mediated SR Ca2 estingly, patients with more severe diastolic dysfunction (E/e’ = 13.452.7 and þ leak. We measured, using confocal microscopy, Ca2 sparks frequency, deceleration time > 305ms) showed increased t-tubule density. Closer exami- þ þ þ Ca2 waves and diastolic [Ca2 ] to assess SR Ca2 leak in isolated mice ven- nation of t-tubule architecture revealed that the observed rise in t-tubule density tricular myocytes under hyperglycemic conditions (300 mg/dl, 500 mg/dl vs. was a result of maintained transversely-oriented tubules and increased levels of þ 100 mg/dl). High glucose enhanced SR Ca2 leak, as observed by an increase longitudinally-oriented tubules. In contrast, t-tubules were lost and disorga- þ in Ca2 spark frequency (n/100mm/s: 0.0950.04 at 100 mg/dl vs. 2.451.1 at nized in patients with dilated cardiomyopathy. To investigate if t-tubule main- þ 500 mg/dl), Ca2 waves (0% at 100 mg/dl vs. 52.4% at 500 mg/dl) and diastolic tenance or proliferation are general features of non-dilated hypertrophic hearts 2þ [Ca ] (as F0: 30.952 at 100 mg/dl vs. 59.2511) (p<0.05). Both pharmaco- we investigated the t-tubule network in patients with hypertrophic cardiomyop- logical (ESI-05) and genetic deletion of Epac2 (Epac2-KO) reduced high athy or aortic stenosis. In both conditions, t-tubule density was increased due to þ glucose-mediated SR Ca2 leak (0.1350.08 vs. 100 mg/dl: 0.0650.03 for a larger fraction of transversely-oriented tubules. We thus conclude that t-tu- ESI-05þ500 mg/dl and 0.2150.07 for Epac2-KO at 500 mg/dl, p=N.S.) and bule density is maintained or even increased in the non-dilated hypertrophic hu- þ restored diastolic [Ca2 ]. Furthermore, the increase of Epac2-based FRET man ventricular myocardium. These findings are consistent with a key role of biosensor activity in high glucose concentration confirms that Epac2 is indeed increased wall stress, a hallmark of dilated ventricular remodeling, in triggering activated in hyperglycemic condition. Interestingly, specific O-GlcNAcylation t-tubule degradation in HFrEF. inhibition by diazo-5-oxonorleucine (DON) fully blocks FRET signal. To conclude, our work suggest that hyperglycemia activates Epac2 by O-GlcNA- 3069-Pos Board B277 cylation to mediate SR Ca2þ leak and arrhythmia ranking it as a potential target Cholesterol Protects Against Acute Stress-Induced T-Tubule Remodeling in the treatment of diabetic cardiomyopathy. in Mouse Ventricular Myocytes Azadeh Nikouee, Keita Uchida, Anatoli N. Lopatin. 3067-Pos Board B275 Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Interplay between Triadin and Calsequestrin in the Pathogenesis of CPVT MI, USA. Marine Cacheux1,Jeroˆme Thireau2,Jeremy Fauconnier2, Alexis Osseni1, Efficient excitation-contraction coupling in ventricular myocytes depends Nathalie Roux-Buisson1, Julie Brocard1, Julien Faure1, Alain Lacampagne2, critically on the presence of the t-tubular network. It has been recently Isabelle Marty1. demonstrated that cholesterol, a major component of the lipid bilayer, plays 1Grenoble Institut des Neurosciences, Inserm U1216, Grenoble, France, an important role in maintaining the integrity of t-tubular system although 2Universite de Montpellier, CHRU Montpellier, Inserm U1046, Montpellier, mechanistic understanding of underlying processes is essentially lacking. France. In this study we tested the hypothesis that membrane cholesterol protects Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare against various acute stresses, including osmotic challenge.Experiments and inherited sudden cardiac death syndrome predominantly affecting chil- were performed using isolated left ventricular cardiomyocytes from adult dren or young adults. This heart rhythm disorder is characterized by bidirec- mice. Depletion and restoration of membrane cholesterol was achieved by tional/polymorphic ventricular tachycardia triggered in condition of intense applying methyl-b-cyclodextrin (MbCD) and water soluble cholesterol physical or emotional stress, without any associated morphological or struc- (WSC), respectively. T-tubule remodeling in response to acute hyposmotic tural alterations of the heart neither any electrocardiogram abnormalities at stress (2115 2 mOsm; 0.7 Na) was assessed using fluorescent dextran trap- rest. The mortality rate by sudden cardiac death reaches 30% of the patients ping assay and by measuring t-tubule dependent IK1 tail current. Myocytes under the age of 30 when the CPVT is not promptly diagnosed and treated. werefirsttreatedwith3mMMbCDfor1hatRTand0.7Naosmoticstress

BPJ 8731_8734 Wednesday, February 21, 2018 619a applied in the presence of extracellular fluorescent dextran. The amount of after TAC surgery. Thus, maintaining NCX1 activity may be a potential dextran trapped in t-tubules sealed in response to stress was increased by therapeutic strategy for preventing the progression of HF, through the mainte- z25% when compared to control cells. Reintroduction of cholesterol by nance of T-tubule architecture. application of 5 mg/ml WSC to cells treated with MbCD restored the amount of trapped dextran to control values. Alternatively, application of 5 mg/ml 3072-Pos Board B280 WSC to normal cells reduced the amount of trapped dextran by z50%, con- Recovery of Cardiac T-Tubules after Hyposmotic Shock firming significant protective effect of cholesterol. Unexpectedly, depletion Greta Tamkus, Keita Uchida, Anatoli N. Lopatin. and enrichment of membrane cholesterol (without osmotic stress) accelerated University of Michigan, Ann Arbor, MI, USA. Cardiac t-tubules are significantly remodeled in various disease states. and decelerated, respectively, the kinetics of IK1 tail current suggesting signif- icant t-tubule remodeling prior to osmotic stress. Results of this study demon- Although certain experimental models have shown that the integrity of the t- strate that modulation of the level of membrane cholesterol has significant tubular system can be restored upon removal of underlying stress, little is effects on the properties of cardiac t-tubules and on t-tubule response to acute known about this process. In this study, we employed a model of hyposmotic stress. osmotically-induced t-tubular remodeling in order to get insight into the mech- anisms involved in the process of t-tubule recovery. Experimentally, sealing of 3070-Pos Board B278 t-tubules in isolated mouse ventricular cardiomyocytes was induced by hypo- T-Tubular Constrictions Promote T-Tubule Sealing smotic shock and the fate of sealed t-tubules was studied using fluorescent Keita Uchida, Azadeh Nikouee, Greta Tamkus, Anatoli N. Lopatin. dextran trapping and diffusion accessibility assays. Cardiomyocytes with trap- Molecular and Integrative Physiology, University of Michigan, Ann Arbor, ped dextran showed no significant changes in fluorescence over 2 hours when MI, USA. stored on ice (p =NSvs control). In contrast, at 31 C dextran fluorescence Cardiac t-tubules undergo significant remodeling in various pathological and declined in a single exponential manner to 10 5 7% of control values (p < experimental conditions, which in many instances can be associated with me- 0.001) with time constant of 20 5 6 min, suggesting reopening of sealed t- chanical or osmotic stress, although the exact underlying mechanisms remain tubules. Dextran diffusion measurements were also consistent with t-tubule re- unknown. In this study we employed fluorescent dextran trapping and diffu- opening. However, the time constant of apparent dextran diffusion in t-tubules sional assays to test the central hypothesis that the magnitude of t-tubule sealing was significantly larger (8.9 5 1.8 seconds) when compared to that in normal in response to osmotic stress is dependent on the magnitude of prior t-tubule cells (3.5 5 0.2 seconds, p < 0.05), suggesting that reopened t-tubules still constrictions. First, dextran trapping experiments showed that hyperosmotic retain some structural defects. Furthermore, the fluorescence of trapped dextran cell shrinking was sufficient to induce t-tubule sealing. However, cell shrinking remaining 15 minutes post-hyposmotic shock was significantly increased in the following hyposmotic swelling was strikingly more effective suggesting that presence of the microtubule depolymerizing agents 1 mM nocodazole (71 5 t-tubules become ‘primed’ for sealing by the preceding osmotic stress. Second, 5%) and 10 mM colchicine (67 5 4%) compared to control cells (43 5 3%, measurements of diffusional mobility of dextran in t-tubules showed that cell p < 0.001). Similarly, the mitochondrial uncoupler FCCP (25 mM) significantly swelling in hyposmotic solution (180 mOsm) for 7 minutes led to a 2-3 blunted the decline in fluorescence following hyposmotic stress. In conclusion, fold increase in the apparent diffusion time constant (td). The increase in cell the results of this study show that stress-induced sealing of t-tubules is a revers- width was only 1452%, arguing against a simple dependence of diffusion ible process involving important components of cellular cytoskeleton and rate on cell size. The increase in td was coincident with 20% reduction in mitochondria. peak fluorescence amplitude. Collectively, the data suggest that cell swelling leads to a gradual development of significant constriction of t-tubule lumens. 3073-Pos Board B281 Comparable Calcium Handling and Contractility in Human IPSC Cardi- Finally, the magnitude of the increase in td was dependent on the magnitude of osmotic stress and therefore, the extent of t-tubule constrictions could be omyocyte Models of Three Different Hypertrophic Cardiomyopathy- modulated. To test the central hypothesis, cells were first exposed to hypo- Linked Mutations 1 1 2 3 smotic solutions of varying osmolarity but washed out with solutions having Kyungsoo Kim , Lili Wang , Vasco Sequeira , Joseph C. Wu , Bjorn C. Knollmann1. 90 mOsm more osmolarity (constant difference). The data showed that, despite 1 a similar shrinking force upon removal of stress, dextran trapping was signifi- Vanderbilt Center for Arrhythmia Research and Therapeutics, Vanderbilt University Medical Center, Nashville, TN, USA, 2Laboratory for Physiology, cantly greater in cells that were swollen more, and therefore developed tighter 3 constrictions, prior to cell shrinking. Overall, the data support the hypothesis VU University Medical Center, Amsterdam, Netherlands, Stanford that the magnitude of t-tubule constrictions determines the propensity for t-tu- Cardiovascular Institute, Stanford University School of Medicine, Stanford, bules to undergo sealing upon stress. CA, USA. Familial hypertrophic cardiomyopathy(HCM) is caused by mutation of sarco- 3071-Pos Board B279 mere genes which is associated with an increased risk of sudden cardiac death The Role of NCX1 on the Maintenance of T-Tubule Architecture in due to arrhythmia. Sarcomere gene mutations typically disrupt the highly tuned Pressure-Overloaded Hearts balance between force generation and calcium(Ca) cycling dynamics. Here we Yoshihiro Ujihara1, Satomi Takatsu2, Keiji Naruse2, Satoshi Mohri1, investigated the relationship between Ca handling and contractility properties Yuki Katanosaka2. in human induced pluripotent stem cells(hiPSC) derived cardiomyocyte(CM) 1Kawasaki Medical School, Kurashiki, Japan, 2Okayama University, of three different HCM-linked mutations(Troponin T I79N: TnT-I79N, Okayama, Japan. Troponin T K280N: TnT-K280N, and Myosin heavy chain7 R663H: MHC7- The Naþ/Ca2þ exchanger 1 (NCX1) is an essential Ca2þ efflux system in the R663H). We generated hiPSC lines from healthy donors and two TnT sarcolemma and transverse (T)-tubules of cardiomyocytes. There is growing mutations(TnT-I79N and TnT-K280N) using CRISPR/Cas9. The Matrigel evidence that T-tubule disorganization is a causal event that shifts the transition mattress method was used to generate single rod-shaped cardiomyocytes and from hypertrophy to heart failure (HF). However, the detailed molecular mech- measured Ca transients and contractility at field-stimulation(0.2 Hz) in Fura- anisms have not been clarified. Here, we showed the pathophysiological role of 2, AM loaded iPSC-CMs. In response to the pacing stimulus, all HCM muta- NCX1 on pressure-overloaded hearts. We generated novel transgenic mice in tions significantly increased the percentage of cell shortening. The time to which NCX1 expression is controlled by a cardiac-specific, doxycycline peak of shortening was not changed, whereas relaxation time was significantly (DOX)-dependent promoter. In the absence of DOX, TAC surgery caused sub- prolonged in comparison to their isogenic controls. Although the properties of stantial chamber dilation with a gradual decrease in contractility by 16 weeks. force generation were similar in all HMC-linked mutations, their Ca handling Cardiomyocytes showed a decline in contractility with abnormal Ca2þ handling was quite different. In TnT-I79N and TnT-K280N mutations, despite the during E-C coupling. Reduced NCX1 activity was observed 8 weeks after TAC reduced or unchanged Ca amplitude, they exhibited both enhanced contractility and was still apparent at 17 weeks. Quantitative analysis of NCX1 distribution and delayed relaxation, indicative of increased sensitivity to Ca. However, in demonstrated that release of NCX1 from the T-tubule area occurred before the MHC7-R663H mutation, albeit consistent hypercontractility and impaired onset of T-tubule disorganization and junctophilin-2 (JP2) mislocalization. relaxation similar to other two HCM mutations, Ca amplitude as well as dia- Induced NCX1 overexpression by DOX treatment starting 8 weeks after stolic Ca significantly increased, which reflects the possibility of unchanged TAC returned NCX1 activity to pre-TAC levels and prevented chamber dila- sensitivity of the myofilaments to Ca. Although further studies need to focus tion with cardiac dysfunction. In DOX-treated myocytes, contractility, T-tubule on elucidation of how different Ca handling defect causes common HCM integrity and proper localization of JP2, synchrony of Ca2þ release from the phenotype in these HCM-related mutations, our findings robustly demonstrated SR, and Ca2þ handling during E-C coupling was preserved 16 weeks after key features of HCM(i.e., increased systolic and impaired diastolic function) in TAC surgery. In addition, DOX treatment attenuated the downregulation of various HCM-linked hiPSC-CM mutations using measurement of contractile survival signalling and upregulation of apoptosis signalling 16 weeks and cytosolic Ca.

BPJ 8731_8734 620a Wednesday, February 21, 2018

3074-Pos Board B282 3076-Pos Board B284 Mechanotransduction via No Signaling Auto-Regulates Cardiomyocyte Mechanical Load Effects on Cardiomyocyte Action Potential, Cacium Contractility Transient, and Contraction Revealed by using a Novel Patch-Clamp-in- Rafael Shimkunas1,2, Bence Hegyi2, Zhong Jian2, Zana Coulibaly2, Gel Technology John A. Shaw3, Nipavan Chiamvimonvat4, Kit S. Lam5, Leighton Izu2, Zhong Jian1, Yi-je Chen1, Bence Hegyi1, Tamas Banyasz1, Zana Coulibaly1, Ye Chen-Izu1,2. Rafael Shimkunas1, Nipavan Chiamvimonvat2, Kit S. Lam3, Leighton T. Izu1, 1Biomedical Engineering, University of California, Davis, Davis, CA, USA, Ye Chen-Izu1,4. 2Pharmacology, University of California, Davis, Davis, CA, USA, 1Pharmacology, University of California Davis, Davis, CA, USA, 2Internal 3Aerospace Engineering, University of Michigan, Ann Arbor, MI, USA, Medicine/Cardiology, University of California Davis, Davis, CA, USA, 4Internal Medicine/Cardiology, University of California, Davis, Davis, CA, 3Biochemistry and Molecular Medicine, University of California Davis, USA, 5Biochemistry and Molecular Medicine, University of California, Davis, CA, USA, 4Biomedical Engineering, University of California Davis, Davis, Davis, CA, USA. Davis, CA, USA. Background: The heart must adjust its contractile force to compensate for the Background: In every heartbeat, cardiomyocytes are under mechanical load blood pressure changes in daily life (due to changes in posture, physical activ- when the heart pumps blood against peripheral resistance. However, because ity, emotional state, etc.) in order to maintain adequate cardiac output. Here we previous patch-clamp experiments are mostly done using load-free cells, the investigate the mechano-chemo-transduction (MCT) mechanisms that trans- mechanical load effects on cells were precluded. Method: We have developed duce mechanical load to regulate cardiac Ca2þ signaling and contractility. an innovative Patch-Clamp-in-Gel technique which enables us to patch-clamp Methods: We used a Cell-in-Gel system to embed freshly isolated rabbit ven- cardiomyocytes while they are embedded in a viscoelastic 3-D hydrogel that tricular myocytes in a 3-D viscoelastic hydrogel. The cell in-gel was continu- imposes a controlled mechanical load on cells. Results: We performed ously perfused with Tyrode’s solution and electrically paced at 0.5 Hz. Self- Patch-Clamp-in-Gel experiments using mouse ventricular myocytes and control experiments were conducted by (1) studying the myocytes contracting discovered the following major results. (1) Compared to load-free cells, the my- in-gel under mechanical load, (2) then dissolving the gel to release the cell into ocytes in-gel under mechanical load show prolonged action potential (AP), Tyrode’s solution and (3) studying the cell contracting in solution load-free. early afterdepolarization (EAD), delayed afterdepolarization (DAD) and trig- Results: The cardiomyocytes contracting in-gel exhibited a larger cytosolic gered AP, suggesting that mechanical load significantly increases arrhythmo- Ca2þ transient than load-free cells (Fura-2 ratio 2.7150.07 vs 2.0850.04), genic AP activities. (2) Simultaneous triple-signal recordings of AP, Ca2þ, revealing the MCT effect on regulating intracellular Ca2þ. The SR Ca2þ con- and contraction further reveal that load-induced EAD occurs independently tent was higher and the fractional release of Ca2þ from SR was also larger when while load-induced DAD occurs in conjunction with spontaneous Ca2þ tide the cell was in-gel than load-free (measured with Fluo-5N), which should and contraction. (3) The above load-induced changes can be reversed by spe- contribute to increasing the cytosolic Ca2þ transient. Furthermore, the MCT ef- cific inhibition of the nitric oxide synthase 1 (NOS1 or nNOS), demonstrating fects on the SR Ca2þ and cytosolic Ca2þ were abolished by using 1 mM L- a critical role of nNOS in mediating the mechanotransduction pathway. NAME to inhibit nitric oxide synthases (NOS), suggesting a critical role of Conclusion: Our newly developed Patch-Clamp-in-Gel technology provides NOS-NO signaling in mediating MCT. The cell in-gel contraction was reduced a powerful tool to control mechanical load on single cells for studying mecha- from 13.550.4% in untreated cell to 10.750.6% after L-NAME treatment, notransduction effects on ion channels, action potential, Ca2þ signaling, and showing the MCT effect on enhancing contraction. Conclusion: Mechano- myocyte contraction. We found that mechanical load significantly affects the chemo-transduction in cardiomyocyte is mediated by NOS-NO signaling to three dynamic systems - electrical, Ca2þ signaling, and contractile systems - regulate the SR Ca2þ release to increase the cytosolic Ca2þ transient, which that govern cardiac function and arrhythmogenic activities, and nNOS-NO in turn enhances the contractility in compensatory response to increased signaling mediates the mechanotransduction in cardiomyocytes. load. This MCT mechanism contributes to the Anrep effect and is fundamental to cardiac regulation in health and disease. 3077-Pos Board B285 Super-Resolution (dSTORM) Imaging of Calcium Handling Proteins in 3075-Pos Board B283 Cardiomyocytes Viscoelastic Eshelby Analysis of the Cell-in-Gell System Ornella Manfra1,2, Xin Shen1,2, Johannes W. Hell3, John Shaw. William Edward Louch1,2. Aerospace Engineering, University of Michigan, Ann Arbor, MI, USA. 1Institute for Experimental Medical Research, Oslo University Hospital and Objective: The Cell-in-Gel system, where isolated live myocytes are University of Oslo, Oslo, Norway, 2K.G.Jebsen Center for Cardiac Research, embedded in a constraining hydrogel, is being used to study mechano- Oslo, Norway, 3College of Biological Sciences, University of California chemo-transduction (MCT) mechanisms. The aim is to better understand Davis, Davis, CA, USA. MCT mechanisms at the single cell level under various mechanical scenarios, In cardiomyocytes, excitation-contraction coupling is initiated at functional el- knowing excessive mechanical stress in myocardium leads to arrhythmias and ements called dyads, where L-type Ca2þ channels (LTTCs) within T-tubules heart failure. While cell contractions can be measured directly, the mechanical face ryanodine receptors (RyRs) in the sarcoplasmic reticulum. While this stress on the cell must be inferred by analysis. arrangement allows efficient triggering of Ca2þ-induced Ca2þ release, an un- Methods: Our previous model was based on the classical Eshelby inclusion quantified fraction of LTCCs are thought to be non-dyadic, and play important problem of linear elasticity. Here, the framework is extended to account for vis- roles in signaling and regulation of Ca2þ stores. In addition, RyR Ca2þ release cosities of gel and cell to provide more accurate calculations of time-varying may also be triggered by the Naþ-Ca2þ exchanger (NCX). However, such a stress fields present during Cell-in-Gel experiments. Hydrogels of various role requires close localization of the two proteins, which has been difficult cross-link densities are used during the experiments to systematically vary to ascertain in studies employing diffraction-limited microscopy. We presently the mechanical constraints on the cell. Gel viscoelastic properties are calibrated aimed to gain greater understanding of the arrangement of LTCCs, NCX, and by torsional rheometry measurements. RyRs within the cardiomyocyte by employing dSTORM (direct stochastic op- Results: Mechanical analyses, using the viscoelastic Eshelby model, allow tical reconstruction microscopy) super-resolution imaging, which enables parametric studies to be performed, establishing trends with Gel stiffness and quantification of proteins at nanometer-scale resolution (30nm). Dual- viscosity. For a purely elastic matrix, the stress within the cell is multi-axial colour 2D dSTORM of adult rat cardiomyocytes revealed that in the cell sur- and uniform, yet surface tractions are non-uniform. Without up-regulation of face only 27% of LTCCs and 24% of NCX were colocalized with RyRs. Simi- Ca2þ transients (related to the Anrep effect), a typical cardiomyocyte in a larly, only 20% of LTCCs and 28% of NCX were colocalized with RyRs within gel of similar stiffness can contract only 20% of its load-free fractional short- the cell interior. By comparison, confocal microscopy greatly overestimated the ening. The presence of Gel viscosity, however, adds a time lag between strains dyadic proportion of LTCCs and NCX in both regions of the cell. Of note, and stresses and causes an increase in mechanical power requirements for cell LTCCs and NCX were observed to occur in dyadic junctions with RyRs along contraction. both transverse and longitudinal-oriented T-tubules. These protein fractions Conclusions: The mechanical simulations provide baseline responses of the represent pools of LTCCs and NCX which are likely within close enough prox- myocyte during contraction in-gel. With preload or afterload, Cell-in-Gel imity of RyRs to trigger Ca2þ-induced Ca2þ release. Furthermore, such close data show enhancements in cell contractility beyond baseline predictions proximity of NCX and RyRs implies that Ca2þ extrusion occurs very near (and amplitude of Ca2þ transients) that are non-monotonic with increasing points of dyadic release. These results provide novel insight into the nanoscale gel cross-link density. Thus, our analyses can be used to quantitatively inform organization of cardiomyocyte Ca2þ handling proteins, providing more accu- ongoing MCT studies that link mechanical stress to cardiac function and re- rate assessment of the dyadic and non-dyadic pools of LTCCs and NCX than modeling. has previously been possible based on diffraction-limited imaging studies.

BPJ 8731_8734 Wednesday, February 21, 2018 621a

3078-Pos Board B286 Cardiac ryanodine receptor (RyR2) mediates the sarcoplasmic reticulum (SR) 3D dSTORM Imaging Reveals Disassembly of Ryanodine Receptor Clus- Ca2þ efflux that induces cardiomyocyte contraction, whereas sarcomeric ters in Failing Cardiomyocytes myosin binding protein-C (cMyBP-C) regulates actomyosin cross-bridge Xin Shen1,2, Jonas van den Brink3, Terje R. Kolstad1,2, Einar Norden1,2, cycling. We report the first evidence for direct interaction between these two Andy G. Edwards3, Michael Frisk1,2, Ivar Sjaastad1,2, Christian Soeller4, essential proteins using several complementary methods, suggesting existence William E. Louch1,2. of a RyR2:cMyBP-C complex. Our studies indicate the C-terminus of cMyBP- 1IEMR, Oslo University Hospital and University of Oslo, Oslo, Norway, C binds to the RyR2 N-terminus in mammalian cells with minimal requirement 2K.G. Jebsen Center for Cardiac Research, Oslo, Norway, 3Simula Research of a fibronectin-like type 3 domain. Importantly, we observe that specific bind- Laboratory, Oslo, Norway, 4Biomedical Physics, University of Exeter, ing between both recombinant cMyBP-C and RyR2 also occurs with the native Exeter, United Kingdom. proteins extracted from cardiac tissue. Co-expression of cMyBP-C and RyR2 in Excitation-contraction coupling in the heart critically depends on the function of HEK293 cells alters cellular Ca2þ dynamics, with reduced frequency of RyR2- ryanodine receptors (RyRs). While advances in single-molecule localization mi- mediated spontaneous Ca2þ oscillations, suggesting a potential inhibitory ef- croscopy (SMLM) have allowed 2D visualization of RyRs at the nanoscale level, fect of cMyBP-C on RyR2 Ca2þ release. Our discovery of direct, functional the precise cellular localisation of RyRs in 3D in both health and disease has yet cMyBP-C association with RyR2 provides novel evidence for putative linkage to be characterized. We presently utilised our recently developed 3D direct sto- between sarcomeric cMyBP-C regulation of myofilament contraction and SR- chastic optical reconstruction microscopy (3D dSTORM) method to quantify the mediated Ca2þ release via RyR2. A distinct mechanism for retrograde sarco- subcellular distribution of RyRs in normal and failing (post-infarction) rat cardi- mere regulation of SR RyR2 channels may exist, with significant relevance omyocytes, hypothesizing that breakup of RyR clusters may contribute to dysre- to cMyBP-C and RyR2 dysfunction observed in inherited cardiac disorders. gulated Ca2þ homeostasis during heart failure. We specifically examined the nanoscale arrangement of two subpopulations of RyRs: sarcolemma- 3081-Pos Board B289 associated surface clusters and transverse tubule-associated interior clusters. Us- Trafficking of Phospholamban and SERCA2a Follows the Nuclear ing 3D dSTORM, we observed a clear difference between surface and interior Envelope-to-SR Along T-Tubules (NEST) Pathway Common to Junctional RyR cluster size in normal cardiomyocytes (13.550.6 and 16.050.7 RyRs/ Sarcoplasmic Reticulum (SR) Proteins < Danning Wang1, Juyi Wan1, Steven E. Cala2, Zhenhui Chen1. cluster, respectively, p 0.05). In failing cardiomyocytes, both surface and inte- 1 2 rior RyR cluster sizes were significantly reduced when compared to normal cells Medicine, Indiana University, Indianapolis, IN, USA, Physiology, Wayne (9.750.7 and 10.550.6 RyRs/cluster, respectively, p<0.05). Interestingly, State University, Detroit, MI, USA. post-infarct non-failing cardiomyocytes only exhibited reduced cluster size The pathways and mechanisms by which phospholamban (PLB) and SERCA2a within the cell interior (10.250.6 RyRs/cluster, p<0.05 vs normal), suggesting proteins traffic to SR sarcomeres within cardiomyocytes (CMs) remain un- that RyR dispersion may initially occur at these sites. Ca2þ release units (CRUs), known. Applying a similar approach used to identify trafficking pathways for which are functional groups of RyR clusters responsible for the cooperative gen- the junctional SR proteins triadin and junctin, we analyzed confocal immunoflu- eration of Ca2þ sparks, contained significantly fewer RyRs in failing cells (sur- orescence images of adult rat CMs 24-72hrs post infection with adenoviruses en- face: 20.650.8 vs. 17.051.1 RyRs/CRU in failing, p<0.05; interior: 24.250.9 coding dog isoforms of PLB (dPLB) or dSERCA2a. Newly-made dPLB or vs. 18.351.0 RyRs/CRU in failing, p<0.05) and exhibited more dispersed orga- dSERCA2a proteins in CMs were specifically detected using species-specific nization. These alterations were paralleled by an observed slowing of Ca2þ spark monoclonal antibodies 1F1 or 3F1, respectively. At 24hrs, dSERCA2a (3F1 sig- kinetics and greater RyR Ca2þ leak in failing cells. Together, our results identify nals) were present in the nuclear envelope (NE), where discrete puncta also ap- disassembly of RyR clusters as a novel mechanism underlying impaired cardio- peared, likely reflecting rough ER translocons producing presorted sites of myocyte Ca2þ homeostasis in heart failure. localized biosynthesis. dSERCA2a protein distributed along transverse tracks emanating from translocons, with radially decreased levels. At the 3rd puncta 5 3079-Pos Board B287 away from the NE, 3F1 fluorescence intensity reduced to 52 12% (15 CMs, Super Resolution Imaging of Ryanodine Receptor Cluster Morphology in 4 rats) of that in the NE, indicating movement of dSERCA2a protein. In contrast, Rabbit and Human Atrial Myocytes dPLB, detected by 1F1, was distributed throughout the NE (16 CMs), without Daria Boyd, Antony Workman, Niall Macquaide. detectable transverse/radial trajectory even at 24hrs post infection. By 48- University of Glasgow, Glasgow, United Kingdom. 72hrs post infection, fluorescence signals for both dPLB and dSERCA2a became Ryanodine receptors (RyRs) are the main intracellular Ca2þ release channels in visible in puncta further away from the NE, but still aligned with T-tubules; a cardiac myocytes. Abnormalities in Ca2þ release are known to participate in the pattern previously found for junctin and triadin. Thus, newly-synthesized PLB formation of arrhythmias. Regulation of RyR by phosphorylation has been and SERCA2a travel along the same pathway used to deliver proteins to junc- extensively studied, but little data exists on the organisation and morphology tional SR. This unique protein trafficking pathway is termed the Nuclear of RyRs. Similarly, little is known about why atrial fibrillation (AF) is more Envelope-to-SR along T-tubules (NEST) pathway. After biosynthesis, but common in patients after myocardial infarction (MI). Using super-resolution before trafficking to SR, PLB is preferentially retained in the NE at levels greater microscopy (dSTORM), we examined RyR clusters in rabbit atrial myocytes than SERCA2a, consistent with our recent report of enhanced steady-state PLB from control/sham and MI animals, as well as in human atrial myocytes. localization to the NE in CMs. The NEST pathway may be a common secretory Surface clusters contained 2250.8 RyRs (3552.0 RyRs per calcium release pathway for cardiac SR protein trafficking. unit (CRU)) in control/sham rabbit myocytes (Nanimals=7, ncells=37). This was 3082-Pos Board B290 significantly reduced (p<0.0001) to 1650.9RyRs per cluster (2451.3RyRs Statins Bind to Cardiac Ryanodine Receptor (RyR2) Channels to Alter per CRU) in MI myocytes (Nanimals=4, ncells=34). In MI, surface RyR clusters Opening Frequency were closer together with nearest neighbour distances of 380520nm in Abigail D. Wilson1, Chris Lindsay1,2, Elisa Venturi1, Angela J. Russell1,2, 5 control/sham (Nanimals=7, ncells=37) vs 347 14nm in MI myocytes (Nanimals=4, Rebecca Sitsapesan1. < 1 ncells=34) (p 0.05). We observed no differences in internal cluster and CRU Department of Pharmacology, University of Oxford, Oxford, United size and nearest neighbour distance between control/sham and MI animals. Kingdom, 2Chemistry Research Laboratory, University of Oxford, Oxford, Finally, we found that both surface and internal cluster and CRU size in human United Kingdom. myocytes were comparable to rabbit, with 2550.9RyRs (4152.1RyRs per A significant proportion of statin users are predisposed to Ca2þ-dependent ar- 5 5 CRU) in surface clusters (Nsubjects=4, ncells=39) and 30 2.2RyRs (41 2.7 rhythmias and sudden cardiac death. Clinical trials indicate that statin therapy RyRs per CRU) in internal clusters (Nsubjects=4, ncells=11). RyR clusters in hu- reduces the incidence of arrhythmias but the underlying mechanisms are not 5 man myocytes were found to be 417 27nm apart in surface RyRs (Nsubjects=4, understood. We therefore investigated if widely-used statins could affect car- 5 2þ ncells=39) and 540 66nm apart in internal RyRs (Nsubjects=4, ncells=11). All diac ryanodine receptor Ca -release channel (RyR2) function by incorpo- data are represented as Mean5S.E.M., respectively. rating sheep cardiac sarcoplasmic reticulum (SR) vesicles or purified RyR2 channels into planar phospholipid bilayers under voltage-clamp conditions as 3080-Pos Board B288 previously described (Sitsapesan & Williams (1994) Biophysical Journal Association of Cardiac Myosin Binding Protein-C with the Ryanodine 67:1484-1494). Concentrations of statin (<1 mM) that would be expected to D Receptor-Ca2 Release Channel: Putative Retrograde Regulation? accumulate within the tissues of patients, significantly reduced native RyR2 Paulina Stanczyk1, Monika Seidel2, Judith White1, Cedric Viero3, channel open probability (Po). For example, Po was 0.1850.06 and Chris George2, Spyros Zissimopoulos2, F. Anthony Lai4. 0.0550.02 (SEM; n=8-11; p<0.05), before and after addition of 1 mM cyto- 1Cardiff University, Cardiff, United Kingdom, 2Cardiff & Swansea solic simvastatin respectively. In contrast, statin concentrations above 20 Universities, Cardiff, United Kingdom, 3Cardiff & Saarland Universities, mM, markedly increased Po. RyR2 channels are normally tightly associated Homburg/Saar, Germany, 4Cardiff & Qatar Universities, Doha, Qatar. with many other proteins to which statins could bind. To determine if statins

BPJ 8731_8734 622a Wednesday, February 21, 2018 directly interact with RyR2, we purified RyR2 before reconstituting into bila- 3085-Pos Board B293 D yers. As with native channels, significant increases in Po were observed at Molecular Basis for Ca2 Binding of RyR2 for Channel Activation and high concentrations; atorvastatin increased Po from 0.1750.06 to 0.5850.11 Diseases States (10 mM) and 0.6850.13 (50 mM), (SEM; n=6; p<0.05) indicating that the Takashi Murayama1, Haruo Ogawa2, Nagomi Kurebayashi1, statin binding site is located on RyR2 rather than on an associated protein. Takashi Sakurai1. We suggest that inhibition of RyR2 channel opening by statins may provide 1Department of Pharmacology, Juntedno University School of Medicine, benefit to those patients with increased risk of sudden cardiac death by reducing Tokyo, Japan, 2Institute of Molecular and Cellular Biosciences, The the opportunity for unregulated diastolic SR Ca2þ-leak. Univesity of Tokyo, Tokyo, Japan. þ Funded by the BHF and Oxford BHF CRE. Type 2 ryanodine receptor (RyR2) is a Ca2 release channel in the sarcoplasmic reticulum of cardiac muscle and indispensable to muscle contraction. Although þ þ 3083-Pos Board B291 the RyR2 channel is activated by Ca2 , the actual mechanism of Ca2 binding þ Phosphorylation of the Type 2 Ryanodine Receptors Plays a Role in the remains largely unknown. Here, we report molecular basis for Ca2 binding Organization of their Array of RyR2 for channel activation and its implications in disease states. RyR2 car- þ Parisa Asghari1, David R.L. Scriven1, Hector Valdivia2, Xander Wehrens3, rying mutations in the putative Ca2 and caffeine-binding sites were functionally Edwin D.W. Moore1. analyzed. The results were interpreted by recent near-atomic resolution RyR1 1Univercity of British Columbia, Vancouver, BC, Canada, 2University of structures at various ligand states. We demonstrated that a hydrophobic interac- Michigan, Ann Arbor, MI, USA, 3Baylor College of Medicine, Houston, TX, tion between tryptophan and isoleucine in the caffeine-binding site makes the þ þ USA. Ca2 binding pocket larger and less favorable for Ca2 . Caffeine alters orienta- Various small molecules and proteins are tightly docked to cardiac ryanodine tion of the tryptophan to break the interaction, making the pocket smaller and þ receptors (RyR2) to provide a sophisticated and coordinated mechanism to more favorable for Ca2 . The tryptophan then forms another hydrophobic inter- regulate channel function; Ca2þ release. Recently we discovered that RyR2 tet- action with a phenylalanine that locates opposite side of the tryptophan, which þ ramers are mobile and can interact with their neighbours in multiple ways. Us- also contributes to Ca2 sensitizing effect of caffeine. Thus, the tryptophan res- ing correlation microscopy (line scan confocal imaging of Ca2þ sparks and idue in the caffeine-binding site switches two hydrophobic interactions to regu- þ dual-tilt electron tomography), we discovered that such interactions are highly late the Ca2 sensitivity. We identified three CPVT mutations that break the þ dynamic in response to physiological factors. Importantly, our data indicated a hydrophobic interaction to hypersensitize the channel to Ca2 . Our results eluci- þ correlation between RyR2 Ca2þ spark frequency and the tetramers’ relative po- dated the initial step of activation of the RyR2 channel by Ca2 and explain how sitions: a side-by-side arrangement had the lowest spark frequency and a check- mutations cause disease states at molecular level. erboard the highest. Currently, RyR2 phosphorylation is considered one of the main physiological factors contributing to Ca2þ leak in heart failure. We have Posters: Cardiac, Smooth and Skeletal Muscle shown that activating phosphorylation kinases and inhibiting protein Electrophysiology II phosphatase-1 (PP1) and protein phosphatase-2A (PP2A) can rearrange 2þ RyR2 tetramers and increase Ca spark frequency. To investigate whether 3086-Pos Board B294 phosphorylation of RyR2 itself is sufficient to rearrange the tetramers; we Self-Organization of Functional Coupling between Membrane and Calcium have studied the following available transgenic mice with mutations of Clock in Arrested Human Sinoatrial Nodal Cells in Response to Camp S2030A, S2808A, dual S2808A/S2030A, S2814A and S2814D. Hearts were Kenta Tsutsui1, Oliver Monfredi1, Syevda Sirenko1, Rostialav Bychkov1, either fixed and examined with dual-tilt electron tomography or exposed to Larissa A. Maltseva1, Mary S. Kim1, Bruce D. Ziman1, Kirill V. Tarasov1, 300 nM isoproterenol (b-adrenergic receptor stimulus) for 2 minutes prior to Mingyi Wang1, Alexander V. Maltsev1, Jaclyn A. Brennan2, Igor R. Efimov2, fixation and examination. Our observations indicate that: 1) S2808 residue Michael D. Stern1, Victor A. Maltsev1, Edward G. Lakatta1. has no effect on the tetramer’s distribution and its further phosphorylation is 1The Laboratory of Cardiovascular Science, The National Institute on Aging, not required for a full response to b-adrenergic receptor stimulation, 2) Baltimore, MD, USA, 2Department of Biomedical Engineering, George S2030 and S2814 residues may interact allosterically to regulate tetramers po- Washington University, Washington DC, DC, USA. sition. These results confirmed our hypothesis that the phosphorylation of rya- Normal automaticity of sinoatrial nodal cells (SANC) in animals involves the nodine receptors themselves plays a role in their reorganization. operation of ‘coupled-clock system’: an ensemble of electrogenic surface mem- brane molecules (‘membrane-clock’), and sarcoplasmic reticulum generating 3084-Pos Board B292 spontaneous rhythmic local Ca releases (LCRs - ‘Ca-clock’). We determined Binding and Regulation of the Cardiac Ryanodine Receptor by PKA and whether a coupled-clock system is involved in the operation and failure of normal CaMKII automaticity of single SANC isolated from human hearts devoid of major cardiac Omid Haji-Ghassemi. diseases. LCRs were observed in all human SANC studied (n=18). In SANC that The University of British Columbia, Vancouver, BC, Canada. failed to generate spontaneous action potentials (AP - arrested SANC), mem- Ryanodine Receptors (RyRs) are the largest known (2.2 MDa) ion channels, 2þ brane potential (Vm) hovered around - 40 mV and LCRs were small and disorga- controlling the release of Ca from the sarcoplasmic and endoplasmic reticula. nized. In response to beta-adrenergic receptor stimulation (bARs), LCRs in A previously solved high-resolution crystal structure of a tandem repeat domain arrested SANC self-organized to become synchronized in time and space, and contains multiple phosphorylation sites for kinases PKA and CaMKII, but the concurrent rhythmic de novo Vm oscillations emerged, followed by generation mechanism through which phosphorylation alters channel activity is not under- of bona-fide APs from a maximum diastolic potential near 60 mV. As a slow stood. Aberrant phosphorylation has been linked to a multitude of disorders, AP firing transitioned to a faster rate, LCR size increased, and LCR period short- affecting channel gating in both skeletal muscle (RyR1) and cardiac (RyR2) ened, partially synchronizing LCRs to late diastole. Changes in the LCR period isoforms. Within the RyR2 phosphorylation domain, residues Ser2808 and over the time course of the bARs response were highly correlated with changes in Ser2814 have received extensive attention as targets for PKA and CaMKII. AP firing rate (r2=0.98). In response to cAMP, LCRs in permeabilized human Docking into cryo-electron microscopy maps suggests a location in the turret SANC self-organized in a manner similar to that in response to bARs in intact region, implying that mutations and phosphorylation may affect the allosteric SANC. When bARs was terminated, LCRs again became small and dysrhythmic, motions within this area. However, the cryo-EM density in this area is poorly Vm depolarized to around 40mV, and APs ceased. Thus, human SANC have a resolved, so other methods are required to look at the precise impact of functional Ca-clock that couples to the membrane-clock to generate spontaneous phosphorylation. APs; these clocks become extremely uncoupled in AP arrested SANC; and clock Here we present NMR, ITC, and X-ray crystallographic data on the influence of uncoupling can be rescued by a cAMP-dependent mechanism that involves an PKA and CaMKII binding to RyR2 as well as the effects of phosphomimetics increased synchronization and rhythmicity of LCRs and hyperpolarization suffi- (S2808D, S2814D) on the structure and dynamics of RyR2 repeat domain. In cient to initiate SANC spontaneous APs. agreement with previous mass-spectrometry investigation, multiple sites can be phosphorylated by both kinases. Most of these are located directly within 3087-Pos Board B295 a flexible loop, but another site is located on a different face of the domain. Positive Feedback Mechanisms among Local Ca Releases, NCX, & ICAL Importantly, both S2808D and S2814D show different fingerprints on the Ignite Pacemaker Action Potentials spectra, suggesting that they may have different functional impacts on the Victor A. Maltsev1, Alexey E. Lyashkov1, Joachim Behar2, channel. Finally, we show the preference of PKA for S2808 via ITC studies Edward G. Lakatta1, Yael Yaniv2. and are currently investigating the interplay between PKA and CaMKII where 1Laboratory of Cardiovascular Science, National Institute on Aging, NIH, we test the impact of prior incubation with one kinase on the activity/affinity of Baltimore, MD, USA, 2Biomedical Engineering Faculty, Technion-IIT, the other. Haifa, Israel.

BPJ 8731_8734 Wednesday, February 21, 2018 623a

Recent data suggest that cardiac pacemaker cell function is determined by Background: Synapse-associated protein 97 (Sap97), a membrane associated numerous time-, voltage-, and Ca-dependent interactions of cell membrane elec- guanylate kinase like (MAGUK) protein that clusters ion channels, receptors trogenic proteins (M-clock) and intracellular Ca cycling proteins (Ca-clock), and enzymes in macromolecular complexes in variety of cell types. In this forming a coupled-clock system. Many aspects of the coupled-clock system, study, we have examined the role of Sap97 in the expression and regulation however, remain unknown. The key players of the system are Ca-release chan- of ion channels and beta-adrenergic receptors (B-ARs) in pacemaker cells of nels (RyR), generating local Ca-releases (LCRs) from sarcoplasmic reticulum, the murine sinus node. Methods and Results: A murine model of cardiac tar- Na/Ca exchanger current (INCX), and L-type Ca current (ICaL). We combined nu- geted Sap97 down regulation was generated. Electrocardiographic analyses on merical model simulations with experimental simultaneous recordings of action wildtype (WT) and Sap97 knockdown (Sap97KD) animals showed a variety potentials and Ca to gain further insight into the complex interactions within the abnormalities indicative of irregular excitation in atria and ventricles. system. Numerical simulations revealed a new positive feedback mechanism, Following isoproterenol (ISO) challenge, WT mice showed an increase in heart dubbed ‘‘ignition’’, which accelerates the diastolic depolarization (DD) to reach rate (HR) that returned to baseline with a biphasic profile; an initial rapid excitation threshold. The ignition phase begins when LCRs start to occur and decline, and a longer lasting decline to baseline (20 mins). Sap97KD animals INCX reverses its trend from decreasing to increasing. The increasing inward showed a higher initial HR increase, and a more prolonged decline to base line, INCX together with funny current and T-type Ca current accelerates DD, bringing which remained at 30% above baseline. Similar characteristics were observed the membrane potential to ICaL activation threshold. During the ignition phase, in animals under anesthesia (N=3,3; p<0.05), or in conscious animals (N=4,4; ICaL–mediated Ca influx generates more LCRs via Ca-induced-Ca-release that p<0.05). Analyses were carried out on cells/tissue samples isolated from the activates more INCX, creating a positive feedback. Simultaneous recordings of sinus node region. In Sap97KD animals, compared to WT, Sap97 protein membrane potential and confocal Ca images support the model prediction of was reduced by 86% (0.7þ/- 0.1 vs. 0.1þ/- 0.05 (p=0.008). Moreover, there the positive feedback among LCRs and ICaL, as diastolic LCRs begin to occur was a 133% increase in b1-AR protein (0.3þ/- 0.1 vs. 0.7þ/- 0.1; p=0.003), an below the voltage range of ICaL activation and continue within the activation increase in pCaMKII, but no changes in PKA. Sap97KD did not alter Cav1.2 range. The ignition begins when DD starts to accelerate (0.15 V/s) notably protein levels, or density of L-type calcium current, but significantly blunted above the recording noise. Moreover, the timing of the ignition onset closely current sensitivity to ISO. In contrast, the main protein component underlying predicted the duration of each cycle in the basal state, in the presence of auto- the pacemaker current (IF; HCN4) was downregulated. Conclusion: The mu- nomic receptor stimulation, and in response to specific inhibition of either the rine model of cardiac-targeted Sap97 downregulation is important for under- M-clock or Ca-clock, indicating general importance of the new coupling mech- standing the role of Sap97 in generating and regulating the electrical impulse anism for regulation of the pacemaker cell cycle duration. in the mammalian sinus node. 3088-Pos Board B296 3090-Pos Board B298 Combined Effects of Gap Junctional and Ephaptic Coupling Therapies on L-Type Calcium Channel Gating Modifiers as a New Class of Antiar- Conduction and Arrhythmogenesis during Ischemia/Reperfusion rhythmic Drugs Gregory S. Hoeker1, Carissa C. James1, Sarah H. Barrett2, James W. Smyth1, Marina Angelini1, Arash Pezhouman2, Marvin G. Chang3, Steven Poelzing1. Nicoletta Savalli1, Guillaume Calmettes2, Antonios Pantazis1, 1Virginia Tech Carilion Research Institute, Roanoke, VA, USA, 2Biomedical Hrayr S. Karagueuzian2, James N. Weiss4,5, Riccardo Olcese1,5. Sciences and Pathobiology, Virginia Polytechnic Institute and State 1 Anesthesiology & Perioperative Medicine, UCLA, Los Angeles, CA, USA, University, Blacksburg, VA, USA. 2 Cardiovascular Research Laboratory, UCLA, Los Angeles, CA, USA, Background: Cardiac ischemia leads to abnormal conduction associated with a 3 Anesthesia and Critical Care, Harvard Medical School, Massachusetts loss in gap junction (GJ) coupling (GJc). Despite increasing GJ conductance, 4 General Hospital, Boston, MA, USA, Medicine (Cardiology), UCLA, Los Rotigaptide has shown limited efficacy in patients with heart disease. Elevating 5 þ 2þ Angeles, CA, USA, Physiology, UCLA, Los Angeles, CA, USA. perfusate sodium (Na ) and calcium (Ca ) can promote normal conduction 2þ Pharmacological treatment of cardiac arrhythmias includes the use of Ca velocity (CV) by enhancing ephaptic coupling (EpC). EpC relies on the gener- channel blockers (Class IV antiarrhythmics), that while suppressing arrhyth- ation of extracellular electric fields in the perinexus, an extracellular GJ adja- mias, also block peak I , compromising EC coupling and contractility. To cent restricted cleft. It is unknown whether enhancing both EpC and GJc Ca,L circumvent these limitations, we are studying and testing the properties of synergistically improves conduction and prevents arrhythmias during L-type Ca channel gating modifiers, rather than blockers, to selectively reduce ischemia/reperfusion. Methods and Results: Langendorff-perfused guinea pig the non-inactivating component (late I ), leaving peak I minimally hearts pretreated with Rotigaptide were optically mapped using our historic Ca,L Ca,L þ þ þ affected, thus preserving cardiac contractility. This strategy is based on our lab standard (HLS) solution (146mM [Na ], 1.25mM [Ca2 ]), elevated Na þ þ þ þ þ discovery that the selective reduction of late I potently suppress early after- (155mM [Na ], 1.25mM [Ca2 ]), or elevated Na /Ca2 (155mM [Na ], Ca,L þ depolarizations (EADs) of the cardiac action potential (AP), recognized 2.0mM [Ca2 ]). At baseline, perfusate composition did not change CV. In cellular triggers of arrhythmias (Madhvani et al., 2015). We previously re- contrast, Rotigaptide significantly increased CV with HSL, but not other per- þ ported that roscovitine, a purine analog known to reduce late I , potently fusates. During 30 minutes of no-flow ischemia, elevated Na and elevated Ca,L þ þ þ suppressed H O -mediated EADs in rabbit ventricular myocytes. We are eval- Na /Ca2 delayed the time to conduction block. Importantly, elevated Na / 2 2 þ uating the robustness of this pharmacological intervention in a new model of Ca2 significantly attenuated ischemia-induced CV slowing and ischemia- EAD favoring condition using 600mMHO þ1-2mM Isoproterenol. H O related perinexal expansion (a negative correlate of EpC). Perfusate composi- 2 2 2 2 alone (n=7) caused EADs in 83.255.39% APs and 85.757.83% APs in pres- tion did not affect interstitial volume or total/phosphorylated connexin43 ence of H O þIsoproterenol (n=5); the perfusion of roscovitine (20 mM) effi- expression. Interestingly, Rotigaptide only prolonged the time to block with 2 2 ciently suppressed EAD occurrence restoring normal AP duration in all HLS. Reperfusion led to frequent ventricular fibrillation (VF) for all interven- myocytes tested. This antiarrhythmic effect was achieved without significantly tions, but Rotigaptide reduced VF duration with HLS. In contrast, no additional perturbing calcium transients and cell shortening, indicating maintenance of arrhythmia reduction was observed when Rotigaptide was combined with þ þ normal EC coupling. We also tested roscovitine ability to suppress EAD- elevated [Ca2 ] and/or [Na ]. Conclusions: During ischemia, elevating þ þ triggered ventricular fibrillation (VF) in isolated-perfused aged rat and rabbit perfusate Na and/or Ca2 promotes normal conduction despite reduced hearts. We found that roscovitine suppressed VF induced by H O (8/8 rat GJc, but this effect is not enhanced with Rotigaptide. Importantly, reports of 2 2 hearts, average time: 1352.8 min) and H O þIsoproterenol (4/4 rabbit hearts, enhanced conduction with Rotigaptide during ischemia/reperfusion may be 2 2 average time: 2157 min), restoring sinus rhythm. Roscovitine had no signifi- dependent on choice of perfusate, suggesting that future studies should consider cant effect on the rate-pressure product (RPP) as a measure of myocardial these effects in the context of underlying ionic fluid composition. workload in isolated rat hearts (n=5). We propose that these findings establish 3089-Pos Board B297 roscovitine as a pilot compound to drive the development of a new class of Sap97 Expression is Important for Heart Rate Control in the Murine antiarrhythmics based on Ca channel gating modification. Sinus Node Todd Herron1, Brad Rosinski1, Roberto Ramos Mondragon1, Hassan Musa2, 3091-Pos Board B299 Kamel Aoun1, Lakshmi Mundada1, Steven Whitesall3, Yan Chen1, Defining the Limits of Stem-Cell Derived Cardiomyocytes (SC-CMS) to Nulang Wang1, Guadalupe Guerrero-Serna1, Hector Valdivia1, Detect Cardiac Proarrhythmic Liabilities Justus M. Anumonwo1. Carlos A. Obejero-Paz, Leslie Ellison, James Kramer, 1Internal Medicine/Cardiovascular, University of Michigan, Ann Arbor, MI, Andrew Bruening-Wright. USA, 2Internal Medicine/Cardiovascular, Ohio State University, Columbus, Safety Assessment, Charles River Laboratories, Cleveland, OH, USA. OH, USA, 3Molecular and Integrative Physiology, University of Michigan, Extracellular Field Potential (FP) and impedance recordings of SC-CMs are Ann Arbor, MI, USA. currently used to detect drug induced cardiac arrhythmia liabilities. Major

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endpoints include the field potential duration (FPD), the duration of the imped- concentration – dependent inhibition of ICa. Using current clamp, both paced ance twitch, and the presence of proarrhythmic markers including early and de- (1.5 nA; 1 ms) and spontaneous action potentials (APs) could be elicited. layed afterdepolarizations and fibrillations. We used the cardioECR instrument Here, tetracaine, an inhibitor of INa, and nifedipine caused shortening of the (ACEA Biosciences) and iCell2 cardiomyocytes (Cellular Dynamics Interna- AP while E-4031 that inhibits the hERG channel, prolonged the duration of APs. tional) to 1) identify the FP parameter that best captures the intrinsic Many APC systems rely on the use of fluoride in the internal solution to gain frequency-dependence of repolarization and, 2) assess the extent the system sufficiently high seal resistances. However, fluoride was shown to have many can detect the mitigating effect of drugs with multiple ion channels effects unwanted side effects that might affect the recordings. Comparison of a phys- (MICE) on proarrhythmic activity. Cardiac repolarization in FP recordings is iological and a fluoride – based internal solution. revealed significantly lower encapsulated by one positive (T3) and two negative (T1, T2) peaks. We detected ICa in the presence of fluoride compared with the physiological solution which a linear relationship with comparable slopes of 0.133 and 0.129 respectively was further manifested in a significantly shorter AP duration. (n=2) between T2 and T3 durations and beat period in the spontaneous frequency range. T1 shows a shallower slope (0.031) suggesting that the parameter does not 3094-Pos Board B302 follow closely the repolarization process and consistent with its limited response A New Analysis Pipeline to Improve Assessment of Cardiac Liability in to drugs that prolong the FPD. MICE effects were investigated by assessing the High throughput Electrophysiology Screens with Routine MOA Detection effect of different combinations of pure hERG (E-4031) and hCav1.2 (nifedi- for Slow Onset Compounds 1 2 1 2 pine) channel blockers designed to simulate distinct IC50 hERG/IC50 Stephan Steigele , Ana L. Teixeira , Martin Ginkel , Verity A. Talbot , 3 2 4 hCav1.2 ratios (HCR). The functional relationship between the number of wells Lisa J. McWilliams , Matt Bridgland-Taylor , Leigh Foster , 1 showing proarrhythmic markers and log (HCR) was fitted to a logistic function Stephan Heyse . 1Genedata, Basel, Switzerland, 2AstraZeneca, Cambridge, United Kingdom, with an inflection point at 0.148 and a HCR of 0.71. Notably, this ratio is com- 3 4 parable to the 0.89 cutoff for torsadogenic drugs in the clinic (Kramer, Obejero- AstraZeneca, Cambridge, United Kingdom, Genedata, San Francisco, CA, Paz et al., 2013). SC-CMs are useful tools to investigate the cardiac risks of drugs USA. by using T2 and T3 to evaluate changes in cardiac repolarization and HCR values Automated patch clamp screens generate highly valuable information for drug (% 0.71) to determine proarrhythmic activity. research programs. In particular, in safety pharmacology early testing is nowadays seen as a cornerstone for managing risk of later failure and patients’ health. Thus, 3092-Pos Board B300 more predictive and affordable screens need to be introduced to gain both speci- Action Potential Triangulation and Instability in TnT-I79N Human IPSC- ficity and throughput e.g. for cardiovascular liability detection and resolution. At CMS AstraZeneca, we have implemented the Nanion Syncropatch 384PE electrophys- Lili Wang, Bjorn C. Knollmann. iology platform, delivering higher throughput measurements for hERG and other Vanderbilt University Medical Center, Nashville, TN, USA. key cardiac ion channels in standard 384 well-based format at dramatically Cardiac troponin T (TnT) I79N mutation causes familial hypertrophic cardiomy- reduced consumable costs and experiment time, whilst capturing full kinetics of opathy. Previous studies showed that I79N mutation increases myofilament Ca channel response. Jointly with Genedata we developed a pipeline for processing sensitivity, which contributes to the development of cardiac arrhythmias. Howev- and analysing this complex data, reading binary raw data directly from the Syncro- er, the arrhythmia mechanism remains elusive. Here, we studied the effect of I79N Patch instrument intoGenedata Screener, then assigning and integrating the sweep mutation on action potential (AP) in human induced pluripotent stem cell-derived recordings from cumulative compound addition series and finally subjecting them cardiomyocytes (hiPSC-CMs). APs were recorded in current clamp mode without to scientists’ review using powerful filtering and masking based on quality control any exogenous Ca buffers added to the intracellular solutions. There was no dif- measurements. We further developed methods for normalising the cumulative ference in resting membrane potential, peak amplitude and time-to-peak between curve recordings compensated for signal variation using time-match control and I79N and control CMs. I79N mutation significantly shortened early repolarization for automatically detecting ‘‘slow onset’’ compounds using a sigmoidal fit model (APD30, APD50 and APD70), whereas late repolarization (APD90) did not per concentration step, flagging those compounds which potency against critical change. As a result, APs from I79N CMs exhibited triangulation (APD(90-30)/ cardiac channels has been potentially under-estimated due to their slow onset. 90: I79N 0.3550.03 vs. control 0.2850.01, P<0.01) and beat-to-beat instability This investment in a next-generation automated electrophysiology platform (Average ratio of interquartile range to median APD50: I79N 0.1250.02 vs. con- and an appropriate automated data processing pipeline is resulting in more pre- trol 0.0750.01, P<0.05) compared to control. The AP triangulation and insta- dictive and affordable safety screens at high scalability and rapid turnover for bility were reproduced with 3 mM Ca-sensitizing agent EMD in control CMs. discovery projects across AstraZeneca. Pretreatment with 3 mM Ca-desensitizer blebbistatin or excess cytosolic buffering (by adding 14 mM EGTA to intracellular solution) prevented AP triangulation in 3095-Pos Board B303 I79N CMs. How could the I79N mutation cause AP triangulation in CMs? In ven- Correlation of IKr Block with Action Potential Changes in Human Stem tricular CMs, Ca extrusion via Na-Ca exchanger (NCX) generates inward current Cell Derived Cardiomyocytes 1 1 1 that contributes to repolarization. Since the I79N mutation increases myofilament Brian K. Panama , Mark W. Nowak , Sanjot Singh , Randall L. Rasmusson2, Glenna C.L. Bett2. Ca buffering and causes the smaller Ca transients, NCX currents could be reduced 1 2 and thereby shorten the early repolarization. To test this hypothesis, NCX- Cytocybernetics, Buffalo, NY, USA, Jacobs School of Medicine and mediated Ca extrusion was blocked by replacing extracellular Naþ with Liþ. Biomedical Sciences, SUNY, Buffalo, NY, USA. NCX block with Liþ eliminated the AP differences between I79N and control Drugs that prolong the cardiac action potential (AP) often act through block of CMs. Taken together, our results indicate that I79N mutation shortens the early IKr, the native current mediated by HERG. HERG block in heterologous expres- repolarization of AP via a NCX-mediated mechanism, resulting in AP triangula- sion systems (e.g., HEK cells) is a primary assay for determining FDA safety tion and instability, both of which would increase arrhythmia risk. NIH- screening. However, the relationship between IKr block and AP prolongation is R01HL71670. complex and may vary from cell to cell depending on ion channel expression. Additionally, use dependence may differ significantly in heterologous expression 3093-Pos Board B301 systems. We used cardiomyocytes derived from human pluripotent stem cells Induced Pluripotent Stem Cell-Derived Cardiomyocytes (COR.4U) Char- (hiPSCDCMs) (Cellular Dynamics, WI) to measure drug effects on IKr and the acterized on an Automated Planar Patch Clamp System (QPATCH HT) cardiac AP in the same cell. Cells were cultured according to the manufacturer’s Daniel R.P. Sauter1, Kazuya Tsurudome2, Rasmus Jacobsen3, directions and used 7-25 days after plating. Standard electrophysiology voltage Goeran Mattsson3. clamp (whole-cell rupture or Amphotericin B perforated patch), was used. 1 2 Sophion Bioscience, Woburn, MA, USA, Sophion Bioscience, Tokyo, hiPSCDCMs lack endogenous IK1, so virtual IK1 was added via a Cybercyte Sys- 3 Japan, Sophion Bioscience, Ballerup, Denmark. tem (Cytocybernetics, NY) to obtain stable electrical behavior. IKr was recorded Human- inducedpluripotent stem cell - derived cardiomyocytes(hiPSC – CM) are using a 4 s voltage step from a holding potential of 80 mV to þ30 mV (P1) fol- providing new, highly predictive strategies to assess cardiotoxicity in vitro and can lowed by a single voltage step to 50 mV (P2) for 6 s (30 s inter-sweep interval). thus reduce costs for cardiac safety assessment in drug development. Different Following IKr measurements, cardiac APs were triggered by a 1.5-2.0 nA stim- technologies are available to assess compound effects on cardiomyocytes, out ulus for 1.5 ms at 0.5 Hz. These IKr and AP measurement protocols were then of all, the patch clamp technique allows to both investigate effects on action poten- repeated with dofetilide or loratadine. Dofetilide (300 nM) completely blocked tials in the current clamp mode and on isolated currents using the voltage clamp IKr and loratidine (6 mM) blocked IKr 3651%. After observing IKr block, both technique. In this study, we used the automated patch clamp (APC) set-up QPatch dofetilide and loratadine prolonged the cardiac AP. These results demonstrate HT (Sophion) to characterize hiPSC – CM (Cor.4U; Ncardia). a direct correlation between IKr block and effects on the cardiac AP. In accor- þ 2þ Using voltage clamp, voltage-gated Na (INa)andCa (ICa) currents dance with CiPA, this approach may provide a more robust and comprehensive were isolated. Application of the ICa inhibitor, nifedipine, resulted in a cardiac safety screen as compared to the standard HERG assay.

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3096-Pos Board B304 121 5 13 ms, n=5; P<0.0004). Acute H2O2 challenge abolished or reversed A Fast Optical Ion Channel Assay for Assessing Action Potentials in Hu- the apicobasal APD gradient and prolonged both apical and basal APDs man Induced Pluripotent Stem Cell Cardiomyocytes (apex 352 5 86 < base 401 5 37 ms, n=4; NS). However, H2O2 remodeling Stephen S. Smith, Thomas Lila, Jay Trautman, Andrew Blatz. impact was regionally heterogeneous, affecting the base more than apex. H2O2 Photoswitch Biosciences, Menlo Park, CA, USA. impaired repolarization and induced occasional early after depolarizations and Recent advances in large scale production have made induced pluripotent stem trigger activity. Given that the apicobasal APD and repolarization gradients in cells (iPSCs) differentiated into cardiomyocytes feasible for use in drug discov- normal zebrafish are reverse those in normal humans, one should exert caution ery and safety pharmacology screening efforts. Recently developed partial or when extrapolating drug-induced QT prolongation in zebrafish to humans. fully optical assays have suffered from either low bandwidth, or low Nonetheless, based on the human-like arrhythmogenic response of the zebrafish throughput. We have developed a new high-throughput cardiac excitability heart to acute H2O2 challenge, zebrafish constitutes a relevant model to inves- assay suitable for use in multiwell assay plates using photodynamic genetically tigate repolarization disorders associated with oxidative stress. encoded actuators to control cellular membrane potential and ‘‘molecular wire’’-based fluorescent voltage sensors for high-bandwidth membrane poten- 3099-Pos Board B307 tial monitoring. Here we present data from iPSC cardiomyoctyes (CDI icells) Ring Finger Protein 207 Degrades T613M Kv11.1 Channel 1 2 1 1 showing nicely resolved spontaneous activity. When known modifiers of the Hannah A. Ledford , Seojin Park , Padmini Sirish , Aiyana M. Emigh , 1 1 3 3 cardiac action potential are introduced, such as the hERG-blocking compound Wilson Xu , Valeriy Timofeyev , James R. Priest , Marco V. Perez , 3 1 1 E-4031, the expected dose-dependent lengthening of the AP is seen, as well at Euan A. Ashley , Vladimir Yarov-Yarovoy , Xiao-Dong Zhang , 1 higher concentrations secondary depolarizations, reminiscent of early after de- Nipavan Chiamvimonvat . 1University of California, Davis, Davis, CA, USA, 2University of Nevada, polarizations (EADs) are evident. When an optogenetic actuator is introduced, 3 the cells respond accordingly to short pacing light pulses. Reno, Reno, NV, USA, Stanford University, Stanford, CA, USA. Introduction: 3097-Pos Board B305 Human ether-a`-go-go related gene (hERG, Kv11.1) channel underlies cardiac þ High-throughput Study of Rabbit Ventricle Action Potential Populations rapidly activating delayed rectifier K current (IKr); hERG mutations in MI Model contribute to Long QT Syndrome (LQTS). Additionally, reduced repolarization Quentin Lachaud, Niall MacQuaide, Francis Burton, Godfrey Smith. reserve can arise from not only ion channel mutations, but also mutations in ion University of Glasgow, Glasgow, United Kingdom. channel interacting proteins. RING Finger Protein 207 (RNF207) has been Background Electrical activity in isolated cardiomyocytes has been widely identified as a potential modifier for Kv11.1 function, cardiac excitation, and studied using patch clamping, which offers limited throughput. Using voltage- QT interval. Recent studies suggested a link between hERG and RNF207 mu- and calcium-sensitive dyes, the electrical activity, calcium dynamics and tations in LQTS onset and severity, specifically T613M hERG (hERGT613M) contractility profile of cells can be measured simultaneously in approximately and RNF207 frameshift mutation (RNF207G603fs). However, exact molecular 100 cells per heart. This feature provides further insight into the mechanisms mechanisms remain unknown. We hypothesize that RNF207G603fs fails to of excitation-contraction coupling in the failing heart. Moreover, this high- degrade hERGT613M, allowing a higher population of membrane-bound throughput methodology allows the study of large cell populations in individual hERGT613M mutant subunits, thus decreasing functional IKr. hearts, offering a quantitative measure of cellular and regional heterogeneity, Methods and Results: otherwise obscured by animal variation and undersampling. Methods Adult rab- We confirmed ubiquitinylation activity in wild-type RNF207 (RNF207WT). bit cardiomyocytes were isolated from the left ventricle of infarcted (MI), control Confocal and super-resolution microscopy in murine and rabbit ventricular car- (Sham), and stock rabbits, loaded with the fluorescent, voltage-sensitive dye diomyocytes confirmed co-localization of hERG with RNF207. Functional an- Ò FluoVolt (1:6000), and plated on to 35mm glass-bottomed plates (Krebs solu- alyses revealed that the trafficking-defective hERGT613M failed to produce tion at 37 C; 1.8mM CaCl2). Left ventricle cells were serially paced at 1Hz, 2Hz currents. Wild-type hERG (hERGWT) subunits only partially rescued and 3Hz using field stimulation (2x20mm graphite electrodes; 40V, 2ms pulse hERGT613M with reduced IKr and faster activation and inactivation kinetics. width). Separately isolated cells originating from either epicardium or endocar- RNF207WT co-expression with hERGWT and hERGT613M restored IKr, whereas dium were paced at 2Hz. A 10s action potential train was recorded from each RNF207G603fs failed to rescue IKr. Co-immunoprecipitation of hERGWT pulled cell. Results The methodology corroborates previously published work showing down RNF207WT, but not RNF207G603fs. Further analysis using cytoplasmic N- an increase in mean APD90 in MI at slow rates. Mean APD90 in MI (Nanimals=6, and C-terminal hERGWT deletions (hERGWT-DN, hERGWT-DC) revealed that 5 5 5 ncells=450-800) was 374 11ms, 287 4ms, and 208 4ms at 1Hz, 2Hz and RNF207WT pulled down hERGWT-DN, but not hERGWT-DC. Additionally, 3Hz, respectively. Mean APD90 in Sham was 336516ms, 25256ms, time-dependent degradation assays demonstrated increased hERGT613M degra- 20651ms, at 1Hz, 2Hz, and 3Hz, respectively (Mean5S.E.M). Regional ana- dation by RNF207WT, but not RNF207G603fs, via proteasomal degradation. lyses (Nanimals=9, ncells=554) found AP90 was shortened in the epicardium vs. We further tested changes to RNF207 structure using Rosetta molecular the endocardium in stock (2156ms difference) and sham animals modeling software. (37510ms), while this difference was entirely lost in MI (P=0.323). Moreover, Conclusion: epicardial cell populations showed consistently higher variability than endocar- RNF207WT functions as an E3 ubiquitin ligase for hERG channels. dial cells, across all experimental groups. This technique consequently provides RNF207G603fs, however, failed to degrade hERGT613M and rescue IKr density, accurate quantification of biological variation in individual hearts and heart re- suggesting that direct protein-protein interaction is necessary for observed gions, suggesting a strong potential for drug screening assays. rescue of current. Our data suggest that normal RNF207 function is critical for hERG quality control and, consequently, cardiac repolarization. 3098-Pos Board B306 Oxidative Stress Remodeling of Zebrafish Cardiac Electrical Gradients 3100-Pos Board B308 Nicholas A. James, Ashraf R. Beshay, Eileen Chang, Thao P. Nguyen. Transient Outward K Current Defines Ca Dynamics on Intact Mouse Medicine/Cardiology, UCLA, Los Angeles, CA, USA. Hearts In mammalian hearts, electrical gradients ensure well-orchestrated electrical Micaela Lopez Alarcon1, Juan I. Felice2, Emilino Medei1, Ariel L. Escobar3. stability and mechanical efficiency under physiological baseline. Regional het- 1Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de erogeneities of ion channels and/or calcium handling proteins establish gradi- Janeiro, Rio de Janeiro, Brazil, 2CIC, Universidad Nacional de La Plata - ents of activation, action potential duration (APD), and repolarization that Conicet, La Plata, Argentina, 3Engineering, UC Merced, Merced, CA, USA. are protective at baseline, but susceptible to disruptive remodeling in myocar- In the heart, a Ca2þ influx through L-type Ca2þ channels triggers Ca2þ release dial injuries and failure. However, in zebrafish hearts, physiological electrical from the sarcoplasmic reticulum (SR). In vertebrates, this influx occurs during gradients are unexplored. Considering the important application of zebrafish the ventricular action potential plateau phase (ph2). However, in murine hearts in high-throughput screening for drug-induced cardiotoxicities (such models, this happens on the early repolarizing phase 1 (ph1). The aim of this as QT prolongation) and the role of the apicobasal APD and repolarization gra- work is to assess if changes in the open probability of Kþ channels defining dients in the inscription of the T wave and the QT interval, this study investi- ph1 (Ito) can modulate both Ca2þ currents and Ca2þ release from the SR during gates the apicobasal electrical gradients in normal adult zebrafish and the the ventricular AP. Pulsed local field microscopy (PLFFM), loose patch photol- arrhythmogenic impact of acute H2O2-induced oxidative stress. Following ysis (LPP) and mathematical modeling were used to test the hypothesis that a epicardial optical mapping of intact zebrafish hearts, apicobasal gradients of decrease in Ito will enhance Ca2þ influx and SR Ca2þ release. A mouse myo- activation, repolarization, and APD under normal baseline vs. oxidative stress cyte mathematical model predicted that a decrease in the ph1 repolarization rate 2þ 2þ (H2O2, 100 mmol/L) condition were determined. At baseline, an apicobasal promotes an increase in the amplitude of the L-type Ca current, the SR Ca 2þ 2þ APD90 gradient was evident with longer APD90 in the apex due to earlier acti- load and in the gain of the Ca induced Ca release process. This theoretical vation and later repolarization compared to the base (apex 198 5 23 > base prediction was experimentally evaluated using LPP. Indeed, increasing

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concentrations of 4 aminopyridines (4-AP) slowed down ph1 repolarization and Cardiac excitation-contraction (E-C) coupling depends on Ca influx (ICa) 2þ 2þ increased Ca influx through L-type Ca channels. Furthermore, Ito activa- through L-type Ca channels (LTCC) during the action potential. ICa activates tion with NS-5806 reduced the amplitude of Ca2þ currents. In addition, simul- ryanodine receptors in the adjacent sarcoplasmic reticulum (SR) membrane taneous recordings of Ca2þ transients and APs by PLFFM showed that a to produce the Ca transient required for contraction. Caveolin-3 (Cav-3) is a reduction in the ph1 repolarization rate produced an increase in the amplitude scaffolding protein, unique to muscle, that is implicated in t-tubule formation of Ca2þ transients and that an increase in the repolarization rate promoted by and in the predominant t-tubular localization of LTCC and its regulatory part- NS-5806 led to a reduction of SR Ca2þ release. Finally, the 4-AP effect on ners. Cav-3 expression has been shown to be reduced in failing hearts and acute 2þ AP ph1 was significantly smaller when the L-type Ca current was partially inhibition of protein binding to Cav-3 has been shown to decrease ICa density at blocked with nifedipine. This indicates that not only ph1 repolarization rate the t-tubules, while global knock-out (KO) of Cav-3 impacts skeletal and car- regulates cardiac contractility, but also that the rate of repolarization is defined diac myopathies. Taken together, these data suggest that loss of Cav-3 may be by the competition between an outward Kþ current and an inward Ca2þ current. involved in the heart failure phenotype. We examined in vivo cardiac function, and the structure and function of ventricular cells obtained from hearts of 3 mo 3101-Pos Board B309 old Cav-3 KO mice and wild-type (WT) mice that had undergone transverse Fast Sodium Currents in Rat Atrial and Ventricular Myocytes aortic constriction (TAC) for 8 weeks. Cav-3 KO exhibited some of the features Rachel E. Caves, Stephanie C.M. Choisy, Simon M. Bryant, Jules C. Hancox, of heart failure observed following TAC: reduced in vivo ejection fraction, Andrew F. James. cellular hypertrophy, reduced ICa density at the t-tubules and disrupted t-tubule School of Physiology, Pharmacology & Neuroscience, University of Bristol, structure. However, cells from Cav-3 KO mice did not show any changes in SR Bristol, United Kingdom. Ca release, suggesting that decreased Cav-3 expression contributes to, but is not Activation of voltage-gated sodium channels (VGSC) underlies the generation the only cause of, the changes observed in heart failure. This work was sup- and propagation of cardiac action potentials while their subsequent inactivation ported by the British Heart Foundation. establishes a refractory period. Blockers of VGSC are highly effective as antiar- rhythmic drugs in the treatment of atrial fibrillation. However, their use is limited 3104-Pos Board B312 because ventricular arrhythmias are a common side-effect. Atrial-ventricular dif- Cardiac Action Potential Propagation through Compact Fibroblasts in 3D ferences in VGSC might be exploited to develop atrial-selective antiarrhythmic Cardiac Microtissues Engineered from Self-Assembled Spheroids as action. In this study, atrial-ventricular differences in the steady-state voltage- Building Blocks 1 1,2 1 dependent activation and inactivation of fast VGSC currents (INa) were examined. Tae Yun Kim , Celinda M. Kofron , Michelle King , 1,3 1,4 5 INa was recorded from rat left atrial (n=11) and left ventricular myocytes (n=11) at Alexander R. Markes , Anawon O. Okundaye , Zhilin Qu , room temperature using the whole-cell patch clamp technique. Recording solu- Ulrike Mende1, Bum-Rak Choi1. tions contained low equimolar sodium (5 mM) and Co2þ (1 mM) was used to 1Cardiovascular Research Center, Cardiovascular Institute, Rhode Island block calcium currents. Voltage protocols had a holding potential of 120 mV. Hospital and Alpert Medical School of Brown University, Providence, RI, 2 INa was recorded in response to a range of test pulses to investigate activation USA, Center for Biomedical Engineering, School of Engineering, Brown and at 30 mV following a range of conditioning pulses to investigate steady- University, Providence, RI, USA, 3Division of Biology and Medicine, Brown state inactivation. Currentswere normalized to whole-cell capacitance as an index University, Providence, RI, USA, 4Department of Molecular Pharmacology, of cell size and mean current-density voltage relations fitted by a modified Boltz- Physiology and Biotechnology, Brown University, Providence, RI, USA, mann relation. There was no significant difference between atrial and ventricular 5Department of Medicine, University of California, , Los Angeles, Los cells in current density-voltage relations: half-maximal voltages (V1/2) of activa- Angeles, CA, USA. tion were atrial, 47.851.5 mV, and ventricular, 46.851.1 mV. On the other Cardiac fibroblasts (CFs) may form electrical coupling with cardiac myocytes hand, the V1/2 of inactivation for atrial and ventricular myocytes were 93.450.5 (CMs) and alter conduction to facilitate arrhythmias. To investigate electrical mV and 87.450.3 mV, respectively. Therefore, INa inactivation occurred at coupling and conduction characteristics through compact region of fibroblasts more negative voltages in rat atrial myocytes compared to ventricular myocytes. in 3D environment, we designed elongated microtissues from individual scaffold-free spheroids comprised of CMs or CFs as building blocks. Fusion anal- 3102-Pos Board B310 ysis of CM-CF spheroid pairs showed that CFs migrated into CM spheroids and Selective Chloride Ion Sensing Microelectrodes using a Boron Cluster accelerated fusion kinetics (DL/Dt: 8.7350.65 vs. 2.8050.46 mm/hr in CM- Ionophore CM pairs), suggesting that CMs and CFs favor a cohesive and intermixed distri- Rafal M. Dziedzic1, Marino DiFranco2, Stephen C. Cannon2, bution characteristic for the healthy myocardium. To determine whether the Alexander M. Spokoyny1. observed fusion leads to functional electrical coupling, we constructed elongated 1 Chemistry and Biochemistry, University of California, Los Angeles, Los 3D microtissues from five spheroids building blocks (CM-CM-Z-CM-CM where 2 Angeles, CA, USA, Department of Physiology, David Geffen School of Z = CM or CF spheroids) and assessed AP propagation using optical mapping. Medicine, University of California, Los Angeles, Los Angeles, CA, USA. Most of microtissues with Z=CF (n=26/35) showed discontinuous AP propaga- Chloride ions play an important role in regulating cell volume and membrane po- tion through the center CF spheroid but with significant conduction delay - tential. Measuring chloride ion concentrations, [Cl ], and gradients in cells is (113.7530.8 vs. 5.951.4 ms when Z=CM). CF spheroids express Naþ channels crucial to studying the function of chloride ion channels in cell membranes. (Nav1.2 and Nav1.5) based on qPCR analysis, and computer simulation repli- Both single- and double-barrelled chloride-selective microelectrodes have been cated discontinuous conduction in CF volume when INa was added to CFs (5% used to measure intracellular chloride concentration. However, the utility of cur- of CM INa), emphasizing an important role of INa in CFs in potentiating AP prop- rent chloride ion sensing electrodes is limited due to interference by other physi- agation. In addition, computer simulation of CF migration into CM spheroids that - ologically present anions (e.g. HCO3 ). Previously, the organometallic [9] we experimentally observed revealed potentiated conduction delay via discontin- mercuracarborand-3 (MC-3) ionophore was used in PVC-based membrane elec- uous conduction in the boundary between CMs and CFs. In summary, this study - trodes which exhibited high selectivity for Cl over other physiologically present provides evidence for electrical coupling between CMs and CFs in 3D microtis- ions. Here, we report the use of MC-3 ionophore in Cl-selective liquid ion micro- sues to allow discontinuous AP propagation through compact CF volumes. The - electrodes for Cl sensing. The microelectrodes exhibit a Nerstian response over a suggested roles of Naþ channel expression in CFs and migration of CFs in poten- - wide Cl concentration range (1 mM - 150 mM) in physiologically relevant con- tiating the delayed AP propagation have implications for arrhythmogenesis in - ditions. Importantly, the microelectrodes are insensitive to HCO3 , along with compact fibrosis and border zone such as in myocardial infarction. other biologically relevant anions, which allow accurate measurement of intracel- lular Cl- concentration. A stable measure of intracellular [Cl-] was obtained from 3105-Pos Board B313 dissociated muscle fibers (FBD), by computing the difference of the ISE voltage Cardiomyocyte ZO-1 Regulates Intercalated Disc Organization and Whole minus the resting potential recorded with a second reference microelectrode. Heart Physiology Wenli Dai, Le Shen, Rangarajan Nadadur, Kaitlyn Shen, Margaret Gadek, 3103-Pos Board B311 Michael Broman, Ivan Moskowitz, Christopher Weber. Comparison of Cardiac Cell Function in Caveolin-3 Knock-Out and TAC University of Chicago, Chicago, IL, USA. Mice Intercalated discs (IDs) in the heart provide both electrical and mechanical Cherrie H. Kong1, Simon M. Bryant1, Hanne C. Gadeberg1, Judy J. Watson1, coupling between cells. Abnormalities in ID proteins are associated with ar- David M. Roth2, Hemal H. Patel2, Andrew F. James1, Mark B. Cannell1, rhythmogenic cardiomyopathy, however the regulation of the ID and the role Clive H. Orchard1. of ID proteins in heart physiology is still poorly understood. Tight junction pro- 1 Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, teins such as ZO-1 (Tjp1) are present and can bind to multiple ID proteins. 2 United Kingdom, Anesthesiology, University of California San Diego, San However, its contributions to ID organization and heart function remain largely Diego, CA, USA. undefined. We aim to understand if and how the tight junction protein ZO-1

BPJ 8731_8734 Wednesday, February 21, 2018 627a contributes to ID structure and heart physiology. We find ZO-1 expression was lium. Moreover, aldosterone treatment impaired vascular relaxation to acetylcho- decreased in patients with paroxysmal atrial fibrillation (8159% of control, line. Patch-clamp studies of isolated VSMCs from LADCAs incubated 24-h with P=0.03) by qPCR, suggesting ZO-1 expression change is associated with hu- aldosterone showed higher Ca2þ current density without change in voltage- man disease. We then generated an inducible cardiomyocyte specific ZO-1 dependent properties of the current. This is associated with increased Cav1.2 pro- knockout mouse (ZO-1 KO) was by crossing Tjp1 floxed mice with myosin tein levels related to a MR-dependent increased expression of ‘cardiac’ Cav1.2- heavy chain 6-CreERT2 mice. 8-12 week old mice were injected with tamoxifen LNT mRNA after aldosterone-treatment, whereas, Cav1.2-SNT expression was to induce Tjp1 gene deletion. Immunofluorescent staining showed decreased unaffected. Similar results were obtained on rat aorta and mesenteric arteries. Coxsackie and adenovirus receptor (CAR) (5652% decrease), and connexin These results were further corroborated in hypertensive aldosterone-salt rodent 43 (6753% decrease) distribution at IDs but no decrease of desmosome and models, notably both ex and in vivo in transgenic mice harboring the luciferase adherens junction proteins was observed. Transmission and immunogold elec- reporter gene under the control of the rat ‘cardiac’ P1-promoter. This alternative tron microscopy showed loss of gap junction structures. Surface ECG showed promoter usage switch led to reduced calcium channel antagonist sensitivity. ZO-1 KO caused increased PR interval (28.0 vs. 45.3 ms) and intra-cardiac These data support aldosterone regulation of ‘cardiac’ Cav1.2-LNT as a mech- electrogram showed increased atrial-His interval (24.551.1 vs. 69.553.0 anism underlying alterations in myogenic tone associated with hypertension. ms) and Wenckebach time (68.9520.1 vs. 131.4515.1 ms), without affecting This transcript switch might participate in the beneficial effect of MR antago- His-ventricular interval. ZO-1 KO mice developed 3rd degree heart block 2 nists in resistant hypertension. weeks after ZO-1 deletion. Loss of ZO-1 in adult heart results in altered ID pro- tein composition, heart block, and heart failure. We propose a model in which 3108-Pos Board B316 HAEM Modulation of Arterial Smooth Muscle Cell Large-Conductance ZO-1 stabilizes connexins and CAR to allow efficient cell-cell communication, 2D D and loss of ZO-1 contributes to the pathogenesis of heart disease. Ca -Activated K (BK) Channel Activity Modupe Ayeni. 3106-Pos Board B314 University of Leicester, Leicester, United Kingdom. A Four Microelectrode Method to Study Intracellular Ion Concentration Several hemolytic diseases are associated with vasospasm. Haem release during and Transport in Skeletal Muscle Fibers hemolysis can build up to toxic levels. Under normal conditions haem is Judith A. Heiny1, Stephen C. Cannon2, Marino Di Franco2. degraded by haem oxygenase to carbon monoxide (CO), bilverdin and Fe2þ. 1 Pharmacology and Systems Physiology, University of Cincinnati, The mechanisms causing vasospasm are poorly understood. Arterial smooth 2 Cincinnati, OH, USA, Physiology, UCLA, Los Angeles, CA, USA. muscle cells (SMCs) express BK channels which produce spontaneous transient Steady state myoplasmic ion concentration and slow ion transport across the outward currents (STOCs) when activated by Ca2þ sparks. STOCs hyperpolar- sarcolemma can be quantitatively studied by means of ion sensing electrodes ize SMCs resulting in cell relaxation and vasodilation. Previous studies show (ISE). ISE’s readout represents the sum of the electrochemical potential of the that intracellular haem inhibits whereas CO enhances BK channel activity in ion of interest and the resting membrane potential (Vm). Vm is measured either inside-out patches. We hypothesize that haem interaction with BK channels by using an independent standard microelectrode or a double-barreled capillary. might contribute to the vasospasm. Therefore, the current study aims to investi- These approaches have known limitations. Ion transport and membrane potential gate the mechanism of action of haem by examining the effects of extracellular are intricately connected, the resting potential may be unstable or be far from the haem and CO on whole-cell BK currents. SMCs were isolated from the mesen- desired value, ion transport can be electrically silent, and membrane currents teric artery of male Wistar rats. Depolarizing pulse-induced BK currents and may be carried by more than one ion. Thus, an electrophysiological method STOCs were recorded using whole-cell patch techniques. CO was applied using able to simultaneously measure the concentration of more than one ion, and to CO-releasing molecule 3 (CORM-3) which was prepared before each use. Extra- measure and control Vm and membrane current (Im) is desirable for physiolog- cellular haem (5 mM) increased pulse-induced BK currents and STOC ical and pathophysiological studies. For skeletal fibers, a triple-barreled elec- amplitudes by 2.3 5 0.5 (p%0.01, n=9) and 1.28 5 0.09 (p%0.05, n=5) times trode has been used in the past allowing for current injection to modify Vm respectively. Extracellular CORM-3 (30 mM) had no significant effect under current clamp conditions. Here we describe a novel four-microelectrode on pulse induced BK currents (n=8) but doubled STOC frequency system composed of a two-microelectrode voltage-clamp amplifier (TEV- (2.06 5 0.61, p%0.05, n=6) without affecting STOC amplitude (n=6). In the 200A, Dagan) and a two channel high impedance electrometer (FD223a, presence of the HO-inhibitor, zinc protoporphyrin-IX, haem increased STOC WPI). This approach allows for potentiometric measurements of the concentra- amplitude (1.5 5 0.04, p%0.001, n=4) and frequency (2.4 5 0.5, p%0.05, n=4). tion and transport of up to two ions in short murine skeletal muscle fibers sub- In summary, our results suggest that the stimulatory effect of haem on STOCs jected to either current- or voltage-clamp conditions. Ion translocation by has a component that is independent of its degradation product, CO. Further- primary and secondary active transport mechanisms, ion exchangers or passive more, other intracellular factors could be contributing to the positive haem ion diffusion can be studied. The system performance is exemplified by using effects. Information gained from further studies could improve our understand- Na, K and H selective electrodes made with commercially available ionophores ing of the changes in vascular tone that occur during haemolytic diseases. and fibers enzymatically dissociated from the flexor digitorum brevis muscles. 3107-Pos Board B315 Posters: Muscle Regulation Cardiac CaV1.2 Signature Induced by Mineralocorticoid in Vessels Debora Falco´n Boyano1, Thassio R. Mesquita1,2, Rogelio Salazar-Enciso3, 3109-Pos Board B317 Hussein Kobeissy4, Angelica Rueda3, Natalia Lo´pez-Andres5, Bioengineering and Characterization of Troponin Peptides for use as Ana Maria Go´mez Go´mez1, Jean-Pierre Benitah1. Therapeutic Reagents to Modulate Muscle Contractility 1UMRS 1180, INSERM, Chaˆtenay-Malabry, France, 2Department of Sienna Wong, Hanzhong Feng, Jian-Ping Jin. Physiology, Federal University of Sergipe, Sergipe, Brazil, 3Departamento de Wayne State University, Detroit, MI, USA. Bioquı´mica, Centro de Investigacio´n y de Estudios Avanzados del Instituto Mounting evidence has indicated that the highly conserved C-terminal end Politecnico Nacional, Mexico City, Mexico, 4EA 4043 UBaPS, Univ. Paris- segment of troponin subunit I (TnI) plays an inhibitory function in regulating Sud, Universite Paris-Saclay, Chaˆtenay-Malabry, France, 5Cardiovascular muscle relaxation (Zhang et al., FEBS J 278:3348 2011). Conformational mod- Translational Research, Navarrabiomed (Miguel Servet Foundation), ulations of troponin subunit T (TnT) restores an epitope structure that cross- Instituto de Investigacio´n Sanitaria de Navarra (IdiSNA), Pamplona, Spain. reacts to the C-terminal end segment of TnI (Chong & Jin, J Mol Evol 68:448, Aldosterone and its cognate mineralocorticoid receptor (MR) are key players in 2009), possibly related to adaptive tuning down of contractile velocity (Feng et the regulation of blood pressure. Part of the vascular effects of aldosterone is al., J Physiol 586:3537, 2008). Preliminary data also showed that this C-terminal 2þ due to the regulation of expression of L-type Cav1.2 Ca channels, the main epitope of TnI is recapitulated in the C-terminal end segment of TnT upon Ca2þ entry pathway in vascular smooth muscle cells (VSMC) regulating mul- removal of the evolutionarily added modulatory structures. Here we developed tiple vascular functions and implicated in the pathogenesis of hypertension. a novel expression and purification system using metal ion affinity tag together Cav1.2 channels are expressed as two distinct tissue-specific transcripts of with the small ubiquitin-like modifier (SUMO) fusion strategy to effectively ex- Cacna1c, which encode, a long ‘cardiac’ (Cav1.2-LNT) and a short press and purify the C-terminal end 27 amino acids of cardiac TnI (cTnI-C27) and ‘vascular/brain’ (Cav1.2-SNT) N-terminal region, regulated by two alternative the C-terminal end 15 amino acids of cardiac TnT (cTnT-C15). Preliminary data promoters (P1 and P2). In the present study, we analyze the molecular mecha- have shown that the TnI C-terminal epitope is well preserved in cTnI-C27 when nisms by which aldosterone, through MR, modulates Cav1.2 expression and isolated from the TnI backbone. With a refined multi-purpose expression vector function in VSMCs exposed 24-h to 108 M aldosterone. and a rapid throughput purification and cleavage protocol, cTnI-C27 and cTnT- The contractile responses of left anterior descending coronary arteries (LADCAs) C15 peptides are prepared in large quantity for conformational verification, to the Cav1.2 channel agonist BayK8644 or KCl depolarization were significantly biochemical binding study and functional characterizations on skinned muscle increased after aldosterone treatment, independently of the presence of endothe- fibers for effects on modulating force production and calcium sensitivity. The

BPJ 8731_8734 628a Wednesday, February 21, 2018 results are providing data to determine the potential use for these engineered tiation in culture by using advanced myogenic protocols and altering physical troponin peptides as therapeutic reagents for modulating muscle contractility. environments to promote in vitro myogenesis. In addition to biomarkers of early By either blocking or competing with the sites on endogenous troponin, these myogenesis, the expression of muscle fiber type-specific isoforms of myofila- peptides may provide drug candidates for the treatment of heart failure. ment proteins is monitored to assess the progression of differentiation of ANM iPSC in comparison with normal iPSC for the effect of slow TnT defi- 3110-Pos Board B318 ciency on the differentiation of type I slow muscle fibers. We are also testing a Influence of Residue-77 on Flexibility of an Extensive Portion of Tropomy- novel FRET-actin based tension reporting system to examine force production osin Tpm1.1 (Alpha) at the sarcomere level and assess the differentiation of iPSC-derived myotubes Anthonydura Madhushika M. Silva, David H. Heeley. and impact of the ANM mutation. In parallel, we employed the well- Biochemistry, Memorial University, St. John’s, NL, Canada. characterized C2C12 myoblast line to validate and refine the experimental con- The conformational flexibility of Tpm 1.1, the major tropomyosin isoform in ditions for myogenic differentiation and functional studies. These investigations striated muscle, was investigated by reversing the only charge substitution in provide novel information to understand the pathogenesis of ANM and other the sequences of the protein from Atlantic salmon and the more thermally stable TNNT1 myopathies for the development of therapeutic approaches. mammalian homologue: Thr-77 in salmon, Lys in rabbit. Salmon Tpms were iso- lated without exposure to heat or organic solvent. Intact proteins and CNBr frag- 3113-Pos Board B321 ments thereof were analyzed by electrophoresis, proteolytic digestion, Edman- Gm7325 Transcription Is Regulated by MyoD in Activated Muscle Satellite sequencing, calorimetry, F-actin binding and bioinformatics (PyMOL). The mu- Cells tation (Thr-77 to Lys) is verified by an electrophoretic shift in the presence of Daisuke Takei1, Miyuki Nishi1,2, So-ichiro Fukada3, Masao Doi1, either SDS or urea. Two unfolding transitions are observed in calorimetry (0.1 Hitoshi Okamura1, Akiyoshi Uezumi4, Lidan Zhang3, Morikatsu Yoshida5, M KCl, 1 mM DTT; pH 7.0), the mid-points (Tms) of which are 5 oC higher Mikiya Miyazato5, Atsuhiko Ichimura1,2, Hiroshi Takeshima1. 1 for mutant-77 than the recombinant control, 30 vs. 35 (minor) and 39 vs. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 2 44 oC (major). Unfolding of fragment CNBr (CN1A, residues 11-127) is mono- Japan, Center for the Promotion of Interdisciplinary Education and 3 phasic, Tms 45 (mutant-77) vs. 40 oC (control). Compared to the control, mutant- Research, Kyoto University, Kyoto, Japan, Graduate School of 4 77 (0.1M NaCl, pH 8.5; Temp 10-30 oC) displays a marked resistance to chymo- Pharmaceutical Sciences, Osaka University, Osaka, Japan, Tokyo 5 tryptic digestion at peptide bonds 11 and 169. The main cleavage site of CN1A Metropolitan Institute of Gerontology, Tokyo, Japan, National Cerebral and (between residues 88 and 89) is also more resistant in the case of the mutant. Wild Cardiovascular Center Research Institute, Osaka, Japan. type salmon Tpm 1.1 displays temperature-sensitive affinity for F-actin - tighter The Gm7325 gene product, namely Myomixer, Minion or Myomerger, was at 4 oC than 30 oC. PyMOL indicates that Lys-77 and Glu-82 in the adjacent chain recently identified as a small protein expressed in active myoblasts by three in- are within ion pairing distance. Loss of ionic-interactions in salmon Tpm1.1 is dependent research groups. The reported observations indicate that Gm7325 concluded to destabilize a-more-than 150 amino acid section of the molecule protein, together with Myomaker, contributes to cell fusion to promote myo- and to be a key factor in providing sufficient flexibility for optimum interaction tube formation. In the present study, to clarify Gm7325-expression profiles dur- in a cold marine environment. ing muscle-fiber regeneration, mouse skeletal muscles were injured by cardiotoxin injection and were subjected to RT-PCR and RNAscope in situ hy- 3111-Pos Board B319 bridization. RT-PCR analysis revealed that in injured skeletal muscle tissues, Troponin Bridges and Stretch Activation in Insect Flight Muscle transcription of Gm7325 is remarkably upregulated, even one day after the Demetris Koutalianos, Kate English, Belinda Bullard. injury. RNAscope in situ hybridization showed positive signals for Gm7325 Biology, University of York, York, United Kingdom. mRNA in activated satellite cells and immature myotubes, while no obvious The indirect flight muscles (IFM) of many insects contract asynchronously. Re- signals were detected in quiescent satellite cells and mature myofibers. In the petitive contractions occur when the muscle is stretched periodically at a con- 5’-flanking regions of mouse Gm7325 and its human homologue, conserved stant sub-maximal level of calcium. The alternate activation of opposing E-box motifs for basic helix-loop-helix (bHLH) transcription factors are repeat- muscles can produce high frequency wing beats. The rapid response to stretch edly arranged around the putative promoter regions. One of the bHLH tran- seen in insects with high wing beat frequencies suggests a sensor that is primed scription factors, MyoD, regulates myogenesis as a master switch. To for action. Troponin bridges between thick and thin filaments are possible stretch demonstrate the causal link between MyoD and Gm7325 transcription, we per- sensors, which may directly pull tropomyosin from an inhibitory positon on thin formed reporter gene assays, suggesting that MyoD activates Gm7325 expres- filaments (Perz-Edwards et al, 2011). We have isolated the tropomyosin- sion via the conserved E-box motifs. Therefore, Gm7325, as a novel MyoD- troponin complex (Tm-Tn) from Lethocerus IFM and examined the association target gene, is specifically induced in activated satellite cells. of the complex with thick filaments and myosin. Unexpectedly, we found that tropomyosin bound to myosin-S1. There are two isoforms of tropomyosin in 3114-Pos Board B322 the IFM (Tm1 and Tm2), derived from different genes. Tm1 bound to tropomy- Both Actin Myosin Attachment and Detachment Kinetics Affect Actin osin, but Tm2 did not. Electron micrographs of isolated thick filaments incubated Sliding Velocities and are Influenced by Mechanical Load with a complex containing only Tm1 (Tm1-Tn) showed binding with an apparent Travis J. Stewart, Sam Dugan, Diego Acala, Richard Brizendine, periodicity of about 40 nm. The ratio of actinþarthrin to Tm1þTm2 is 6.9:1 and Christine R. Cremo, Josh E. Baker. Tm1:Tm2 is 1:1, which suggests that both tropomyosin isoforms are associated Pharmacology, University of Nevada, Reno, Reno, NV, USA. with the thin filament in the usual way. Crossbridges that produce force bind to In the standard in vitro motility assay, F-actin slides over surface-attached target sites spaced at 39 nm on the thin filament; troponin bridges are midway myosin with an average velocity, V. As the density of myosin is increased, between these target sites. Our results suggest that non-force producing bridges the number of myosins available to move actin, N, also increases, where veloc- are steered towards Tm1 by the troponin complex. Troponin bridges are present ity, reaches a peak value before decreasing. We propose a simple model in in the relaxed IFM and bear some similarity to MyBP-C, which crosslinks thick which at low N the working step of a myosin, is free to displace actin resulting and thin filaments in cardiac and skeletal muscles. We will describe the biophys- in a V that is largely influenced by actin-myosin attachment kinetics, katt,as $ $ 1 ical properties of Tm1 that are needed for forming a troponin bridge. Vatt=v N d, where v is the actin-myosin ATPase rate (cycles s ) and d is the myosin step size. As N is increased, actin-bound myosin heads begin to 3112-Pos Board B320 impose a mechanical load that resists working steps of other myosin heads, re- Myogenic Differentiation of iPSC Homologous of a Nemaline Myopathy- sulting in V that becomes increasingly influenced by actin-myosin detachment Causing Nonsense Mutation in TNNT1 Gene kinetics, kdet, reaching a detachment-limit at high N, Vdet=L$kdet. Here L de- Anupom Mondal, Jian-Ping Jin. scribes a tether length or the distance that an actin-bound myosin head can be Physiology, Wayne State Univeristy, Detroit, MI, USA. freely pulled by a moving actin filament. The point at which a myosin head rea- A TNNT1 nonsense mutation that truncates the slow skeletal muscle isoform of ches it’s tether length dictates it either detach from the actin filament, or alter- troponin T (slow TnT) at codon 180 causes a recessively inherited nemaline nately, the point at which the bound myosin head begins to resist the actin myopathy in the Amish. More TNNT1 recessive nemaline myopathy mutations filament’s velocity by imposing a mechanical load. The N-dependence of the are found in other ethnic groups. Amish Nemaline Myopathy (ANM) patients relative contribution of Vdet and Vatt can be determined mathematically, and exhibit a complete loss of slow TnT protein in muscle cells with severe muscle the resulting analytical model accurately describes the N-dependence of V weakness, atrophy and diminished slow fibers. Our present study investigates measured in an in vitro motility assay at different katt and kdet as perturbed how the loss of intact slow TnT and the possible presence of the truncated by varying ionic strength, ATP, and blebbistatin. Measuring the effects of a-ac- slow TnT fragment impact on the formation of functional myofilaments during tinin on the N-dependence of V and fitting these data to our model, we show that myogenesis. Using induced pluripotent stem cells (iPSC) derived from ANM pa- a mechanical load imposed against actin sliding slows kdet and to a larger extent tient cells and normal human iPSC control, we pursued skeletal muscle differen- slows katt.

BPJ 8731_8734 Wednesday, February 21, 2018 629a

Posters: Intracellular Transport 3117-Pos Board B325 Optimizing Cationic Liposome Composition for Hydrophobic Drug 3115-Pos Board B323 Loading and Delivery to Human Cancer Cells Uptake and Localization of Aminoglycoside Antibiotics in Live Escherichia Victoria Steffes, Scott MacDonald, Meena M. Murali, Kai K. Ewert, coli Cyrus R. Safinya. Eliza M. Warszawik1, Jochem H. Smit2, Yichen Li2, Mark Loznik1, UC Santa Barbara, Goleta, CA, USA. Avishek Paul1, Thorben M. Cordes2, Andreas Herrmann1. Paclitaxel (PTXL) is a hydrophobic drug that acts as a mitotic inhibitor, inducing 1 apoptosis and halting the proliferation of tumor cells. PTXL is most often admin- Polymer Chemistry and Bioengineering, University of Groningen, Ò Groningen, Netherlands, 2Molecular Microscopy Research Group, University istered in hospitals in the patented formulation Taxol whose solvents cause of Groningen, Groningen, Netherlands. hypersensitivity reactions in addition to the side-effects of the chemotherapeutic Aminoglycosides are clinically important therapeutic agents widely applied to PTXL drug. To improve the therapeutic efficacy of PTXL, it should be admin- treat infections caused by Gram (þ) and (-) bacteria. Understanding of funda- istered by a non-toxic delivery agent with tumor cell specificity. Lipid nanopar- mental mechanisms by which aminoglycosides cross the cell wall and find ticles have many advantages as drug delivery vehicles including a diverse set of their target is important for gaining further insight into their mode of action lipid building blocks to tune physical and chemical properties, low toxicity, and and overcoming bacterial resistance. Although the mechanistic details of their ease of modular processing. Our recent study [1] revealed that loading uptake of aminoglycosides in Escherichia coli (E. coli) are largely unknown, PTXL in cationic liposomes (CLs) at a concentration lower than the membrane it has been demonstrated that the accumulation of these antibiotics occurs in solubility limit (less than 3 mol%) may have short and long-term benefits for Ò three consecutive phases. An initial ionic binding to cells occurs instanta- drug delivery outcomes compared to the CL formulation EndoTAG , which neously when the antibiotic comes into contact with the bacteria and is fol- employs CLs at the PTXL membrane solubility limit. In the work presented lowed by two energy-dependent phases termed energy-dependent phase I here, we consider the effects of lipid curvature, hydrophobic tail saturation, (EDPI) and energy-dependent phase II (EDPII). A duration and a rate of and inclusion of PEG-lipid on PTXL solubility in CLs, and we correlate the phys- EDPI, in which the antibiotic is transported into the bacterial cytosol, depends ical properties of the liposomes to PTXL delivery efficacy observed in in vitro on the concentration of the aminoglycosides. After being transported to the cell studies. [1] Steffes et al. Distinct solubility and cytotoxicity regimes of cytosol, aminoglycosides are able to bind their target (A-site in 16s small paclitaxel-loaded cationic liposomes at low and high drug content revealed by ribosomal subunit). Binding causes translation mismatches, blocks transloca- kinetic phase behavior and cancer cell viability studies. Biomaterials 2017, tion and results in synthesis of misfolded proteins, which being inserted to the 145, 242-255. DOI: 10.1016/j.biomaterials.2017.08.026. bacterial membrane result in the membrane leakage and a rapid uptake during EDPII leading to bacterial cell death. Here, we employed a series of 3118-Pos Board B326 fluorophore-labelled aminoglycoside probes for live-cell fluorescence micro- Actin Corrals G-Protein Coupled Receptors in Ciliary Membrane scopy imaging to study the uptake process of aminoglycosides in live Sungsu Lee, Peter Calvert. E. coli cells. Since the uptake of aminoglycosides is driven by the electrical Department of Ophthalmology, SUNY Upstate Medical University, potential difference across the membrane, we investigated how the charges Syracuse, NY, USA. of those conjugates influences this process. We have developed an algorithm Current understanding of the mechanisms underlying G-Protein Coupled Re- that enables a quantitative analysis of fluorescent probes in bacterial mem- ceptor (GPCR) transport within primary cilium is limited and several brane and cytosol. We showed that uptake of aminoglycosides can be tuned competing models exist, including motor-driven transport, local binding and by addition of adjuvants, what can be a new strategy to fight bacterial resis- free diffusion. Our analysis of the mean square displacement (MSD) of single tance to this class of drugs. GPCRs within ciliary membranes as a function of time step magnitude showed predominant mode of transport is diffusion with transient confinement to mem- 3116-Pos Board B324 brane sub-regions. The mechanism of this confinement is not well understood, Hydrophobic-Mediated Assembly of Lipid-Coated Building Blocks by and we hypothesize that actin filaments delimit the membrane sub-regions. We Double-End Anchored Polymers performed super-resolution high speed imaging of single GPCRs transfected Emily Wonder1, Chenyu Liu1,2, Kai K. Ewert1, Phillip Kohl1, Youli Li1, into murine inner-medullary collecting duct (IMCD3) cells using quantum- Weihong Qiao2, Cyrus R. Safinya1. dots. Latrunculin A, Cytochalaisin D, and Jasplakinolide was used to alter actin 1University of California, Santa Barbara, Santa Barbara, CA, USA, 2Dalian dynamics in primary cilium. The MSD(tau) analysis revealed free and fast local University of Technology, Dalian, China. diffusion, followed by confinement to membrane sub-regions, slowing diffu- In this work, we introduce PEG (polyethylene glycol) molecules with double- sion 15 fold. The average diffusion coefficient of the free local diffusion end hydrophobic anchors (DEAs) that allow for hydrophobic-mediated was [2.53 5 0.42 micro m2 s1], which is an order of magnitude higher than assembly of lipid-coated building blocks. By varying the PEG length, anchor previous measurements of GPCR diffusion coefficient inside cilium. The hydrophobicity, and molar fraction of DEA-PEG molecules within a mem- average diffusion coefficient of the slow confined diffusion was [0.18 5 brane, we can alter the assembly properties of lipid-based systems. This is 0.03 mm2 s1]. The size of the corral is calculated to be approximately affected through a change in the equilibrium balance of looping (both anchors 260nm. Disruption of actin by Latrunculin-A and Cytochalasin D resulted in in one membrane) and bridging (anchors spanning two membranes) conforma- increased diffusion coefficient and MSD. Latrunculin A treatment resulted in tions. Using differential interference contrast microscopy, dynamic light scat- a 1.5350.19 fold increase in the diffusion coefficient above vehicle control. tering, and small angle x-ray scattering, we have demonstrated an ability to Enhancing actin polymerization with Jasplakinolide significantly decreased tune DEA-PEG-mediated behavior in both pure lipid and lipid-nucleic acid the diffusion coefficient and MSD(tau). Fast imaging and the MSD analysis systems. following altered actin dynamics demonstrates that the predominant modes Cationic liposomes (CLs) are a common synthetic carrier of nucleic acids of GPCR movement within the ciliary compartment is free local diffusion in for gene delivery and silencing. Optimization of DNA delivery and expres- highly fluid membrane, followed by transient confinement to membrane sub- sion requires understanding of the interactions between cellular membranes regions delimited by actin. Ongoing studies aim to understand the physiological and CL-nucleic acid nanoparticles (CL-NA NPs), affecting NP binding, role of the ciliary membrane compartmentalization. uptake, endocytic trafficking, and endosomal escape. PEGylation is neces- sary to sterically stabilize the NPs but also has the effect of inhibiting 3119-Pos Board B327 cell-nanoparticle interactions. Adding DEA-PEG molecules to our NPs al- Retrograde Diffusion of Kinesin-II Facilitates Flagellar Length Control in lows for the formation of polymer bridges between the NP and endosomal Chlamydomonas membranes. This increases the rate of collisions between the cationic NPs Alexander Chien1, Sheng Ming Shih1, Raqual Bower2, Douglas Tritschler2, and anionic cellular membranes and promotes membrane fusion and endo- Mary E. Porter2, Ahmet Yildiz1. somal escape. Confocal imaging of a human prostate cancer cell line 1UC Berkeley, Berkeley, CA, USA, 2University of Minnesota, Minneapolis, (PC3) has shown evidence that NPs modified with DEA-PEG molecules MN, USA. have altered interactions with plasma and endosomal membranes. Colocali- To assemble and maintain functional cilia, kinesin-II and dynein-1b motors po- zation of NPs with recycling (Rab11-GFP) and late endosome/lysosomal wer intraflagellar transport (IFT) of axonemal precursors and sensory proteins (LysoTracker) pathways indicate a change in endosomal trafficking that between the cell body and the ciliary tip. IFT motors have been implicated in mimics the effects of very high membrane charge density [Majzoub, ciliary elongation and length control, but the underlying mechanism for deter- Wonder, et al. J. Phys. Chem. B, 2016]. This demonstrates the possibility mining the flagellum’s final length remained unclear. We observed that, unlike of using DEA-PEG molecules to tune NP-cell membrane interactions in dynein-1b, kinesin-II detaches from IFT trains at the flagellar tip and returns to order to promote endosomal escape and exogenous gene expression. the cell body by diffusion in Chlamydomonas flagella. Retrograde diffusion of

BPJ 8735_8739 630a Wednesday, February 21, 2018 kinesin-II delays its recycling to the basal body and kinesin-II accumulates in a from the a4b2Rs not in the membrane of the acidic vesicles. The slow phase concentration gradient in the flagellum. As a result, kinesin-II is depleted at the is caused by combination of trapping of the protonated ligands inside the acidic flagellar base as the flagellum elongates. When the flagellum is elongated via vesicles, and the a4b2R high-affinity binding sites in the vesicle lumen keeping Liþ treatment, or when the cell regrows flagella in the presence of protein syn- the ligands out of the vesicle solution. This interpretation is supported by the thesis inhibitor, the base is similarly depleted of kinesin-II. These results sug- experimental data and can help us further understand the mechanism of nico- gest that accumulation in the flagellum acts as a negative-feedback mechanism tine addiction and smoking cessation drugs. to limit the number of kinesin-II available for moving cargo on IFT trains, and limits the growth of flagella when they reach longer lengths. 3123-Pos Board B331 Intracellular Transport Characterization of the Transcription Factor Gli2 3120-Pos Board B328 by Fluorescence Correlation Spectroscopy Approaches Regulation of Rab5 in its Effector Binding and Guanine Nucleotide Con- Belen Torrado1, Leonel Malacrida2, Jose Luis Badano1, Florencia Irigoı´n3, version by Site-Specific Monoubiquitination Enrico Gratton2. Sangho Lee. 1Institut Pasteur de Montevideo, Institut Pasteur de Montevideo, Montevideo, Biological Sciences, Sungkyunkwan University, Suwon, Republic of Korea. Uruguay, 2LFD, University of California Irvine, Irvine, CA, USA, 3Dpto. Rab GTPases are involved in intracellular trafficking pathways and have Hisologı´a y Embriologı´a, Universidad de la Repu´blica Uruguay, Montevideo, recently been reported to be ubiquitinated. However, the functions of Uruguay. ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 Gli2 is the principal transcriptional activator of the Hedgehog signalingin mam- is monoubiquitinated on K116, K140, and K165. Upon co-transfection mals. Upon pathway activation, Gli2 moves into the cilium before reaching the with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and nucleus. While two conserved classical nuclear localization signals (cNLS) EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants mediate Gli2 nuclear localization via importin (Imp)-a/b1, these sequences are restored these abnormalities, whereas K116R did not. We derived structural not required for Gli2 ciliary import (Torrado et al, 2016. PLoSONE;11(8)). How- models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by ever, Importin-b2 is involved in Gli2 ciliary entry, and might also collaborate in solution scattering and observed different conformational flexibilities in Gli2 nuclear entry (Torrado et al, 2016. PLoSONE;11(8)). Several questions a site-specific manner. Structural analysis combined with biochemical data about Gli2 intracellular traffic still remain opened. To analyze the nucleo- revealed that interactions with downstream effectors were impeded in cytoplasmic transport of Gli2 we used fluorescence correlation spectroscopy mUbRab5K140, whereas GDP release and GTP loading activities were altered methods, in particular pair correlation function and Number & Brightness ap- in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We proaches. We compared Gli2 nuclear transport when the pathway is on and propose a regulatory mechanism of Rab5 where monoubiquitination downre- off. Gli2 ciliary transport was analyzed by fluorescence correlation spectroscopy gulates effector recruitment and GDP/GTP conversion in a site-specific approaches combined with 3D-particle tracking so we could avoid the out of manner. focus produced by the movement of the cilium. We applied these methods to evaluate Gli2 diffusion through and interaction with the transition zone and along 3121-Pos Board B329 the cilium. The results obtained so far and further analysis will give us a better Resolving Endosome Rotation in Intracellular Trafficking understanding about the intracellular movement of this key protein. We will pre- Yan Yu. sent the results and some technical limitations in our measurements due to bleach- Indiana University, Bloomington, IN, USA. ing. Work supported in part by NIH grants P50 GM076516 and P41 GM103540, Intracellular cargos are known to be transported by molecular motors, but the The Company of the Biologists, PEDECIBA (Uruguay) and ANII (Uruguay). mechanisms by which different groups of motor proteins maneuver cargos through the complex microtubule network remain elusive. In this study, we 3124-Pos Board B332 investigated this long-standing question by analyzing the rotational dynamics All-Atom Structure and Ionic Conductivity of the Nuclear Pore Complex of cargos at microtubule intersections in unprecedented detail. By using a novel David Winogradoff1, Christopher Maffeo1, Wei Si1,2, Aleksei Aksimentiev1. single-particle rotational imaging method, we simultaneously resolved the rota- 1University of Illinois at Urbana-Champaign, Champaign, IL, USA, 2School tional and translational motion of endosomes in living cells, and determined the of Mechanical Engineering, Southeast University, Nanjing, China. correlation of their dynamics with underlying microtubule structures. While it The nuclear pore complex (NPC) serves as a gatekeeper, regulating biomole- was previously known that cargos pause and switch directions at microtubule cular transport across the nuclear envelope. The key barrier to nuclear transit intersections, this study provides the first evidence demonstrating rotational dy- is the central channel of the NPC, composed of disordered nucleoporins. namics of intracellular cargos at microtubule intersections and elucidating the Several mutually exclusive theoretical models have been proposed to account underlying driving forces behind such movements. for selective transport across NPCs. Recent advances in cryo-electron micro- scopy have elucidated the structure of the NPC’s outer scaffolding whereas 3122-Pos Board B330 measurements of ionic current have been used to monitor molecular transport Kinetic Modeling of Weak Base nAChR Ligand Selective Trapping within through solid-state nanopores coated with the disordered inner core nucleopor- Intracellular Acidic Vesicles: Insights into Mechanisms Underlying ins. Yet, the microscopic mechanism of selective transport through the NPC Nicotine Addiction and Smoking Cessation and the architecture of the NPC’s central channel remain largely unknown. Yuqi Liu1, Stefan Trapp2, William N. Green3, Esmael J. Haddadian4. 1 2 Here, we report the results of all-atom molecular dynamics simulations that The University of Chicago, Chicago, IL, USA, Environmental Engineering, have characterized the structural and electrical properties of the NPC diffusion Technical University of Denmark, Kongens Lyngby, Denmark, 3Department 4 barrier. The ionic conductivity of an electrolyte solution containing disordered of Neurobiology, The University of Chicago, Chicago, IL, USA, Biological nucleoporins was found to non-linearly depend on the nucleoporin concentra- Sciences Collegiate Division, The University of Chicago, Chicago, IL, USA. tion, approaching zero as the protein content reaches 70%. Brownian dynamics Recent work from our group has characterized selective trapping of nicotinic simulations of the entire biological NPC produced convergent configurations of acetylcholine receptor (nAChR) ligands within neurons in the brain (Govind 1 the NPC’s disordered central channel, which were used to build an ensemble of et al. eLife 2017 ). The ligands are weak bases that bind with high-affinity to all-atom models. The all-atom models of the entire NPC, each containing about a4b2-type AChRs (a4b2Rs), such as the anti-smoking drug varenicline (Chan- 140 million atoms, were validated through measurement of the NPC’s ionic tix) and epibatidine. Selective trapping occurs within a4b2Rs-containing acidic conductivity. Our study sets the stage for subsequent investigation of selective vesicles of cells and neurons. Slow release of trapped varenicline reduces the molecular transport through the NPC. effects of long-term nicotine exposure. Selective trapping is further regulated by nicotine exposure, which increases the number of acidic vesicles. Nicotine, 3125-Pos Board B333 also a weak base, is not trapped because its lower pKa and lower affinity to Synthetic Mimics of the Nuclear Pore Complex a4b2Rs1. Here, we present a kinetic model that accounts for the biphasic Laura Maguire1, Michael Stefferson2, Katherine Rainey1, Nathan Crossette1, behavior of these weak bases leaving the cell after accumulating in vesicles Eric Verbeke3, Meredith Betterton2, Loren Hough1. and binding to the nAChRs on the cell-surface and within intracellular vesicles. 1BioFrontiers Institute and Department of Physics, CU Boulder, Boulder, CO, Accumulation and release of molecules in the cell by diffusion into and out of USA, 2Department of Physics, CU Boulder, Boulder, CO, USA, cytosol and vesicles is calculated with the Fick’s Law of diffusion (neutral 3BioFrontiers Institute and Department of Chemical and Biochemical molecule) and the Nernst-Planck equation (ion, Trapp and Horobin 2005). Engineering, CU Boulder, Boulder, CO, USA. The ligand binding to a4b2s is modeled using empirical receptor-ligand ki- Transport between the nucleus and cytoplasm of eukaryotic cells occurs netics. Solving our coupled differential equations numerically, we found that through the Nuclear Pore Complex, a remarkable filter that blocks the passage the fast release phase is mainly caused by unbinding of protonated ligands of most macromolecules while allowing passage of transport factors and their

BPJ 8735_8739 Wednesday, February 21, 2018 631a cargo. A class of intrinsically disordered protein domains called the FG Nups the voltage-dependence of activation and slow inactivation. This change, how- act as a barrier to the transport of most molecules, while passage of transport ever, has minimal effects on sodium ion selectivity or permeability. Other factors requires their binding to these same proteins. There is not consensus shared amino acid changes are compensatory and recover functional disrup- on either the nature of the barrier nor the means by which binding overcomes tions in the voltage-dependence of slow inactivation without altering TTX the barrier. We create tunable mimics of the NPC using FG Nups, the intrinsi- resistance. These results suggest that adaptive evolution in the nervous system cally disordered proteins that line the NPC channel in vivo. Using this platform, is constrained by functional costs but that extreme phenotypes can evolve we are able test potential mechanisms of selective transport. through combinations of progressive substitutions that increase resistance and compensatory substitutions that mitigate costs. Intriguingly our results sug- 3126-Pos Board B334 gest that biophysical properties associated with the excitability of VGSCs may Monitoring the Tagged mRNA Export Rate via Nuclear Pore Complex in be more evolutionarily labile that other core properties of the channels and may Live Cells with a Snapshot provide insights into the evolution of the diverse functional capabilities seen in 1 2 1 1 2 1 Yueyue Jing , Jingya Ye , Longfang Yao , Lan Mi , Biao Dong , Jiong Ma . extant vertebrate VGSC isoforms. 1Department of Optical Science and Engineering, Fudan University, 2 Shanghai, China, State Key Laboratory of Biotherapy, Sichuan University, 3129-Pos Board B337 Chengdu, China. Enhanced Tetrodotoxin-Sensitive Neuronal NaD Channel Activity Associ- As mRNA conveys genetic information from DNA to the ribosome, its ated with Arrhythmogenic Calmodulin Mutation N98S export rate changes under DNA manipulation and further affects hence pro- Przemyslaw Radwanski1, Jonathan Davis2, Sandor Gyorke1. tein translation. In live cells, the rate of a specific kind of mRNA imply the 1Pharmacy, The Ohio State University, Columbus, OH, USA, 2Physiology cells’ future growth tendency or even differentiation direction on molecular and Cell Biology, The Ohio State University, Columbus, OH, USA. level. Due to its easy degradation, the gross of mRNA was usually semi- Cardiac arrhythmias, often stemming from abnormal impulse generation, are quantified from the extracts of a large amount of cells by using techniques one of the leading causes of mortality in the United States. Recent evidence like RT-PCR. However, monitoring a specific mRNA rate requires single- þ suggests that tetrodotoxin-sensitive neuronal Na channels (nNav) are an in- live-cell synchronous quantification. Therefore, in this study, we developed tegral part of catecholaminergic polymorphic ventricular tachycardia (CPVT) an intravital fluorescent detectable system for the export of mRNA through as well as long QT (LQT)-associated arrhythmias. With the advent of nuclear pore complexes (NPC) with laser scan confocal microscopy. By calmodulin (CaM)-dependent disorders manifesting overlap in CPVT and adapting a mRNA motion model and NPC active transportation model to long QT syndrome (LQT), there is surprisingly little evidence linking ar- the fluorescence images, tagged mRNA transport rate was calculated for rhythmogenic CaM mutations with abnormalities in Naþ handling. Here each single live cell. With this method, it would help us to better understand we investigated recombinant CaM mutant associated with LQTS/CPVT the regulation of gene expression and even to visualize cells’ differentiation (N98S). We performed simultaneous recordings of membrane potentials direction. 2þ þ and intracellular Ca handling as well as assessed Na currents (INa) during various pharmacological interventions. Electrophysiological analysis re- Posters: Voltage-gated Na Channels vealed that dialysis of CaM-N98S (6.5 mM) induced a gain-of-function in INa by altering steady-state inactivation which manifested in non- inactivating persistent I in intact wild type cardiomycyotes. Surprisingly, 3127-Pos Board B335 Na CaM mutant-mediated alterations in I function were abolished by nNa in- Elucidating the Specificity and Binding Rate of a Subtype Selective Sodium Na v hibition with 4,9-anhydro-tetrodotoxin (4,9-ah-TTX; 300nM). In line with Channel Inhibitor with Molecular Dynamcis Simulations these findings, myocytes dialyzed with N98S displayed prolonged action po- Ben Corry. tential duration, aberrant Ca release events and arrhythmogenic after depolar- Research School of Biology, Australian National University, Acton, ACT, izations that were abolished with 4,9-ah-TTX treatment. Taken together, Australia. these findings identify a previously unexplored mechanism for arrhythmo- Nerve and muscle signalling is controlled by voltage-gated sodium (Nav) chan- genic CaM mutations based on altered nNa function. Furthermore, this study nels which are the targets of local anaesthetics, anti-epileptics and anti- v indicates that nNa inhibition may be a viable therapeutic modality for man- arrhythmics. Current medications do not selectively target specific types of v agement of CaM-related arrhythmias. Nav found in the body, but compounds that do so have the potential to be break- through treatments for chronic pain, epilepsy and other neuronal disorders. We use long computer simulations to show how a promising lead compound can 3130-Pos Board B338 target one Nav implicated in pain perception and specific channels found in Characterization of a NaV1.4 Hypokalemic Periodic Paralysis Mutation in bacteria, and accurately predict the affinity of the compound to different chan- Domain I nel types. Most importantly, we explain the slow kinetics of this class of com- James R. Groome1, Landon Bayless-Edwards1, Paula Arinze1, pound that currently limits their therapeutic utility. The negative charge on the Frank Lehmann-Horn2, Karin Jurkat-Rott2. 1 2 compound is essential for high affinity binding but is also responsible for ener- Biological Sciences, Idaho State University, Pocatello, ID, USA, Division getic barriers that slow access to the site. This knowledge aids the design of of Neurophysiology, Ulm University, Ulm, Germany. compounds affecting specific eukaryotic and bacterial channels and assists We characterized the clinical effects and biophysical properties of hypokale- future drug development. mic periodic paralysis mutation, R222W, initially identified in Korean pa- tients. The heterozygous mutation was also found in a 52 year-old patient 3128-Pos Board B336 with weakness beginning at age 18 and associated with mild hypokalemia, Understanding Nervous System Evolution through Natural Experiments: and associated with myalgia. R222W muscle fibers exhibited action poten- Tetrodotoxin Resistance in Snakes tials with attenuated height, and slowed rate of rise compared to those Shana L. Geffeney1, Gabriela Toledo2, Charles T. Hanifin1. from a control patient. Membrane potential recordings in normal potassium 1Biology, Utah State University-Uintah Basin, Vernal, UT, USA, 2Biology, showed that R222W fibers exhibited a greater density of depolarized poten- University of Virginia, Charlottesville, VA, USA. tials (P2) compared to controls. R222W was functionally characterized with A diverse array of species has evolved resistance to toxins that target proteins cut-open oocyte voltage clamp recordings at 20 C. The mutation produced a critical for nervous system function such as ion channels. Understanding the depolarizing shift of the activation midpoint; this effect was shown to be in- mode and tempo by which ion channels evolve resistance to toxins can inform direct by comparing wild type and R222W channels in the IFM/QQQ inac- our understanding of the key functional properties of these proteins. Tetrodo- tivation deficient background. R222W produced several effects on toxin (TTX) is a well-studied example of a toxin that cripples nervous system inactivation consistent with hypoexcitability. Compared to wild type function by binding to and blocking the outer pore of voltage-gated sodium ion hNaV1.4, R222W produced a left-shift of the steady-state fast inactivation channels (VGSCs). Many different species have independently evolved resis- curve, accelerated entry, and slowed recovery. R222W also produced more tance to TTX, including newts and octopuses that use it as a defense as well complete slow inactivation and slowed its recovery. At hyperpolarized volt- as garter snakes that consume TTX-defended prey. These species show a ages, R222W promoted a cationic omega current, which may explain the par- remarkable number of shared substitutions in their VGSCs as they evolve adoxical depolarization of muscle fibers. This work was supported by NIH TTX resistance suggesting that common structural and functional constraints 1R15NS093579-01A1 to JRG, NIH NIGMS P20GM103498 to ISU, the shape evolutionary change of VGSCs. Our data gathered from a constructed non-profit Else-Kroner-Fresenius Foundation, German DGM Muscle Disease native snake channel demonstrate that a single shared change in the outer Society, Taro Pharmaceuticals, and the German BMBF Ministry for IonNeur- pore causes the greatest increase TTX resistance and dramatically disrupts Onet project jointly to KJR and FLH.

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3131-Pos Board B339 tein is critical to the propagation of skeletal muscle fiber action potentials. Our Biophysical Characterization of Two NaV1.4 Mutations Making a Clinical recent work characterizes the biophysical effects of R222W, the first hypokale- Overlap between the Myotonia-Hyperkalemic and Hypokalemic Periodic mic periodic paralysis mutation described in DIS4-hNaV1.4. Two computational Paralysis Clusters of Disorders approaches were used to determine how this mutation influences biophysical Mohamed Chahine1, Hugo Poulin1, Pascal Gosselin-Badaroudine1, properties of this channel and how these parameters affect action potential initi- Savine Vicart2, Karima Habbout3, Damien Sternberg2, Serena Giuliano3, ation in skeletal muscle fibers. First, we ran molecular dynamics simulations to Bertrand Fontaine2, Saı¨d Bendahhou3, Sophie Nicole2. compare the translation of the S4 segment and residue-residue interactions that 1Medicine, Laval University, Quebec, QC, Canada, 2Groupe Hospitalier Pitie occur in R222W and wild type voltage sensors. When entering the gating pore, Salpeˆtrie`re, Paris, France, 3CNRS, University Nice Sophia-Antipolis, Nice, R222W rotates further away from the tyrosine (Y168) of the gating charge trans- France. fer center than does the native arginine. Second, we used a two-compartment We report here the functional characterization of two dominant SCN4A mathematical model of an action potential in a muscle fiber to simulate the effects missense mutations targeting the R1451 residue located in transmembrane of R222W on action potential initiation. Using cut-open voltage clamp electro- segment S4 of DIV. The R1451L is a novel mutation found in two unrelated physiology, we found that the kinetics of activation and fast inactivation are patients. The first patient was diagnosed with SMC, and the second with a each affected by this mutation. With that data, a barrier model was constructed mixed hyperPP-hypoPP phenotypes. The R1451C mutation was previously re- to determine transition rates to and from the inactivated or activated states. ported by our group in one patient with a single attack of PP induced by gluco- When applying these rate equations to a Hodgkin-Huxley model of an action po- corticoids. To elucidate the mechanism underlying the phenotypes caused by tential in a skeletal muscle fiber, we found that the action potential in an R222W the R1451C/L mutations and their roles in different types of sodium channelo- fiber was attenuated compared to the control. This comparison in simulation mir- pathies, we used the whole-cell patch-clamp technique to study tsA201 cells rors the difference in action potentials in control and R222W patients. This work expressing WT, R1451C or R1451L channels. Our results showed that both was supported by NIH 1R15NS093579-01A1 to JRG, the EKF Foundation, mutations shifted the steady-state inactivation to hyperpolarized potentials, German DGM Muscle Disease Society, Taro Pharmaceuticals, and the German reduced the current density, slowed the recovery from slow inactivation and ex- BMBF Ministry for IonNeurOnet project to KJR and FLH. hibited a slow of the overall kinetics of fast inactivation. Cooling further en- hances the abnormalities of fast inactivation kinetics in R1451L channels. 3134-Pos Board B342 Homology modeling revealed a dissimilar disruption of hydrogen bonds in NaV1.4 Loss of Function Changes for Recessively Inherited Myopathy the VSD of R1451C/L mutant channels.The altered biophysical properties of with Fluctuating Weakness Nathaniel Elia1, Perry Shieh2, Marbella Quinonez1, Stephen Cannon1. R1451C/L channels account for the clinical phenotypes seen in our patients 1 2 but that additional factors are likely to play a role in the diversity of symptoms. Physiology, UCLA, Los Angeles, CA, USA, Neurology, UCLA, Los Angeles, CA, USA. 3132-Pos Board B340 Mutations in SCNA4, coding for the skeletal muscle specific NaV1.4 sodium Physiological Tradeoffs of TTX Resistance in NaV1.4: Whole Cell Electro- channel, are associated with a variety of dominantly inherited disorders in physiology and Tissue Myography Reveal Reduced Tetrodotoxicity at the which gain-of-function changes cause myotonia or periodic paralysis. In Cost of Channel Function contrast, very few loss of function NaV1.4 mutations have been identified, Robert E. del Carlo1, Jessica S. Reimche2, Michael T.J. Hague3, and these cause recessively inherited myopathy and fluctuating strength that Edmund D. Brodie, Jr.4, Normand Leblanc5, Chris R. Feldman2. has features of myasthenia or a variant of periodic paralysis. 1University of Nevada, Reno, Reno, NV, USA, 2Biology, University of We report on the functional characterization of two mutations at the same res- Nevada, Reno, Reno, NV, USA, 3Biology, University of Virginia, idue in DIVS4 that both produce recessive muscle disorders. R1460W was iden- Charlottesville, VA, USA, 4Biology, Utah State University, Logan, UT, USA, tified in a 6 y.o. with myopathy, episodes of weakness and breathing difficulties. 5Pharmacology, University of Nevada, Reno - School of Medicine, Reno, The patient was homozygous for R1460W, and the parents were both asymp- NV, USA. tomatic carriers. A heteroallelic mutation R1460Q/null was reported in a child The interaction between toxic newts (Taricha) and several resistant garter with congenital weakness, ptosis, plus intermittent laryngospasm and painful snake species (Thamnophis) is a model system of predator-prey coevolution. muscle stiffness suggestive of myotonia. Voltage clamp studies in HEK cells re- Pacific newts defend themselves by secreting the potent neurotoxin, tetrodo- vealed loss of function deficits with a decreased current density and a left shift toxin (TTX), from glands the skin. TTX specifically binds to the outer pore for the voltage-sensitivity of fast inactivation for both mutant channels. A of voltage-gated sodium channels, NaV proteins. These transmembrane pro- modest gain-of-function change was also detected, as an accelerated rate of re- teins form a sodium-selective pore surrounded by four voltage-sensitive do- covery from fast inactivation. Use-dependent inactivation for high frequency mains that rapidly open and close the channel with changes in membrane pulse trains was reduced for mutant channels, which may explain the myotonic potential. As such, NaV proteins are responsible for the first electrical event features. Remarkably, the three prior cases of recessive NaV1.4 myopathy / initiating every action potential and every skeletal and cardiac muscle contrac- myasthenia plus the two reported here are all in the DIVS4 voltage sensor tion. TTX ligation to the channel pore prevents sodium ion movement through domain and all cause a prominent left shift of inactivation. the membrane, thereby abolishing excitability, and leading to numbness, paral- ysis, and eventually death by respiratory arrest. TTX has served as an agent of 3135-Pos Board B343 selection on at least three species of Thamnophis that prey on sympatric Tari- A Computational Model of the Cardiac Sodium Channel DIII Voltage cha. Thamnophis atratus, T. couchii, and T. sirtalis have independently evolved Sensor: Connecting Molecular Movements to Tissue Dynamics 1 2 2 adaptive mutations within the pore of the skeletal muscle channel variant, Jonathan Moreno , Wandi Zhu , Jonathan Silva . 1Cardiology, Washington University in St. Louis, St. Louis, MO, USA, NaV1.4. The amino acid substitutions are hypothesized to reduce the affinity 2 of TTX to the pore, thereby providing physiological resistance to TTX. Here, Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA. we discuss how these same mutations may additionally reduce sodium channel þ function provided that animals carrying these mutations display diminished The cardiac Na channel is a macromolecular protein complex consisting of 4 skeletal muscle performance. This hypofunction is likely explained by alter- domains, each composed of 6 transmembrane subunits. The voltage sensing do- ations to biophysical properties of the channel, such as reduced I magnitude mains (VSD) on DI - DIV, and in particular the DIII VSD has been shown to be Na critical in activation, inactivation, channel opening, and local anesthetic antiar- and depolarized steady-state activation and inactivation curves. Whole cell þ rhythmic drug action. To date, computational models of the cardiac Na chan- electrophysiology performed on a TTX-sensitive rat NaV1.4 ortholog as well as site-directed mutants mirroring the resistance-conferring mutations found nel only account for current kinetics, which limits their ability to predict how in snakes reveal interesting support for this biophysical tradeoff. perturbations by mutations and drugs at the molecular level may affect higher dimensional properties in the heart. We began by expanding a computational 3133-Pos Board B341 model of the cardiac Naþ channel, and incorporated voltage clamp fluorometry Investigating a Domain I Hypokalemic Periodic Paralysis Mutation in data on DIII VSD movement; this technique allows one to fluorescently track hNa 1.4: A Computational Approach VSD conformation and correlate VSD kinetics with ionic currents. Our V þ Landon J. Bayless-Edwards1, James R. Groome1, Frank Lehmann-Horn2, expanded 12-state Markov model of the Na channel recapitulates a wide range Vern Winston1, Karin Jurkat-Rott2. of current kinetics including steady state availability, steady state activation, 1Biological Sciences, Idaho State University, Pocatello, ID, USA, 2University deactivation, recovery from inactivation, and mean open time, as well as of Ulm, Ulm, Germany. DIII fluorescent activation and deactivation. Next, we examined two LQT3 mu- Hypokalemic periodic paralysis is a muscular disease characterized by episodes tations, R1626P, and M1652R, which have opposing effects on DIII and have þ of flaccid paralysis associated with low serum potassium. Mutations linked to this differing sensitivities to the Na channel blocker mexelitine. We built disease have been found in the voltage-gated sodium channel, hNaV1.4. This pro- computational models of each, and their unique interaction with mexelitine.

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Our results show that a hyperpolarizing shift in DIII can account for the into lipid bicelles will then be used for NMR structural studies and for the dis- increased sensitivity of R1626P to mexelitine, and the depolarization seen covery of toxins targeting this particular domain of the channel. with M1652R renders it mexelitine insensitive. Our computational models were then used to characterize and track these differential effects on DIII move- 3139-Pos Board B347 ment on the action potential and coupled virtual tissue. We conclude that DIII Sequence and 3D Alignments of the Pore-Lining Helices in P-Loop Chan- represents a novel molecular target and plays a primary role in determining dif- nels Reveal their Conserved and Variable Features Denis B. Tikhonov1, Boris S. Zhorov1,2. ferential sensitivity of certain LQT3 mutations to the local anesthetic 1 mexelitine. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS, St. Petersburg, Russian Federation, 2Biochemistry and Biomedical Sciences, 3136-Pos Board B344 McMaster University, Hamilton, ON, Canada. Intracellular Calcium Alters Sodium Channel Kinetics to Influence The family of P-loop channels is characterized by the tetrameric or pseudo- Neuronal Firing tetrameric structure with extracellular membrane-reentering P-loops, which Marco A. Navarro, Jenna L. Lin, Benton R. Berigan, Mirela Milescu, include selectivity-filter residues. This very diverse family includes, but not Lorin S. Milescu. limited to voltage-gated potassium, sodium and calcium channels, glutamate- University of Missouri-Columbia, Columbia, MO, USA. and cyclic nucleotide-gated channels, TRP and two-pore channels. The channels Intracellular calcium can modulate the kinetics of voltage-gated sodium (Nav) play key roles in physiology and are targets for many toxins and medicinally and other channels. Existing studies on Nav channels report effects on intrinsic important drugs. Numerous channelopathies are described for human P-loop kinetic properties, including inactivation. Here, we show evidence that calcium channels. The X-ray and cryo-EM structures of P-loop channels in the open may also modulate the interaction between Nav channels and fibroblast growth and closed states show conserved state-dependent folding despite the channel se- factor-homologous factors (FHFs). Certain FHF subtypes bind to the C-termi- quences are very diverse. Here we compared 3D structures of different P-loop nus of Nav channels and result in an open-state block that competes with channels and found that the inner helices in many channels adopt classical endogenous fast inactivation. This binding is fast (ms) at depolarizing voltages, alpha-helical conformations, while in TRPV1, NavPaS, and two-pore channel but unbinding is very slow (100s of ms). Neurons that express these FHF sub- TPC1 they have a short p-helical bulge in the middle of the helix. Due to the var- types have characteristically low firing rates. The calcium-dependent effect that iations in the inner helices folding, residues, which appear in matching positions we observe is mostly a change in the kinetics of recovery from the FHF-induced of the sequence alignment, may have different orientations in 3D aligned chan- long-term inactivation. This calcium dependence may serve as an additional nels and different contacts with neighboring segments. In sodium, calcium, feedback mechanism for regulating neuronal firing rates. TRPV and two-pore channels, the pore-lining helices contain conserved aspar- agines, which are lacking in potassium channels. Comparison of the sequence- 3137-Pos Board B345 and 3D-alignemnts suggests that the asparagines appeared in evolution as Synthetic Batrachotoxin Derivatives as Molecular Probes of Voltage- insertions. Each insertion is accommodated by one of two ways. The first way Gated Sodium Ion Channel Function is a p-helix bulge immediately upstream the insertion with preserving most of Timothy M.G. MacKenzie, Justin Du Bois. inter-segment contacts. The second way is a twist of the helix C-terminal third Chemistry, Stanford University, Stanford, CA, USA. with switching its inter-segment contacts. The two possibilities should be Batrachotoxin (BTX) is a potent and selective agonist of voltage-gated sodium considered in homology modeling of P-loop channels and in structure-based in- channels (NaVs). Toxin binding to the inner pore of the channel (Site II) elicits a terpretations of numerous experimental data on physiology, pathophysiology, multitude of effects, including a change in activation potential, inhibition of pharmacology and toxicology of the channels. Supported by RFBR. inactivation at all potentials, and a loss of ion selectivity. De novo synthesis of BTX has enabled structure-activity studies with toxin derivatives to develop 3140-Pos Board B348 a molecular picture of how BTX alters NaV function. Substitution of the natu- Mechanism of Selective Resistance of the Bumble Bee Sodium Channel rally occurring C20-pyrrole ester with other acyl groups has been explored. BiNaV1 to TAU-Fluvalinate Replacement of the rigid ring with a conformationally flexible C20- Ke Dong1, Shaoying Wu2, Yoshiko Nomura1, Yuzhe Du1, Boris Zhorov3. heptynoate ester results in a compound that demonstrates unusual effects on 1Michigan State Univ, East Lansing, MI, USA, 2College of Plant Protection, channel gating, most markedly on steady-state inactivation. Channels altered Henan Agricultural University, Zhengzhou, Henan, China and Department of by BTX fail to inactivate over a range of depolarizing potentials. By contrast, Entomology, Michigan State University, East Lansing, MI, USA, 3McMaster the heptynoate derivative does not completely abrogate channel inactivation at University, Hamilton, ON, Canada. weakly depolarizing potentials; however, at membrane potentials >-70 mV, Pyrethroid insecticides, which target insect voltage-gated sodium channels, are channel inactivation is inhibited. To our knowledge, this is the first report of widely used to control pests in agriculture and insect vectors that transmit human a BTX derivative that decouples the effects on threshold activation and diseases. However, some insecticides have negative impact on beneficial organ- steady-state inactivation, two hallmarks of BTX binding to NaVs. A channel isms including bees. Honey bees and bumble bees are highly sensitive to most py- state model to explain the observed differences is proposed. rethroids, but are resistant to a particular pyrethroid, tau-fluvalinate (t-FVL). The mechanism of bee resistance to t-FVL remains elusive. Here we functionally 3138-Pos Board B346 characterized the sodium channel BiNav1-1 from the common eastern bumble Recombinant Expression of a Voltage Sensing Domain from Human bee (Bombus impatiens)inXenopus oocytes. We found that the channel is highly NaV1.7 sensitive to six commonly used pyrethroids, but is resistant to t-FVL. Our phylo- 1,2 1 1,2 Ryan V. Schroder , Ping Wang , Sebastien F. Poget . genetic and mutational analyses revealed that three residues, which are conserved 1Department of Chemistry, CUNY College of Staten Island, Staten Island, t 2 in sodium channels from 12 bee species, underlie resistance to -FVL or hyper- NY, USA, Ph.D. Program in Biochemistry, Graduate Center of the City sensitivity to other pyrethroids. We further have built a NavMs-based model of University of New York, New York, NY, USA. the open BiNav1-1 channel and used Monte Carlo energy minimizations to Voltage gated sodium channels (VGSCs) are involved with excitatory cell dock deltamethrin DMT and t-FVL in the channel. The computer modeling signal response and propagation. The human voltage-gated sodium channel and model-driven mutagenesis uncovered four additional residues in the PyR1 NaV1.7 has been shown to be involved with neuronal pain response. The cur- and PyR2 pyrethroid receptor sites that contribute to the unique selectivity of rent standard for pain relief medications involves opiates, derivative molecules the bumble bee sodium channel to t-FVL versus other pyrethroids. Our results of opium, which have a high risk of addiction and misuse. Therefore, drugs that suggest that, unlike most pyrethroids, t-FVL lacks strong interactions with can inhibit NaV1.7 as an alternative target could have great potential as a new PyR1 and PyR2. Our study contributes to understanding of a long-standing class of pain medications that would not pose the problem of addiction. The enigma of selective pyrethroid toxicity in bees and may be used to assist in future three-dimensional structure of NaV1.7 is currently unknown. Knowledge of modifications of pyrethroids to achieve selective control of pests with minimal such a high-resolution structure could lead to major breakthroughs in the field effects on non-target organisms. Supported by NIH, NSERC and RSF. of rational drug design for pain therapeutics. In this study, we intend to gain structural information about this channel via NMR spectroscopy. Due to the 3141-Pos Board B349 size constraints of solution NMR, we are focusing our investigation on the iso- Selective Conduction in a Human Sodium Channel Controlled by Ion- lated voltage sensing domain (VSD) from repeat II of the channel. We have Carboxylate and Lysine Interactions successfully expressed this VSD using the Trp-D-LE inclusion body system. Emelie Flood, Celine Boiteux, Toby W. Allen. We designed a fusion construct containing an Asn-Gly sequence in between School of Science, RMIT University, Melbourne, Victoria, Australia. the fusion partners for hydroxylamine cleavage. We are currently working Bacterial and human voltage-gated sodium channels exhibit similar ion se- on refolding the cleaved VSD by exchanging denaturing detergent with lectivities, despite distinct EEEE and DEKA signature sequences. The phospholipids as membrane mimetics. The NaV1.7 repeat II VSD reconstituted recent solution of high-resolution structures for simpler bacterial sodium

BPJ 8735_8739 634a Wednesday, February 21, 2018 channels has allowed us to learn much about ion conduction mechanisms, 3144-Pos Board B352 but our understanding of their mammalian counterparts remains limited. A The Role NaV1.9 in Somatosensory Signaling 1 1 1 model of the human Nav1.2 channel has been constructed by grafting resi- Juan Salvatierra , Frank Bosmans , Marcelo Diaz-Bustamante , dues of its selectivity filter and external vestibular region onto the bacterial James Meixiong2, Xinzhong Dong2. 1 NavRh channel. Multi-microsecond fully atomistic simulations, using the Physiology Department, Johns Hopkins University, Baltimore, MD, USA, DE Shaw Anton supercomputer, capture long time-scale ion and protein 2Neuroscience Department, Johns Hopkins University, Baltimore, MD, USA. movements associated with ion permeation. We observe Naþ knock-on con- Our bodies are continuously bombarded by a deluge of different sensory stimuli, duction facilitated by low energy multiple carboxylate-Naþ complexes, akin from the light touch of a lover to the harsh scalding of boiling water. As such, our to the bacterial channels. These complexes draw carboxylates from both the somatosensory system is fine tuned to perceive and transmit these stimuli with DEKA and vestibular EEDD rings. We observe that lysine, when charged, exquisite fidelity at millisecond time scales. Due to their importance in action po- actively participates in ion movements, with the critical step involving the tential generation, voltage-gated sodium (Nav) channels are key players in this þ formation of a complex that collectively binds Na and lysine in a high field signal transmission process. Of the nine Nav channel subtypes, Nav1.7, þ þ strength site. In contrast, multiple K complexes are disfavored, and K - Nav1.8, and Nav1.9 are particularly important for the transmission of sensory lysine complexes nonexistent. Instead, lysine acts as an plug that attenuates modalities, such as pain and itch, due to their strategic expression in the somato- þ þ þ the flow of K ions. These mechanisms, controlled by different Na and K sensory nervous system. Although Nav1.7 and Nav1.8 have been extensively þ affinities for high field strength complexes, helps explain Na selectivity studied with respect to specific modalities, the role of Nav1.9 remains unclear. across all sodium channels. However, accumulating evidence suggests a prominent role for this channel sub- type in nociception and pruriception. Challenges in expressing this channel in a 3142-Pos Board B350 heterologous system and a poor understanding of its tissue distribution compli- Thermal Melt Circular Dichroism Spectroscopy of Membrane Proteins as cate the formation of a hypothesis concerning the transmission of sensory modal- a Tool for Cryo-EM Preparations ities by Nav1.9. To overcome these obstacles, we developed a Nav1.9 stable cell Altin Sula, Sam M. Ireland, Jennifer Booker, B.A. Wallace. line and an sfGFP-tagged Nav1.9 mouse line, which allows us to refine tissue dis- Institute of Structural and Molecular Biology, Birkbeck College, University tribution and function of this channel. Based on reports of patients with a gain-of- of London, London, United Kingdom. function mutation in this channel that experience complete absence of pain, yet Detergents, amphiphiles, and other amphipathic environments are commonly unrelenting itch, we sought to explore the role of Nav1.9 in these sensory modal- used for cryo-electron microscopy sample preparations of membrane proteins. ities using our newly generated mouse and cell lines. Although Nav1.9 KO mice As such preps often require substantial amounts of effort to produce and survey show mild reduction in inflammatory pain, they show drastic reductions in itch samples suitable for cryoEM, any method that can decrease the amount of mi- behaviors. We are currently using several genetic mouse lines and known pruri- croscope time and effort can be of value for structure determination studies. In togens to investigate the link between pruriception and Nav1.9 activity. Our goal this study, circular dichroism (CD) spectroscopy as the method for testing all is to determine how modulation of Nav1.9 via these pruritogens alters somato- these potential types of sample preparations, and the NavMs voltage-gated so- sensory perception. dium channel (whose high resolution we have determined [Sula, Booker et al (2017) Nature Comms. 8:14205]) was used as the exemplar protein for the tests. 3145-Pos Board B353 The first comparison made was of the CD-determined secondary structure with Pyrroline Derivatives of Mexiletine-Like Compounds Have Dual Activity the crystal structure. In nearly all sample types examined, at room temperature as Use-Dependent Sodium Channel Blockers and Antioxidant the structures were identical, and so did not provide any basis for selection of Michela De Bellis, Francesca Sanarica, Alessia Carocci, Giovanni Lentini, the amphiphile to use. Then we examined their thermal stabilities by CD, both Sabata Pierno, Diana Conte Camerino, Annamaria De Luca. in terms of the Tm of the sample and the principal component analyses of the Dept Pharmacy - Drug Sciences, University of Bari, Bari, Italy. unfolding pathways. This enabled us to identify environments in which the pro- A focused screening of synthesized analogues of mexiletine (Mex) helped us to tein was most stable, and we have used them to produce single particle clarify the molecular requisites to modulate Nav1.4 channel, disclosing a key (initially, negative stain) samples. role of substituents on the asymmetric center and on the pharmacophore amino- Supported by grants from the BBSRC. terminal group. We investigated further structural changes by introducing a pyrro- line ring on the amino-terminal group of Mex (VM11) and of its potent 3143-Pos Board B351 use-dependent isopropyl derivative (CI16). The pyrroline group is expected to Development of High throughput Electrophysiology Assays of Recombi- change the physicochemical properties (logP and pKa) improving both the hydro- nant Nav1.9 Channels phobic interaction with the binding-site and the use-dependent behavior, while Matthew D. Fuller1, Chris Mathes1, Zhixin Lin1, Mark L. Chapman1, possibly conferring an additional antioxidant action. Vaseline gap voltage- Nina Brinkwirth2, Claudia Haarmann2, Michael George2, Niels Fertig2, clamp recordings on adult frog myofibers were performed to assess the tonic Andrea Bruggemann€ 2. 1 2 and use-dependent block of peak INa by pyrroline derivatives. VM11 and CI16 Icagen, Durham, NC, USA, Nanion, Munich, Germany. were 3 and 6-fold more potent than Mex in producing a tonic-block The Nav1.9 voltage dependent sodium channel has long been a desirable target (IC50=23.450.9mM and 12.650.2mM, respectively). Both have increased use- for potential pain therapeutics owing to the highly restricted expression in pe- dependent behavior, with CI16 showing a 40-fold increase of potency with respect ripheral sensory neurons. More recently human genetic evidence has been pro- to Mex during 10Hz stimulation (IC50=0.650.1mM), resulting the strongest duced which directly links Nav1.9 to human pain disorders, increasing the use-dependent Mex-like compound so far. The derivatives behaved as bothresting motivation to develop pharmacological screening methodologies. The primary and inactivated channel blockers, as estimated by the weak voltage-dependent ef- barrier to assay development has been removed with the generation of robust fect and by the shift of the hN-curves. The experimental data fitted with the mo- recombinant Nav1.9 cell lines which recapitulate the unique biophysical prop- lecular modeling simulation based on previously proposed interaction of main erties of the channel. We have previously reported the generation and charac- pharmacophores with NaV1.4 binding site. Both VM11 and CI16 showed a terization of human, mouse and rat Nav1.9 channels stably expressed in human remarkable cytoprotection in C2C12 myoblasts against H2O2-dependent damage, HEK-293 cells which exhibit the slowly activating and inactivating inward so- at concentrations close to the IC50 for blocking Nav1.4, resulting more potent than dium channel currents that are characteristic of native Nav1.9 (Lin et al, 2016). their parent compounds. Then, the novel pyrroline compounds have increased Nanion’s SyncroPatch 384PE high throughput electrophysiology (HTEP) sys- therapeutic features as sodium channel blockers and an interesting cytoprotective tem allows for high fidelity assessment and detailed biophysical characteriza- activity. The overall profile enlarges the pharmacological potential from channe- tion of NaV channels with up to 384 simultaneous recordings. To accelerate lopathies to myopathies in which alteration of excitation-contraction coupling is the pharmacological characterization of NaV1.9 we have sought to develop paralleled by oxidative-stress, i.e. muscular dystrophies (supported by DPP-NL). HTEP assays on the Syncropatch platform. Here we report that SyncroPatch re- cordings of recombinant Nav1.9 channels can recapitulate the pharmacological 3146-Pos Board B354 properties of Nav1.9. Our results show stable recordings of human and rat or- Population-Based Mathematical Modeling to Deduce Disease-Causing D thologs of Nav1.9. Recordings in the absence and presence of known Nav1.9 Cardiac Na Channel Gating Defects inhibitors (lidocaine, tetracaine, TC-N 1752) were robust and stable, allowing Chiara Campana1, Ivan Gando2, Reina Bianca Tan2, Frank Cecchin2, for accurate measurements of compound IC50s. Additionally, biophysical William A. Coetzee2, Eric A. Sobie1. properties of voltage-dependent activation and inactivation properties were 1Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New characterized. Collectively, these results show that Nav1.9 channels can stud- York, NY, USA, 2Pediatrics, NYU School of Medicine, New York, NY, USA. þ ied on high throughput electrophysiological systems, facilitating the search for Mutations in the SCN5A gene, which encodes the primary cardiac Na channel novel pain therapeutics. (Nav1.5), can cause arrhythmic disorders such as Type 3 Long QT Syndrome

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(LQT3). A pediatric patient with severe ventricular arrhythmias was recently based on the NavAb crystal structure (PDB ID: 3RVY). To capitalize on recent determined to carry a missense mutation in SCN5A that putatively leads to a advances, we have updated our hNav1.7 homology model using the recent replacement of glutamine by proline at residue 1475 (Q1475P). We sought to cryo-EM structure of the electric eel Nav1.4 channel (PDB ID: 5XSY) as a tem- uncover, through population-based mathematical modeling of ion channel plate. To test predicted pairwise contacts between toxin and channel, point mu- gating: (1) what defects in gating at the molecular level could account for tations of hNav1.7 and conotoxin KIIIA in the putative binding site have been cellular electrophysiological recordings; and (2) whether these alterations to generated. We have identified several residues that alter the kinetics of toxin channel gating could reproduce the patient’s LQT3 phenotype. Electrophysi- binding, notably acidic residues on the DII pore-helix. Our experimental and ology experiments, performed in HEK293 cells, demonstrated that, compared modeling results may be useful for rational design of novel peptides to inhibit with Wild Type (WT) Nav1.5 channels, Q1475P channels exhibited: (1) a pos- hNav1.7 with high affinity and selectivity. itive shift in the activation curve; (2) a positive shift in the inactivation curve; (3) faster inactivation; and (4) faster recovery from inactivation. Simulations 3149-Pos Board B357 attempted to recapitulate these results by randomizing parameters in a pub- Intermediate States and Structural Ensembles of Calmodulin Bound to the NaV1.2 IQ Motif lished Markov model of Nav1.5 gating, generating a large (n=10,000) popula- 1 2 1 1 tion of channel variants, filtering to match WT and Q1475P channel behaviors. Ryan Mahling , Adina M. Kilpatrick , Holly M. Isbell , Madeline A. Shea . 1Biochemistry, University of Iowa Carver College of Medicine, Iowa City, This produced subpopulations of channel variants describing the gating of 2 either genotype. The analysis suggested that the channel gating steps most IA, USA, Physics, Drake University, Des Moines, IA, USA. likely to be affected by the mutation include transitions from: (1) open to The voltage-gated sodium channel 1.2 (NaV1.2) is critical for initiating and closed, (2) inactivated to closed; and (3) closed to inactivated. In addition, propagating action potentials. Multiple auxiliary proteins, including calmodulin (CaM), regulate NaV1.2. An IQ motif (residues 1901-1927: IQP) in the C-termi- when simulated Nav1.5 channels were incorporated into a model of the human ventricular action potential, Q1475P channels were more likely than WT chan- nal tail of NaV1.2 binds to the C-domain of CaM (CaMC) tightly (Kd 6 nM) un- der apo (calcium-free) conditions. Calcium binding to CaM lowers its affinity nels to produce arrhythmogenic early afterdepolarizations (EADs). The results 2þ provide important insight into this patient’s LQT3 phenotype and demonstrate (Kd 20 nM) for NaV1.2 IQP. Solution structures of apo (2KXW) and (Ca )2- how population-based modeling can generate mechanistic hypotheses about al- CaMC (2M5E) bound to NaV1.2 IQP showed calcium reversed the orientation terations in ion channel gating that result from mutations. of CaMC on nested, anti-parallel sites within the IQ motif (Biophysical Chem- istry (2017) 224:1-19). Backbone NMR assignments of apo 13C,15N-CaM and 13 15 13 15 3147-Pos Board B355 C, N-CaMC bound to C, N- NaV1.2-IQP showed the CaMC-IQ interface was identical, while CaMN was unaffected by IQP (Biomol NMR Assts (2017) INA Loss-of-Function by Compound Variants in SCN5A from a Large Founder Population with Excess Sudden Cardiac Death 11:297–303; BMRB 27094, 27095). Using solution NMR, an ensemble of struc- Cristina Altrocchi1, Roel R.L. Sp€atjens1, Henry Sutanto1, tures of apo CaM bound to NaV1.2 IQP were determined with CYANA, and 2 1 1 refined in Amber; NaV1.2 IQP was bound to CaMC but no NOEs were observed Rachel M.A. ter Bekke , Sandrine Seyen , Jordi Heijman , 15 Cristina Moreno3, Paul G.A. Volders2. between NaV1.2 IQP and CaMN. Stoichiometric calcium titrations of N-CaM- 1 2 IQP monitored by NMR, and equilibrium titrations monitored by fluorescence Cardiology, Maastricht University, Maastricht, Netherlands, Cardiology, 2þ 3 demonstrated all 4 Ca -binding sites in CaM saturated simultaneously, indi- Maastricht University Medical Center, Maastricht, Netherlands, National 2þ Institute of Neurological Disorders and Stroke (NIH), Bethesda, MD, USA. cating a reduction in the Ca -binding affinity of sites III and IV in CaMC to Recent clinical investigations in a Dutch-German founder population with excess match the intrinsic calcium-binding affinity of CaMN. Both domains retained cooperative calcium binding. Consistent with CaMN being equivalent, but inde- sudden cardiac death, revealed striking phenotypic heterogeneity: long QT- 15 pendent, to CaMC, residues in CaM- N-IQp responded monotonically to addi- syndrome, cardiac conduction disease, (drug-induced) Brugada syndrome, iso- 2þ rhythmic atrioventricular dissociation and overlap. Ventricular tachyarrhythmia tion of 4 Ca ions. NMR data suggested calcium-triggered reversal of CaM on often occurred during mental or physical stress. DNA sequencing identified the NaV1.2 IQP. The roles of NaV1.2 sequences flanking the IQ motif, and allosteric pathogenic SCN5A deletion mutation c.4850_4852delTCT, encoding for effects of additional auxiliary proteins are being explored. NIH R01 GM57001 Nav1.5-DelF1617, and a common polymorphism c.1673A>G, Nav1.5-H558R. (MAS), NIH T32 NS045549 (RM), UI CCOM FUTURE in Biomedicine (AMK). In the present study, we performed whole-cell patch-clamping on Chinese ham- 3150-Pos Board B358 ster ovary cells, transiently transfected with wild-type (WT) Nav1.5, Nav1.5- Calmodulin Regulation of Nav1.8 Channel DelF1617 or Nav1.5-DelF1617-H558R, combined with in-silico action potential Liang Hong, Meihong Zhang, Erin Lambers, Arvind Sridhar, Ambili Menon, (AP) simulations. Nav1.5-DelF1617 showed a significant peak INa density reduc- Dawood Darbar. tion compared to Nav1.5-WT, without changes in the steady-state activation and University of Illinois, Chicago, Chicago, IL, USA. channels availability. The time constant of inactivation was slower in Nav1.5- The voltage-gated Na channels play a key role in the generation and propagation DelF1617 and recovery from inactivation faster. The addition of H558R partially of the cardiac action potential (AP). Emerging data show that Nav1.8 channel is rescued the peak current density, without altering the decay rate of the fast inac- a modulator of cardiac conduction and plays a pivotal role in cardiac repolari- tivation, and slowing recovery from inactivation. No TTX-sensitive persistent zation. We and others have shown that Nav1.8 mutants are associated with atrial current was detected. AP simulation confirmed a reduction of the phase 0 upstroke fibrillation (AF) and Brugada syndrome (BrS). The voltage gated sodium chan- velocity for Nav1.5-DelF1617 and Nav1.5-DelF1617-H558R, due to a decreased nels contain a calmodulin(CaM)-binding IQ domain involved in channel inacti- peak INa. AP duration was slightly prolonged, especially at low pacing rate (1Hz). vation, here we examined the role of in CaM modulating Nav1.8 channel gating Sudden change in pacing rate or hypokalemia in mutant conditions did not evoke function, and found that CaM enhances slow inactivation of the Nav1.8 channel proarrhythmic responses. Taken together, our results demonstrated an INa loss-of- and hyperpolarizes steady-state inactivation curve of Na currents. The addition function by the compound variants Nav1.5-DelF1617 and Nav1.5-DelF1617- of intracellular 290-309, a CaM peptide antagonist, has opposite effects and H558R, which fits partially with the clinical phenotype. Investigations on proar- attenuates slow inactivation. In addition, the effects of CaM on the Nav1.8 chan- rhythmic triggers, such as cAMP stimulation of mutant INa, are ongoing. nel gating are disrupted in the Nav1.8 channel truncated IQ domain. We further tested the consequences of CaM on Nav1.8 mutants within the IQ domain asso- 3148-Pos Board B356 ciated with AF and BrS. Our findings suggest that CaM plays an important role Assessing the Structural Basis of m-Conotoxin KIIIA Inhibition of the in the regulation of Nav1.8 channel function in cardiac arrhythmia. Voltage-Gated Sodium Channel Nav1.7 Ian H. Kimball, Phuong T. Nguyen, Jon T. Sack, Vladimir Yarov-Yarovoy. 3151-Pos Board B359 Physiology & Membrane Biology, University of California, Davis, Davis, Calmodulin Recognition of Voltage-Gated Sodium Channels NaV1.1, CA, USA. NaV1.4 and NaV1.7 The voltage-gated sodium channel subtype Nav1.7 has been shown to play a Holly M. Isbell1, Adina M. Kilpatrick2, Zesen Lin1, Ryan Mahling1, critical role in pain signaling, making it an important drug target. A number Madeline A. Shea1. of peptide toxins from cone snails (conotoxins) inhibit sodium conduction 1Biochemistry, Univ. of Iowa Carver College of Medicine, Iowa City, IA, through Nav1.7 with high affinity, and make potential scaffolds for selective USA, 2Physics and Astronomy, Drake University, Des Moines, IA, USA. channel inhibitors. However, the atomistic details of protein-protein interac- Voltage-gated sodium channels (Nav) are responsible for initiating and propa- tions between conotoxins and Nav channels necessary for rational design of gating action potentials by allowing inward sodium flux. Although the topol- such inhibitors remain unclear. Our study aims to further define the molecular ogies of NaV1.1 (CNS), NaV1.4 (skeletal) and NaV1.7 (PNS & CNS) are determinants of m-conotoxin KIIIA action against the human Nav1.7 channel similar, their roles differ. Defects in NaV1.1 cause Dravet syndrome, severe ep- (hNav1.7). We have previously used Rosetta computational modeling software ilepsy, sleep disturbance and cognitive impairment, while defects in NaV1.7 to generate preliminary molecular models of the hNav1.7 pore-forming domain alter pain perception and response to anesthetics (e.g., Lidocaine). Recently,

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NaV1.7 also was recognized to act in the central nervous system and play a role to mexiletine. We hypothesized that altered voltage-sensing domain (VSD) in weight regulation. Calmodulin (CaM) binds to an IQ motif (IQBXFRGFX, function determines mutation-dependent mexiletine blockade. B = basic, F = aromatic) found in the C-terminal tail of all NaV isoforms, and Methods:NaV1.5 contains four domains (DI-DIV), each with a VSD. We pre- contributes to the calcium-dependent inactivation of some. The free energies of viously created four channel constructs that can track each VSD’s conformation 2þ apo (calcium-free) CaM and (Ca )4-CaM binding to the isolated IQ motif of changes when tethered with a fluorophore. VSD-tracking fluorescence and NaV1.1, NaV1.4 and NaV1.7 all had Kd values < 100 nM. However, for NaV1.1 ionic currents were recorded simultaneously for WT and LQT3-linked channels 2þ and NaV1.7, binding to apo CaM was slightly favored over (Ca )4-CaM, while with or without mexiletine. no calcium-dependent difference was seen for NaV1.4. Thermal denaturations, Results: Mexiletine blockade of WT channel stabilized the DIII-VSD activated calcium titrations, and solution NMR studies showed that each IQp only per- conformation. Among 14 common LQT mutations, 10 showed varied DIII- turbed properties of the C-domain of CaM (CaMC), as seen for NaV1.2 under VSD activation voltage-dependence, despite distal locations of some, e.g. the same conditions (Biophysical Chemistry (2017) 224:1-19, Biomol NMR R1626P(DIV-VSD) and E1784K(C-terminus). DIII-VSD activation shifts Assts (2017) 11:297–303). The CaM N-domain (CaMN) retained characteristics correlate strongly with tonic block by mexiletine(R=0.91), suggesting that of free CaM while CaMC was bound to the IQp. To gain structural and thermo- the DIII-VSD conformation determines mexiletine blockade at the cardiomyo- dynamic insight into separable roles of CaMN and CaMC in regulating confor- cyte resting potential. Decoupling the DIII-VSD from the pore eliminated mational change of the NaV C-terminal tails, we are determining solution NMR mutation-specific mexiletine sensitivity, suggesting that the activated DIII- structures of each, and studying effects of mutations in both CaMN and CaMC, VSD facilitates mexiletine block by promoting pro-binding conformations as well as the roles of auxiliary proteins. Support: UI ICRU Fellowships (HI within the DIII pore. Unlike WT, mexiletine blockade of the inactivation- and ZL), NIH T32 NS045549 (RM), UI CCOM FUTURE in Biomedicine impaired channel (F1486Q) did not shift the DIII-VSD, implying that during (AMK), and NIH R01 GM57001 (MAS). the action potential plateau, the DIII-VSD and inactivation gate coordinate to regulate mexiletine blockade. To predict use-dependent block (UDB) and 3152-Pos Board B360 QT-interval shortening (DQT) from channel gating parameters, we applied a Hypokalemic Periodic Paralysis Cases with Substitutions from Arginine to machine learning approach, partial least-square regression(PLSR). Our PLSR Lysine in the Voltage Sensor model showed that changes in DIII-VSD activation, steady-state inactivation Maki Nakaza1, Tomoya Kubota1, Savine Vicart2, Daisuke Watanabe3, and slow recovery from inactivation parameters are strongly predictive of 4 5,6 2 5 Norito Kokubun , Mitsuru Furuta , Damien Sternberg , Yosuke Kokunai , mexiletine-linked changes in UDB and DQT (RUDB=0.92, RDQT=0.82). Tatsuya Abe3, Bertrand Fontaine2, Masanori P. Takahashi1. Conclusion: Traditional anti-arrhythmic block models have focused on the role 1Department of Functional Diagnostic Science, Osaka University Graduate of channel inactivation. We have revised this model to include an essential role School of Medicine, Suita. Osaka, Japan, 2Departement de Neurologie, CMR of the DIII-VSD. Canalopathies Neuromusculaires, ICM-UMR 1127, INSERM, UPMC, CNRS, Hoˆpital Universitaire Pitie-Salpeˆtrie`re, Paris, France, 3Department of 3154-Pos Board B362 Neurology, National Hakone Hospital, Odawara. Kanagawa, Japan, Effects of Cannabidiol on Human Nav Channels 4Department of Neurology, Dokkyo Medical University, Tochigi, Japan, Mohammad-Reza Ghovanloo1,2, Noah Gregory Shuart2, 5Department of Neurology, Osaka University Graduate School of Medicine, Janette Mezeyova2, Peter C. Ruben1, Samuel J. Goodchild2. Suita. Osaka, Japan, 6Department of Neurology, Kansai Rosai Hospital, 1Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, Amagasaki, Hyogo, Japan. BC, Canada, 2Cellular and Molecular Biology, Xenon Pharmaceuticals, Familial hypokalemic periodic paralysis (HypoPP) is a rare skeletal muscle dis- Burnaby, BC, Canada. ease caused by the dysregulation of excitability in salcolemma. HypoPP is Cannabis sativa contains many related compounds known as phytocannabi- characterized by repeated episodic paralytic attacks with hypokalemia and noids. The main psychoactive and non-psychoactive compounds are 9-tetrahy- several mutations in CACNA1S gene coding Cav1.1 and SCN4A gene coding drocannabidiol (THC) and cannabidiol (CBD), respectively. Unlike THC, CBD Nav1.4 have been identified as causative mutations. Most of the mutations has a low affinity for Cannabinoid receptors. Cannabis products are receiving are substitution to non-charged amino acid from the positively charged arginine attention as potential therapeutics for neurological conditions including epi- (R) in segment 4 (S4) of the voltage sensor in either Cav1.1 or Nav1.4. Bio- lepsy. Much of the evidence for clinical efficacy has been from case reports physical experiments demonstrated that the loss of the charge in S4 by the or smaller surveys until a recent phase III trial of CBD treatment for Dravet arginine-to-non-charged substitution creates a leak current called ‘‘gating syndrome demonstrated efficacy. The mechanisms for the anticonvulsant ef- pore current’’. Since the discovery, ‘‘the gating pore current theory’’ has fects of CBD are unclear and likely involve both cannabinoid and non- been suggested as the common pathogenic feature in HypoPP. However, the cannabinoid receptor pathways. CBD is reported to modulate several ion chan- pathological mechanism from generation of ‘‘gating pore current’’ to paralytic nels, including: TRP, T-type calcium, and sodium channels (Nav). Evaluating attack is still unknown. In this report, we have identified five cases with an therapeutic mechanisms and safety of CBD demands a richer understanding of arginine(R)-to-lysine (K) substitution in voltage sensor, which retained positive its interactions with CNS targets. We used the Qube-384, a high-throughput net charge of S4 ; one is a Japanese boy with R219K mutation in SCN4A gene automated patch-clamp platform, to characterize the functional effects of and the other four are one Japanese man, one French man and two French CBD on Nav’s. Our results show that CBD non-selectively inhibits Nav1.1- brothers with R897K mutation in CACNA1S gene. All cases presented typical 1.7, with 1.9-3.8 mM potency, suggesting that CBD-Nav interactions occur at paralytic episodes with hypokalemia and, in four of five cases, significant physiologically relevant concentrations (in vivo brain exposures 10 mM). decrement in the compound action potential was observed with prolonged ex- Cooperative block is indicated by a steep Hill slope of 3, suggesting multiple ercise test. From Nav1.4-R219K mutant channel expressed in vitro,we interaction sites. CBD exhibits resting-state block (IC5012 mM, 100 mV), measured the ionic current by patch-clamp technique and the gating pore cur- and becomes more potent at depolarized potentials (IC502 mM, 45 mV). rent by cut-open voltage clamp technique. Nav1.4-R219K revealed gating pore This state-dependence has similarities to classic pore-blocking local anesthetics current, but the size of the conductance was smaller compared to that observed (LAs); CBD also slows recovery from slow and fast inactivation, supporting in other mutant HypoPP-Nav1.4 channels previously reported. Our data might that binding preferentially stabilizes inactivated states. To examine whether provide a new insight in pathogenic mechanism of HypoPP. CBD interacted with the canonical LA binding site we made a point mutation in the Nav1.1 pore, F1763A, which only mildly decreased potency (2-fold) 3153-Pos Board B361 suggesting that the CBD mechanism of inhibition involves more delocalized Molecular Basis of Mexiletine Response Variability in Sodium Channels pore interactions than LAs. Lastly, the CBD block of Nav’s is temperature- with Long QT Mutations dependent, as its potency increases at lower temperatures. Wandi Zhu1, Taylor L. Voelker1, Jonathan D. Moreno1,2, Andrea Mazzanti3, Silvia G. Priori3,4, Jonathan R. Silva1. 1Biomedical Engineering, Washington University in St. Louis, Saint Louis, Posters: Voltage-gated Ca Channels MO, USA, 2Cardiology, Washington University in St. Louis, Saint Louis, MO, USA, 3Molecular Cardiology, IRCCS Salvatore Maugeri Foundation, 3155-Pos Board B363 Pavia, Italy, 4Department of Molecular Medicine, University of Pavia, Pavia, Gating Defects of a CACNA1D Missense Mutation Linked to a Develop- Italy. mental Disorder of Unknown Cause Background: Long-QT syndrome type-3 (LQT3) is a life-threatening disease Nadja Hofer, Joerg Striessnig. caused by mutations in the cardiac sodium channel (NaV1.5). The class-Ib Pharmacology and Toxicology, Institute of Pharmacy, Innsbruck, Austria. 2þ anti-arrhythmic mexiletine was proposed as an effective therapy for LQT3. Background: Low voltage-gated Cav1.3 voltage-gated L-type Ca -channels However, clinical studies showed that LQT3 variants have distinct sensitivities are key regulators of neuronal excitability controlling neuronal development

BPJ 8735_8739 Wednesday, February 21, 2018 637a and different types of learning and memory. Recently, large-scale genetic anal- Activation of voltage-gated calcium channels is controlled by four separate ysis revealed de-novo missense mutations in their pore-forming a1-subunit voltage sensing domains (VSD I-IV). Among the members of the CaV channel (CACNA1D gene) in 6 patients associated with a neurodevelopmental syn- family the skeletal muscle CaV1.1a is atypical because of its low voltage sensi- drome including varying degrees of sporadic autism spectrum disorder tivity, low current density, and its slow activation kinetics. Alternative splicing (ASD, G407R), intellectual disability (A749G), neurological manifestations of exon 29, located in the extracellular IVS3-S4 linker, results in dramatically (including seizures, V401L) and endocrine symptoms (G403D, I750M). A increased voltage sensitivity and current density, demonstrating the importance typical hallmark of these mutations are severe gating changes compatible of VSD IV for controlling CaV1.1 gating properties. Using structural modeling with a gain-of-channel-function. Here we investigated if similar gating changes combined with mutagenesis and electrophysiology, our team recently discov- are observed in a de-novo CACNA1D mutation (IIS4-S5 linker, Cav1.3 a1mut) ered that inclusion of exon29 in CaV1.1a disrupts the interactions between which could explain symptoms in a patient diagnosed with a severe develop- the two outermost gating charges (R1, R2) in IVS4 and a countercharge (D4) mental disorder of unknown cause. in IVS3. Our model predicts that in the absence of exon 29 these interactions Methods: Mutant (Cav1.3 a1mut) and wild-type Cav1.3 a1 were co-expressed facilitate the transition from intermediate state two into the activated state, re- together with b3 and a2d-1 subunits in tsA-201 cells and calcium currents sulting in increased voltage sensitivity, and stabilize the activated state, leading (15mM) were measured using the whole cell patch-clamp technique. to an increased opening probability. Although the countercharge D4 as well as Results: Very similar to the previously characterized mutation V401L (IS6), alternative splicing of the S3-S4 linker in VSD IV are conserved in all L-type A749G and I750M (IIS6), Cav1.3 a1mut dramatically shifted the voltage- calcium channels, our results show that, while in CaV1.1 this mechanism deter- dependence of Cav1.3 steady-state activation and inactivation to more negative mines its characteristic gating properties, in CaV1.2 and CaV1.3 D4 is involved voltages (20 mV) without slowing of inactivation. A complete biophysical in fine-tuning voltage sensitivity. Applying patch-clamp analysis of CaV1.1 analysis revealed that these changes are compatible with a mutational gain- with and without exon 29 we characterized the single-channel properties of of-function phenotype. CaV1.1a and CaV1.1e and refined our mechanistic model of the VSD IV tran- Conclusion: By demonstrating the typical gating changes previously shown by sition into the activated state. Predictions of the model are being tested by us for CACNA1D de-novo missense mutations we propose that Cav1.3 a1mut single-channel analysis of mutated CaV1.1 constructs in order to reveal how also explains the symptoms in this patient with a severe developmental disor- alteration of the D4-R1/R2 interactions affected the channel gating properties. der. Patients carrying such mutations may benefit from treatment with already þ available L-type Ca2 -channel blockers, such as nimodipine. Such CACNA1D 3158-Pos Board B366 missense mutations are likely underreported in large-scale genetic analyses. RGK Proteins Preferentially Inhibit Fast-Inactivating Voltage-Gated Support: Austrian Science Fund (FWF F4402, W1101). Calcium Channels: Implications for Human Disease Salma Allam1, Rose Levenson-Palmer2, Zuleen Chia Chang1, 3156-Pos Board B364 Kaur Sukhjinder1, Scott Dobbins2, Jian Yang2, Zafir Buraei1. 1 2 DHEA-Induced Inhibition of ICaL in Arterial Smooth Muscle Cells. Pace University, New York, NY, USA, Columbia University, New York, Involvement of Glucose-6-Phosphate Dehydrogenase and GPCR Signaling NY, USA. Rikuo Ochi1, Sukrutha Chettimada2, Sachin A. Gupte1. Voltage-gated calcium channels (VGCC) are critical for nerve, heart, and skel- 1Biochemistry, Molecular Biology and Pharmacology, New York Medical etal muscle function, and their mutations can cause neurological and cardiovas- College, Valhalla, New York and University of South Alabama, Mobile, AL, cular disease. Some of these mutations alter channel inactivation, leading to USA, 2Biochemistry and Molecular Biology, Harvard Medical School, aberrant Ca2þ influx into cells. For example, Timothy syndrome (TS) is char- Boston, Mass and University of South Alabama, Mobile, AL, USA. acterized by severe cardiomyopathy, and is often accompanied by autism spec- Dehydroepiandrosterone (DHEA) is a hormone abundantly released from trum disorder. The abnormalities arise from a point mutation in the L-type adrenal cortex as DHEA-sulfate (DHEAS). DHEA is uncompetitive inhibitor VGCC that slows channel inactivation. On the other hand, some types of of glucose-phosphate-dehydrogenase (G6PD) which reduces NADPþ to migraine and epilepsy are associated with point mutations that speed the inac- NADPH. Epiandrosterone (EPI), a metabolite of DHEA, inhibits ICa,L in tivation of P/Q-type VGCC. RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) ventricular myocytes. Mechanism and modulation of DHEA-induced inhibi- are small GTPases that strongly inhibit L-, N-, P/Q- and R-type VGCC. 2þ tion of ICa,L werestudiedbyrecordingwhole-cellICa,L (Ba current) in They are difficult to study because their overexpression nearly completely abol- A7r5 arterial smooth muscle cells (ASMCs) and bovine coronary ASMCs. ishes VGCC currents. Thus, we studied RGK-mediated inhibition using two- DHEA produced voltage-dependent (VDI) and voltage-independent inhibi- electrode voltage clamp (TEVC) in Xenopus oocytes, where we titrated the tion (VIDI) of ICa,L. VDI decreased ratio (r500)ofICa,L amplitude at 500 amount of injected RNA to achieve a more physiological level of VGCC inhi- ms (I500) to peak amplitude, and shifted quasi-steady state inactivation curve bition of 50%. Under these conditions, we discovered that mutations that (fN-V) to the left. Dose- and voltage-dependence of DHEA-induced inhibi- speed inactivation, such as those that cause familial hemiplegic migraine, tion of ICa,L was analyzed by repetitive pulses from holding potentials (HPs) turn channels hypersensitive to Gem inhibition. Remarkably, when we chal- from 80 to 20 mV. IC50 of DHEA-induced inhibition decreased with de- lenged the non-inactivating TS channel with Gem, we found that it was insen- polarization of HP. fN-HP relationship was more negative than fN-V ob- sitive to inhibition. Furthermore, expressing VGCC with the b2a subunit, a tained by 2 s-pre-pulse and both were shifted to the left by DHEA. Ca2þ channel auxiliary subunit known to slow channel inactivation, also Window ICa,L (IWD) calculated from I-V and fN-HP fittedwellwithrecorded weakened Gem inhibition.This could potentially occur if Gem were to trap IWD. DHEA strongly inhibited these IWDs. Depletion of cholesterol by channels in the inactivated state, thereby inhibiting inactivating channels, while MbCD shifted fN-V to the right and decreased DHEA-induced VDI. Intra- sparing non-inactivating channels. Our additional findings corroborate this cellular dialysis of GDP-b-S, a blocker of GPCR signaling, shifted I-V since channel recovery from inactivation is much slower in the presence of and fN-V relationships to the right. GDP-b-S significantly decreased efficacy Gem.These results shed light on a new type of VGCC regulation. In addition, of DHEA to induce inhibition of ICa,L. 6-Aminonicotinamide (6-AN), an in- our results point to RGKs as potential players in calcium channelopathies. hibitor of G6PD, induced progressive and irreversible inhibition of ICa,L by inducing VIDI. In the presence of 6-AN-induced VIDI, EPI induced addi- 3159-Pos Board B367 tional inhibition by VDI. High concentrations of DHEAS produced weak A Complex of RIM2alpha and RIM-Binding Protein 2 Stabilizes Slow Voltage-Dependent Inactivation of Cochlear Inner Hair Cell Cav1.3 VDI. DHEA-induced inhibition of G6PD underlies DHEA-induced VIDI 2D probably via thiol oxidation. GPCR signaling regulates voltage-dependent L-Type Ca Channels Nadine J. Ortner1, Alexandra Pinggera1, Anita Siller1, Nadja Hofer1, gating of ICa,L and shift of fN-V relationships to the right by GDP-b-S down- Niels Brandt2, Andrea Raffeiner3, Isabelle Lang2, Eduard Stefan3, regulates DHEA-induced inhibition of ICa,L. Gerald J. Obermair4, Jutta Engel2,Jo¨rg Striessnig1. 3157-Pos Board B365 1Department of Pharmacology and Toxicology, University of Innsbruck, 2 Probing the Voltage-Sensing Mechanism of CaV1.1 Calcium Channels at Innsbruck, Austria, Department of Biophysics, Saarland University, Single Channel Resolution Homburg, Germany, 3Institute of Biochemistry, University of Innsbruck, Pierre Coste de Bagneaux1, Bruno Benedetti2, Petronel Tuluc3, Innsbruck, Austria, 4Division of Physiology, Medical University Innsbruck, Marta Campiglio1, Bernhard Flucher1. Innsbruck, Austria. 1Section of Physiology, Medical University Innsbruck, Innsbruck, Austria, Cav1.3 L-type Ca2þ-channels (LTCCs) in cochlear inner hair cells (IHCs) 2Institute of Experimental Neuroregeneration Spinal Cord Injury and Tissue exhibit a unique slow current inactivation to enable tonic sound-induced neuro- Regeneration Center Salzburg (SCI-TReCS)Paracelsus Medizinische transmitter release. The slow Ca2þ-dependent inactivation can be explained by Privatuniversit€at, Salzburg, Austria, 3Department of Pharmacology and the calmodulin-competing binding of CaBP2, whereas the molecular mecha- Toxicology, University of Innsbruck, Innsbruck, Austria. nism underlying slow voltage-dependent inactivation (VDI) remains

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unexplained. Here we investigated if slow VDI is stabilized by the formation of gating normally, but molecular mechanism of PIP2 regulation remains unclear. a large signaling complex of Cav1.3 with the presynaptic scaffolding proteins It was recently reported that subcellular localization of b subunit is a key factor RIM and RIM-binding-protein (RBP). for the control of PIP2 sensitivity of CaV channels. Here we found that the intra- The expression of the putative interaction partners in IHCs was investigated via cellular movement of I-II linker in a1 subunit is important for determining the nested-PCR. RBP2 interaction with the C-terminus of the full length (Cav1.3L) PIP2 sensitivity of CaV channels. When the I-II linker was shifted to the plasma or short Cav1.3 isoform (Cav1.3S) was determined using GST-pulldowns and membrane, current inhibition by PIP2 depletion significantly decreased as like co-immunoprecipitations. The functional impact of coexpressed RIM and/or the responses triggered by membrane-tethered b subunit. Consistently we also RBP on VDI of Cav1.3 Ba2þ currents (15 mM,b3orb2a,a2d1) was evaluated found that inserting a flexible linker between membrane-tethered Lyn and GK via whole-cell patch-clamp recordings in tsA201-cells. domain of b subunit increased the PIP2 sensitivity of CaV channels. Polybasic We detected RIM2a and RBP2þ3 transcripts in mature IHCs and showed for motif at the C-terminal end of the I-II linker of CaV channels is a potential PIP2 the first time that b2e is the predominant IHC b2-splice variant. RBP2 could interaction site. Neutralization of the polybasic motif of I-II linker abolished only interact with the full-length Cav1.3 C-terminus. Using b3, coexpression PIP2 sensitivity of CaV channels. Together, our results indicate that the confor- of RIM2a and RBP2 significantly slowed Cav1.3L VDI to a similar extent mational shift of I-II linker to the plasma membrane is the key mechanism for than observed in IHCs, while RIM2a or RBP2 alone had less (RIM2a)orno decreasing the PIP2 sensitivity of CaV channels and this shift is mainly regu- effect (RBP2). This effect was splice variant-specific because when co- lated by auxiliary CaVb subunit in physiological condition. expressed with Cav1.3S RBP2 even antagonized the slowing of VDI by RIM2a. The palmitoylated b2a-isoform slowed VDI by its own preventing 3162-Pos Board B370 an additional effect by RIM2a and/or RBP2 (b2e is currently investigated). STAC Proteins Associate to the IQ Domain of CaV1.2 and Inhibit Calcium- We show that different mechanisms contribute to the slow VDI of IHCs. Dependent Inactivation Marta Campiglio1, Pierre Coste de Bagneaux1, Nadine J. Ortner2, RIM2a/RBP2 stabilize slow VDI in Cav1.3L-b3 LTCC complexes while 2 1 membrane-associated b2 variants (b2a and most probably also b2e) generate Petronel Tuluc , Bernhard E. Flucher . 1Physiology and Medical Physics, Medical University of Innsbruck, slow VDI without further modulation by RIM/RBP. 2 Supported by Austrian Science Fund (FWFP27809,W11010,F44150). Innsbruck, Austria, Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria. 3160-Pos Board B368 STAC proteins form an adaptor proteins family of three members (STAC1, Function of L-Type Calcium Channel Microdomain in Human Myocytes STAC2, and STAC3). The skeletal muscle isoform STAC3 was found to be from Hearts with Ischemic versus Dilated Cardiomyopathies essential for excitation-contraction (EC) coupling, and more specifically for Jose L. Sanchez-Alonso, Sophie Schobesberger, Claire E. Poulet, the function of CaV1.1 and for its mechanical coupling to the RyR. Recently Navneet Bhogal, Rasheda Chowdhury, Julia Gorelik. two distinct molecular domains in STAC3 were identified as necessary for National Heart and Lung Institute, Imperial College London, London, United its functional interaction with CaV1.1 : the C1 domain, which recruits STAC Kingdom. proteins to the calcium channel complex in skeletal muscle triads, and the Introduction: Recently we have revealed how the imbalance of L-type cal- SH3-1 domain, involved in EC coupling, which interacts with the CaV1.1 II- cium channels (LTCC) at the cell membrane could lead to arrhythmia in the III loop. These interaction sites are conserved among the three STAC proteins whole heart, in a rat model of myocardial infarction as well as in a group of and L-type calcium channels. However, the region in CaV1 channels interacting patients with dilated cardiomyopathy (DCM). In this study, we conduct a with the STAC C1 domain and the possible role of this interaction in neuronal more extensive comparison of myocytes from hearts with ischemic (ICM) CaV1 channels remained unidentified. Here, by testing CaV1.2/2.1 chimeras in -/- versus DCM. Also, the LTCC function is assessed in myocytes from patients dysgenic (CaV1.1 ) myotubes, we identified amino acids 1641-1668 in the C- with Left Ventricular Assistance Device (LVAD). terminus of CaV1.2 as necessary for association of STAC proteins. This Methods: Cardiomyocytes were isolated from ventricular tissue of donor sequence contains the IQ domain and alanine mutagenesis revealed that the normal hearts and hearts of patients with DCM or ICM with and without amino acids important for STAC association overlap with those binding the LVAD. LTCC current was recorded in distinct cellular compartments: T-tu- C-lobe calmodulin and mediating calcium-dependent inactivation (CDI) of bules (TT) and the areas between them or ‘‘crests’’, using super-resolution CaV1.2. Patch clamp recordings revealed that coexpression of STAC proteins scanning patch-clamp technique. Cell size and T-tubules density were results in CDI inhibition of CaV1.2 currents and that this inhibition depends measured with confocal microscopy under Di-8-ANEPPS staining. on the IQ domain of CaV1.2. Our data demonstrate that STAC proteins asso- Results: Cardiomyocytes from failing hearts develop a hypertrophic pheno- ciate to the IQ domain of CaV1.2, that this interaction results in an inhibition type, more severe in DCM than in ICM patients, which is partially reversed of CDI, and suggest a role of STAC1 and STAC2 as modulators of calcium en- with LVAD. In addition, these cells have less Z-grooves, lower TT density try through CaV1 channels. This work was supported by research grants from and a significantly increased LTCC density in the crest microdomains. Func- the Austrian Science Fund (FWF) T855 to M.C., P27031 and W1101 to tionally, LTCCs open probability revealed to be abnormally high in failing pa- B.E.F. and P27809 to Jo¨rg Striessnig. tients. These pro-arrhythmic LTCCs, as it has been proposed in relation with the AP prolongation, are, interestingly, in the crests of DCM and in the TT 3163-Pos Board B371 of ICM myocytes. Inhibitor analysis showed that hyperphosphorylation of Voltage-Gated Calcium Channel a1-Subunits Regulate Cardiac Function D. melanogaster LTCC occurs via calmodulin-dependent kinase II in DCM and via protein ki- of the Aging Heart of nase A in ICM. Implantation of LVAD was found to reduce the LTCC activity Alexander Lam, Priyanka Karekar, Girija Hariharan, Michelle Fleyshman, independently of the disease. Kajol Shah, Harpreet Singh, Shubha Gururaja Rao. Conclusion: These findings suggest that both ICM and DCM myocytes contain Drexel University, Philadelphia, PA, USA. pro-arrhythmic hyper-active LTCCs but their increased activity occurs as the Ion channels are essential in regulating cellular function and cellular signaling result of two different phosphorylation pathways targeting different pathways; disruption of these ion channels often contribute to dysfunction in microdomains. cellular processes. Ion channels are the key targets for cellular reactive oxygen species (ROS), in which these channels function as signaling switches between 3161-Pos Board B369 ionic homeostasis and ROS. A paraquat screen was carried out in D. mela- Molecular Mechanism of Voltage-Gated Ca2D Channel Regulation by nogaster to identify the ion channels regulating ROS handling and survival; Membrane PIP2 this revealed that voltage-gated calcium channels (VGCCs) handle the medi- Cheon-Gyu Park, Byung-Chang Suh. ated ROS stress differently based on the expression of a1-subunits of VGCCs Brain and Cognitive Sciences, DGIST, Daegu, Republic of Korea. (D-type, T-type, and cacophony) and gender. In aging, the lifespan of the T- Voltage-gated calcium (CaV) channels play essential roles in adjusting calcium type mutants was shorter, as their 50% survival was 2252 and 5056 days influx upon membrane depolarization. CaV2 (N-, P/Q- and R-type) channels are for males and females compared to 4951 days and 6651 days for males concentrated in the presynaptic nerve terminals and important for the neuro- and females in wild-type (n>25 flies). Similarly, a decrease in lifespan was transmitter release. Adjusting the presynaptic calcium channel gating exerts observed in cacophony mutants of 4452 for females; however, the lifespan potent influence on synaptic plasticity. CaV channels need auxiliary subunits in D-type mutant males increased to 7351 days. VGCCs are known to for proper trafficking to the plasma membrane and the channel gating. Espe- contribute to cardiac function, such as pacemaking and heart rate determina- cially CaVb subunit plays crucial roles in the surface expression of CaV chan- tion. Therefore, optical coherence tomography (OCT) was carried out to char- nels and fine-tuning of channel gating. It has been known that CaV channels are acterize the role of VGCCs in cardiac function of the aging heart in VGCC modulated by membrane phosphatidylinositol 4,5-bisphosphate (PIP2). The mutants. All three mutants showed a significant decrease in heart rate of binding affinity between ion channel and PIP2 is important for the channel 31854, 34757, and 33059 beats per minute (bpm) by week 3 in D-type,

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T-type, and cacophony mutants, respectively as compared to wild-type of Surgery, University of California, San Diego, La Jolla, CA, USA, 36258 bpm (nR11 flies). T-type mutants showed a significant decrease in 4Department of Anatomy, Physiology and Genetics, Uniformed Services fractional shortening (8251%) as compared to wild-type (8650.5%) in University, Bethesda, MD, USA. week 1. Our results implicate VGCCs are vital in maintaining heart rate and Alpha calcium channel blockers (CCB) are widely prescribed for the treat- with age, and the absence of these channels can have detrimental effects on car- ment of hypertension. In the present study, we found that Nitrendipine diac rhythm. (NTD), which belongs to the family of alpha CCB, affects the proliferation of murine neuroblastoma cells (N2A) as detected by the XTT method. In 3164-Pos Board B372 2D addition, NTD induced apoptosis of N2A cells in a concentration- A Novel Form of CaV1.4 Ca Channel Regulation Revealed by Alterna- dependent manner as detected by flow cytometry using annexin 5 staining tive Splicing and a Mutation Causing Congenital Stationary Night and propidium iodine permeability. The anti-hypertension effect of NTD is Blindness related to binding and blocking the activity of the voltage-dependent Brittany Williams, Amy Lee. L-type calcium channels. These channels consist of four subunits (a1, Molecular Physiology and Biophysics and Interdisciplinary Graduate a2/d, g and b subunits), localized in the plasma membrane. NTD specifically Program in Neuroscience, University of Iowa, Iowa City, IA, USA. binds with high affinity to the a1c subunit, which is critical for ion conduc- To support its role in mediating tonic exocytosis at the photoreceptor synapse, 2þ 2þ tivity. To further investigate if the toxic effect of NTD is related to voltage- Cav1.4 L- type Ca channels undergo weak Ca -dependent inactivation dependent L-type calcium channel binding, the gene encoding for the a1c (CDI). The mechanism involves a C-terminal automodulatory domain (CTM) subunit (cacna1c) was knocked down by siRNA in N2A cells. We observed that competes with calmodulin (CaM) binding to the channel. A mutation that that the decrease in cacna1c expression did not have a significant effect on causes congenital stationary night blindness (CSNB2), K1591X, causes prema- NTD toxicity in N2A cells. Therefore, we concluded that the toxic effect ture truncation of the channel just downstream of the CaM binding region and of NTD on neuroblastoma cells is independent of the binding of the drug removal of the entire CTM. The mutant channels (Cav1.4K1591X) exhibit strong to the L-type calcium channel. Although further clinical studies are required CDI and activation at more negative voltages than full-length channels similar to to evaluate the contribution of the toxic effect of NTD to the health condi- a splice variant (Cav1.4Dex47) that lacks just a portion of the CTM. To determine tion, it is important to notice the potential toxic effect of this drug that is if selective deletion of exon 47 causes distinct regulation of CDI and activation widely prescribed for the treatment of hypertension. as compared to the K1591X mutation, we compared the properties of Cav1.4K1591X and Cav1.4Dex47 in electrophysiological recordings of trans- 3167-Pos Board B375 fected HEK293T cells. We found key differences in how CaM regulates CDI Carboxyl Terminus as a Key Regulator of Gating and Signaling of L-Type of these channels. First, Cav1.4K1591X undergoes significantly faster CDI Calcium Channels than Cav1.4Dex47. Second, CDI of Cav1.4Dex47 can be suppressed by dominant 2þ Yaxiong Yang, Yuanyuan He, Xiaodong Liu. negative expression of CaM mutants that cannot bind Ca in either the C-termi- Biomedical Engineering, Tsinghua University, Beijing, China. nal (CaM34) or N-terminal (CaM12) lobe, whereas only CaM34 blunts CDI of L-type calcium channels (CaV1) play vital roles in critical biological processes Cav1.4K1591X. Third, CaM12 reverses the negative shift in activation including calcium dynamics and gene regulation, where the carboxyl terminus of Cav1.4Dex47 but not of Cav1.4K1591X. We conclude that the deletion of of CaV1 gets closely involved. In this presentation, we are introducing some of exon 47 alters how CaM functionally interacts with the channel in a way that the recent progress and findings about C-tail’s roles in Ca 1 gating and is not reproduced by the K1591X mutation, which may contribute to the patho- V 2þ signaling. First, we report C-terminus mediated inhibition (CMI) for CaV1.3 logical effects of K1591X for Ca signaling at the photoreceptor synapse. that multiple motifs coordinate to compete calmodulin (CaM) off the channel, 2þ 2þ 3165-Pos Board B373 and tune down Ca current and Ca influx toward the lower limits deter- mined by end-stage CDI (Ca2þ-dependent inactivation). Acute CMI by Gating Pore Currents in DIII Hypopp Mutations of CaV1.1 Fenfen Wu, Marbella Quinonez, Steve C. Cannon. rapamycin-inducible heterodimerization helps reconcile the concurrent activa- Physiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, tion/inactivation attenuations to ensure CMI as a new modality of channel in- hibition (Liu N, et al., eLife, 2017). Data suggest that CMI could also USA. 2þ profoundly affect the downstream Ca signaling especially the CaV1-depen- Mutations in the voltage sensor domains (VSD) of CaV1.1 are an established cause of hypokalemic periodic paralysis (HypoPP). Remarkably, 8 of 9 pub- dent gene regulation. Such importance of C-tail is in part demonstrated by the work dealing with the side-effects of CaM-based GCaMP, one of the lished mutations are at arginine residues in S4 segments. The prevailing view 2þ is that R/X mutations cause gating pore ‘‘leakage’’ currents that increase the sus- most popular types of Ca probes. We unveil the intrinsic problem prevailing over different versions and applications, that GCaMP acts as impaired CaM of ceptibility to depolarization and weakness in low K. Prior attempts to experimen- 2þ apo and Ca forms both critical to C-tail functions in CaV1, resulting into dis- tally confirm this hypothesis were limited by poor expression of CaV1.1 at the 2þ membrane. We recently showed co-expression of Stac3 dramatically increases torted Ca dynamics and altered gene expression. We then design and imple- > ment GCaMP-X, which successfully resolves the problems of detrimental expression ( 100x current amplitude) in Xenopus oocytes. We now report on 2þ the functional consequences of HypoPP mutations in the DIII VSD. nuclear accumulation, acute and chronic Ca dysregulation, and abnormal The HypoPP mutant constructs (R897S, R900S, R900G) were co-expressed cell morphogenesis. The interplays between the C-terminus and signaling pro- teins well demonstrate the ultimate and synergistic tasks of CaV1 complex: to with a2/d, b1a, and Stac3 subunits in oocytes. Cut-open voltage-clamp record- 2þ ings demonstrated an anomalous inward-rectifying current in R897S channels deliver Ca of appropriate spatiotemporal patterns (gating) to the right lis- (R1 position). At 80 mV, the amplitude of this gating pore current was com- teners and actuators (signaling). parable to the peak inward Ca2þ current at þ40 mV. Currents recorded from HypoPP mutations in the DIII R2 position (R900S and R900G) were more 3168-Pos Board B376 linear at hyperpolarized potentials, and therefore difficult to distinguish Probing the Pathogenic Mechanisms Underlying CaV 1.2 Channelopathies from a non-specific leak. Block by divalent cations, however, was much Moradeke A. Bamgboye1, Maria K. Traficante1, David T. Yue2, Ivy E. Dick1. greater than for WT channels. We interpret this difference as evidence of a 1 2 gating pore current also for R2 HypoPP mutant channels. Deactivation of Physiology, University of Maryland, Baltimore, MD, USA, BME, Johns Hopkins, Baltimore, MD, USA. the R2 gating pore current presumably occurs at more depolarized potentials 2þ 2þ than for the R1 mutant, and therefore the nonlinear inward rectification is Voltage gated Ca channels are the main conduit for Ca entry into most obscured by incomplete block of ionic current through the conventional pore excitable cells, and the CaV1.2 L-type channel is among the most widespread, (alpha current). These data support the notion that all HypoPP mutations in existing in cardiac, neuronal, and smooth muscle cells. These channels are pre- cisely tuned to function within each of these systems, enabling the same chan- CaV1.1 or NaV1.4 create a deleterious gating pore current that underlies sus- ceptibility to transient depolarization and weakness. nel type to coordinate neuronal excitability, vasoconstriction and cardiac excitation-contraction coupling. This tuning is accomplished in part by the nu- 3166-Pos Board B374 ances of channel gating and voltage dependence in each channel subtype, and The Antihypertensive Calcium Channel Blocker Nitrendipine Displays a by two major forms of feedback regulation: Ca2þ dependent inactivation (CDI) Cytotoxic Effect on Neuroblastoma Cells, Which is Independent of Binding and voltage dependent inactivation (VDI). A growing number of genetic muta- to L-Type Voltage-Gated Calcium Channels tions in CaV1.2 are now recognized as the cause of severe cardiac and neuronal Antonio De Maio1, Isabel Rivera2, David M. Cauvi3, Nelson Arispe4. syndromes (channelopathies), which are frequently resistant to conventional 1Departments of Surgery and Neurosciences, University of California, San treatment options. Biophysical studies have shown that these disease-causing Diego, La Jolla, CA, USA, 2Initiative for Maximizing Student Development, mutations can often impact channel gating properties including CDI and University of California, San Diego, La Jolla, CA, USA, 3Department of VDI. However, as the number of known channelopathies continues to expand,

BPJ 8735_8739 640a Wednesday, February 21, 2018 so do the mechanistic differences among different mutations, resulting in Posters: TRP Channels II increasing numbers of patients for which standard therapies are inadequate. Here, we aim to connect genetic mutations within CaV1.2 to specific effects 3171-Pos Board B379 on channel gating and regulation, in order to improve our understanding of Drosophila-Inspired Molecular Thermosensors the pathogenesis of these disorders. Moreover, these biophysical alterations Marzie Amirshenava, Benjamin Zars, Benton Berigan, Paige Martinez, likely underlie the lack of efficacy of calcium channel blockers in the treatment Troy Zars, Lorin S. Milescu, Mirela Milescu. of these channelopathy patients. Thus, characterization of these channel deficits University of Missouri Columbia, Columbia, MO, USA. will increase our understanding of disease pathogenesis and redirect treatment Gustatory receptors (Gr) are a family of transmembrane proteins that have strategies. been extensively studied in the context of insect taste and odor sensory sys- 3169-Pos Board B377 tems. A recent study identified Gr28bD, a member of the Drosophila Gr family, as a key player in thermosensation. Very little is known about Direct Inhibition of CaV2.3 by Gem Does Not Requiere a Direct Alpha1E/ Beta Interaction the structure and function of Gr28bD, other than having a putative Gustavo F. Contreras1,2, Nieves Navarro2, Guido Mellado2, seven-transmembrane domain architecture. In a previous study, we showed Daniela De Giorgis2, Carlos Gonzalez2, Alan Neely2. that Gr28bD exhibits a non-inactivating, temperature dependent, cation 1University of Chicago, Chicago, IL, USA, 2Universidad de Valparaiso, non-specific current, when expressed in Xenopus laevis oocytes. Here, we Valparaiso, Chile. identified and tested Grs from other Drosophila species and discovered The Rem, Rem2, Rad, and Gem/Kir (RGK) sub-family of small GTP-binding that their temperature response varies across species. By comparing the protein include e the most potent endogenous inhibitor of High-Voltage Acti- Gr28bD sequence with these orthologs, we identified functional domains. vated (HVA) calcium channels. RGK proteins use several mechanisms to We then generated various chimeric constructs and obtained proteins with distinct properties, including temperature dependence. We expect inhibit calcium current (ICA): i) they promote dynamin- dependent endocyt- ocys; ii) the lower the Po of the channel: ii) and they voltage sensor or reduction these results to aid in the design of new thermogenetic tools for extrinsic of charge. All RGK proteins associate directly with Ca2þ channel b subunits neuronal control. (Cavb), and the binding between a / Cavb has been shown to be essential CaV 1 3172-Pos Board B380 for their inhibitory action for CaV1.2 and CaV2.1 and CaV2.2. In this study, 2þ IP6 Does Not Induce Binding between Coiled-Coil Helices or between the we investigated inhibition of CaV2.3 Ca channels by RGK proteins. We found that when Xenopus laevis oocytes expressing Cav2.3 channels were in- N-Term ARDS and Coiled-Coil jected with purified Gem protein, but not Rem, calcium currents where signif- Gilbert Q. Martinez, Sharona E. Gordon. icantly decreased. This reduction was accompanied by a right shift in the Physiology and Biophysics, University of Washington, Seattle, WA, USA. conductance-voltage (GV curve) relationship of the channel. Furthermore, Transient Receptor Potential Ankyrin type 1 (TRPA1) is a cation-selective Gem also decreases the number of channels in the plasma membrane, evi- ion channel that is activated by several noxious compounds and by reduced denced by a reduction in maximal charge movement after injection of purified temperature. Given its role in noxious sensation and inflammation, TRPA1 protein. The kinetic and voltage dependence of the reduced charge movement has become an active drug target for the treatment of pain and was not affected and thus immobilization of a subset of voltage-sensor seems inflammation-related ailments such as itch and cough. Although the cryo- unlikely. Surprisingly both effects are not dependent on the binding between EM structure of TRPA1 has provided a starting point in understanding CaVa1 to Cavb since CaV2.3 were expressed alone. Thus unlike than other channel activation, the structural mechanisms remain elusive. In particular, neuronal calcium channels, Gem inhibit Cav2.3 channels in a CaVb-indepen- an N-terminal Ankyrin Repeat Domain (ARD) contains a highly reactive dent manner and through two synergistic mechanisms: a increase in voltage cysteine that activates TRPA1 demonstrating the importance of this region for activation and a decrease in the number of channels. in channel function. Furthermore, it was hypothesized that a tetrameric C- Acknowledgement: FONDECYT 1161672 to A.N, FONDECYT 1161672 to terminal coiled-coil was stabilized by a polyphosphate compound, IP6, G.F.C and The Centro Interdisciplinario de Neurociencia de Valparaı´so is a which would account for the requirement of polyphosphates in excised Scientific Millennium Institute. patch electrophysiology experiments recording TRPA1 activity. Here, we used biochemical and biophysical approaches of isolated cytoplasmic do- 3170-Pos Board B378 mains from human TRPA1 to probe the role of IP6 on the oligomerization A Skeletal Muscle L-Type Calcium Channel with a Mutation within the of N-terminal ARD and the C-terminal coiled-coil. Surprisingly, we found Selectivity Filter Conducts Potassium that oligomerization of the C-terminal coiled-coil was indistinguishable in 1 2 Roger A. Bannister , Donald Beqollari . the absence and presence of IP6.IP6 similarly had no discernable effect 1Medicine, University of Colorado School of Medicine, Aurora, CO, USA, on binding of the ARD to the C-terminal coiled-coil. Our data suggest 2 Surgery, University of Rochester, Rochester, NY, USA. that the role of IP6 in TRPA1 activation likely involves mechanisms not A glutamate to lysine substitution at position 1014 within the selectivity filter of yet considered. 2þ 2þ the skeletal muscle L-type Ca channel (CaV1.1 E1014K) abolishes Ca flux through the channel pore. Mice engineered to express exclusively the mutant 3173-Pos Board B381 CaV1.1 E1014K channel display accelerated muscle fatigue, changes in muscle Distinctive Drug Binding Sites and Gating Mechanisms of the Nociceptive composition and altered metabolism relative to wild-type littermates. In Ion Channel TRPA1 contrast, mice expressing another mutant CaV1.1 channel that is impermeable Jun Chen, Tania Chernov-Rogan. 2þ to Ca (CaV1.1 N617D) have shown no detectable phenotypic differences Genentech, South San Francisco, CA, USA. from wild-type mice to date. The major biophysical difference between the As an irritant sensor, TRPA1 channel has emerged as a therapeutic target for CaV1.1 E1014K and CaV1.1 N617D mutants is that the former channel is pain, itch and respiratory diseases. It has been reported that a plethora of elec- known to conduct robust Naþ and Csþ currents in patch-clamp experiments. trophilic agonists, such as allyl isothiocyanate, activate TRPA1 through cova- However, neither of these monovalent conductances seems to be of relevance lent modification of cysteine residues (e.g., C621); and A-967079, an in vivo; the channel opens much too slowly to conduct Naþ during the upstroke antagonist, binds to the pore domain through induced fit. Despite these pro- of the action potential and Csþ is not present in muscle fibers under physiolog- gresses, it remains largely unknown how covalent modification leads to channel ical circumstances. In this brief study, we used the recently developed tsA-201 opening, and how non-reactive agonists and other antagonists interact with the cell-CaV1.1 expression system to show that a YFP-fused CaV1.1 E1014K chan- channel. From high throughput screening and structure-function studies, we nel conducts robust 1,4-dihydropyridine (DHP)-sensitive Kþ currents at depo- have discovered several classes of TRPA1 modulators and determined critical larizing test potentials whereas YFP-fused wild-type CaV1.1 and CaV1.1 residues/domains, including N-ter, PreS1, TM1-4, pore and C-ter domain of the N617D channels do not. Our observations, coupled with a very large body of channel. For examples, G6828, a reactive agonist, modifies C621 residue but its work by others regarding the role of Kþ accumulation in muscle fatigue, raise functional effect is mediated by PreS1 -helix, indicating that PreS1 domain the possibility that the introduction of an additional Kþ flux from the myoplasm couples covalent modification (in the N-ter) to channel opening (pore). into the restricted space of the transverse tubule lumen accelerates the onset of G1243, a non-covalent agonist, activates TRPA1 by binding to TM1-4 domain, fatigue and precipitates the metabolic changes observed in CaV1.1 E1014K a site equivalent to the capsaicin binding site in TRPV1. Therefore, TRPA1 and muscle. Thus, the results of this study may provide useful information towards TRPV1 may share similar gating mechanism despite the lack of sequence ho- understanding the divergent phenotypes of CaV1.1 E1014K and CaV1.1 N617D mology. Together, these data shed light on the structure-function of TRPA1 and mice. Supported by a generous grant from the Boettcher Foundation to R.A.B. provide insights to rational drug design.

BPJ 8735_8739 Wednesday, February 21, 2018 641a

3174-Pos Board B382 ical severity. However, the mechanistic insight of Ca2þ-dependent inactivation TRPC3 Underlies GABAB Receptor-Mediated Augmentation of Type-1 (CDI) is unveiled yet. Here, we firstly confirmed that Calmodulin (CaM) effect Metabotropic Glutamate Receptor-Coupled Slow Excitatory Postsynaptic to CDI, however its lobe specificity is distinctive compared to VDCC channels. Potential in Cerebellar Purkinje Neurons CDI of TRPC6 can be affected by both lobes of CaM and almost no effect from JinBin Tian, Michael X. Zhu. the dominant negative CaM. To obtain mechanistic insights of CDI, binding Integrative Biology and Pharmacology, University of Texas McGovern stoichiometry of CaM and TRPC6-Calmodulin binding domain (CBD) were Medical School at Houston, Houston, TX, USA. determined by NMR and ITC titration experiments, and both experiments Transient Receptor Potential Canonical 3 (TRPC3) channels have been demon- concluded to a 1:2 stoichiometry (CaM : TRPC6-CBD). These results indicated strated to underlie a slow excitatory postsynaptic current (sEPSC) that is that CDI could be explained by a close proximity of two TRPC6-CBDs by a coupled to the activation of type-1 metabotropic glutamate receptor (mGluR1) ‘bridge’ mechanism of CaM. Furthermore, we found that coiled-coil segment by glutamate release at the parallel fiber -Purkinje cell synapses of the cere- in the C-terminal contributes to the proximal distance of two CBD’s by its bellum. On the other hand, Purkinje neurons receive extensive inhibitory syn- self-assembly, and deletion of this segment caused a marked delay of CDI. aptic inputs from interneurons located in the molecular layer of cerebellar Deletion or point mutations of coiled coil in TRPC6 are associated in nephrotic cortex and GABAB receptors (GABABRs) are expressed on the dendrites of syndrome caused by focal segmental glomerulosclerosis (FSGS). All coiled- Purkinje cells and often co-localized with mGluR1. It has been shown that acti- coil mutants found in FSGS patients delayed CDI, would lead to proceed podo- 2þ vation of postsynaptic GABABRs potentiates mGluR1-coupled sEPSCs in Pur- cyte damage due to overload of Ca . Thus our model provides mechanistic in- kinje cells, but the underlying molecular mechanism remains elusive. Here we sights of CDI by CaM, CBD with coiled-coil of TRPC6 channels. report that the potentiation of mGluR1-coupled sEPSCs in Purkinje cells by 3177-Pos Board B385 stimulating postsynaptic GABABRs was completely absent in TRPC3 knockout mice, but persistent in TRPC1-, TRPC4- and TRPC1/4/5/6- Ischemic Neuronal Cell Death Mediated by TRPC Channels knockout mice, suggesting that TRPC3 is the only TRPC isoform that mediates Jaepyo Jeon, Sun Guanghua, Jinbin Tian, Sung-Ming Ting, the potentiation. Moreover, our results indicate that the potentiation was a pure Jaroslaw Aronowski, Michael X. Zhu. postsynaptic event because it was not affected by blocking neurotransmission The University of Texas Health Science Center at Houston, Houston, TX, with tetrodotoxin. Furthermore, we found that the potentiation was blocked USA. Brain ischemia causes damage to neurons due to reduced oxygen and glucose by antagonists of either GABABRs or mGluR1, suggesting that it may originate supply, i.e. oxygen and glucose deprivation (OGD). Cell death after cerebral directly from the integration of synaptic signals at the receptor level and the 2þ strengthened signal was mainly mediated by mGluR1-TRPC3 coupling instead ischemia may result from elevation in intracellular calcium (Ca ), loss of cell ion homeostasis and glutamate-induced excitotoxicity, which can all result of any other pathways involved in GABABR signaling. Since mGluR1-TRPC3 from activation of Transient Receptor Potential (TRP) proteins. During coupling is essential in cerebellar long-term depression, synapse elimination, 2þ and motor coordination, our findings that TRPC3 is also responsible for the ischemia/reperfusion to the brain, Ca influx to neurons may be induced by the elevation of extracellular glutamate concentration, which elicits several synaptic integration of GABABR-mediated inputs may suggest a novel mech- anism that underlies essential cerebellar functions, such as motor learning. neurotoxic events through activation of glutamate receptors. Among them, the metabotropic glutamate receptors are known to couple to TRPC channel 3175-Pos Board B383 activation, leading to membrane depolarization and sustained Ca2þ influx. Photoswitchable Diacylglycerols Identify a Novel Lipid-Gating Mecha- To determine the contribution of TRPC channels in glutamate-induced intracel- 2þ 2þ 2þ nism in TRPC3 Channels lular Ca overload, We measured intracellular Ca concentration ([Ca ]i) Oleksandra Tiapko1, Michaela Lichtenegger1, Gema Guedes de la Cruz2, changes in response to glutamate stimulation in cultured cortical neurons. In 2 1 1 -/- 2þ Toma N. Glasnov , Barbora Svobodova , Wolfgang Schreibmayer , neurons prepared from TRPC4 mice, the glutamate-induced [Ca ]i increase Dieter Platzer1, Sarah Krenn1, Niroj Shrestha1, Rainer Schindl1, was also significantly less than in that from the wild type (WT) controls. These Thomas Stockner3, Christoph Romanin4, Klaus Groschner1. results indicate the likely involvement of TRPC4 channels in glutamate- 1 2 2þ Medical University of Graz, Graz, Austria, University of Graz, Graz, induced [Ca ]i rise, a condition commonly associated with OGD. To test if Austria, 3Medical University of Vienna, Vienna, Austria, 4University of Linz, the TRPC channel activity is linked to ischemic neuronal death, we used Linz, Austria. both an in vivo model and an in vitro model. Transient middle cerebral ar- Transient receptor potential canonical channels TRPC3/6 and 7 are known to tery/common carotid artery occlusion resulted in large infarct areas in WT sense diacylglycerol. The recognition machinery for membrane lipids in these mice. The infarct areas were significantly smaller in the brains of TRPC4-/- channels remains unknown. We synthetized a DAG derivative containing two mice. In cortical neuron cultures prepared from TRPC1-/-, TRPC4-/- and arachidonic acid-mimetic photoswitches (OptoDArG), which enabled efficient TRPC1/C4/C5/C6-/- mice, OGD-induced neuronal death was significantly light-mediated control of TRPC3. This tool was employed to gain an understand- less than that from WT mice. These data suggest that TRPC channels play a ing of the molecular process of lipid sensing in TRPC3 channels. Structure- significant role in neuronal death induced by OGD. Our study reveals that guided mutagenesis screen in the TRPC3 pore domain uncovered a single glycine TRPC channels significantly contribute to ischemic cell death through calcium residue within the TRPC3 S6 helix (G652) as a determinant of lipid recognition. overload and that inhibition of TRPCs may be neuroprotective against brain This residue is part of a lateral fenestration within the pore domain and hence pre- damage following ischemic stroke. dicted to be exposed to membrane lipids. Mutation of G652 to alanine (G652A) or larger residues eliminated PLC-mediated activation and distinctly changed the 3178-Pos Board B386 sensitivity profile to lipid activators, including enhanced efficacy and potency Functional Charaterization of Zebrafish Transient Receptor Potential of OptoDArG. Optical ‘lipid-clamp’ experiments allowed detailed insight into Melastatin 2 the kinetics of channel activation/deactivation and suggested the existence of a Ha Nam Tran1, Jure Hederih2, Tomohiro Numata3, Masayuki X. Mori2, Shingo Maegawa4, Hiroshi Hosokawa4, Yasuo Mori2. lipid recognition/interaction site in close proximity to the channels selectivity fil- 1 ter, which controls gating movements in the pore domain of TRPC3. We report Graduate School of Global Environmental Studies, Kyoto University, Kyoto University, Japan, 2Engineering, Kyoto University, Kyoto University, Japan, successful utilization of an optical ‘lipid clamp’ approach to examine molecular 3 4 recognition and gating processes in TRPC3 channels. School of Medicine, Fukuoka University, Fukuoka, Japan, Graduate School of Informatics, Kyoto University, Kyoto, Japan. 3176-Pos Board B384 Releasing pollutants into the aquatic environments causes serious damage to D Ca2 -Dependent Inactivation Mediated by Calmodulin in TRPC6 Chan- small life such as fish. In the case of fish, transient receptor potential (TRP) pro- nel Underlies FSGS Channelopathy tein can sense pollutants such as hydrogen peroxide and changes of environ- Masatoshi Uno1, Onur K. Polat1, Tran Nam Ha1, Shota Yamaji1, ment factors such as nitrogen cycle. To date, mammalian TRPM2 is well Yasuo Mori1, Hidehito Tochio2, Masayuki X. Mori1. characterized to sense these factors, however the functional properties of zebra- 1Engineering, Kyoto University, Kyoto, Japan, 2Dept of Biophysics, Kyoto fish TRPM2 still lacking. In this research, we show the characterisation and University, Kyoto, Japan. functional role of zebrafish TRPM2 (zTRPM2) heterologuosly expressed in þ TRPC6 channel contributes to Ca2 influx in a variety of tissues such as brain, HEK293T cell. lung, brain, heart and kidney and its excessive channel activity is known to be The result showed that cDNA of zTRPM2 has an open reading frame encoding associated with pathophysiological changes such as vascular remodeling, pul- a protein of 1470 amino acids , it expressed in broad-range of organs such as monary hypertension, and chronic kidney diseases. Inhibitory regulation of this heart, kidney, spleen, gills, and well-expressed in nervous system such as tri- þ channel by Ca2 is thus expected to have a profound influence on the patholog- geminal ganglion. Intriguingly, functional analysis by calcium imaging and

BPJ 8735_8739 642a Wednesday, February 21, 2018 electrophysiological approach revealed that zTRPM2 is activated by pollutants, 3181-Pos Board B389 such as hydrogen peroxide (H2O2), nitrite (NO2-) a pollutant in nitrogen cycle G-Protein Beta-Gamma Subunits Inhibit the Heat-Sensitive TRPM3 Ion and NO which is released from SNAP (s-nitroso-n-acetyl-dl-penicillamine) or Channels SNAC (s-nitroso-n-acetylcysteine). However, temperature, pH conditions and Tibor Rohacs1, Yevgen Yudin1, Doreen Badheka1, Istvan Borbiro1, hypoxia are not effect to zTRPM2 channel activity. The activation of zTRPM2 Aysenur Yazici1, Siyuan Zhao1, Cassandra Hartle2, Tooraj Mirshahi2. was inhibited by mammalian TRPM2 inhibitor econazole and clotrimazole. 1Rutgers - New Jersey Medical School, Newark, NJ, USA, 2Weis Center for These results suggest a functional role of zTRPM2 in the detection of oxidants Research, Geisinger Clinic, Danville, PA, USA. that are known as pollutants and zTRPM2 inhibitors may assists in protecting Transient Receptor Potential Melastatin 3 (TRPM3) is a heat-activated non-se- þ fish from the polluted environments. lective, Ca2 permeable cation channel also stimulated by chemical agents such as pregnenolone sulphate and CIM0216. Here we show that activation 3179-Pos Board B387 of Gi-coupled cell surface receptors inhibits TRPM3 currents in a mammalian Biochemical Characterization of the Interaction of TRPM3 with Gbg expression system, which was alleviated by co-expression of proteins that bind Proteins bg subunits of heterotrimeric G-proteins (Gbg). Co-expression of Gb1g2, Fabian Gruss1, Marc Behrendt2, Mieke Nys1, Johannes Oberwinkler2, Gb3g2, Gb4g2, but not Gb5g2 or constitutively active mutants of Gaoor Chris Ulens1. 1 Gai, inhibited pregnenolone sulphate-induced TRPM3 currents. Purified Gbg Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, proteins applied to excised inside out patches also inhibited TRPM3 activity, 2 € Leuven, Belgium, Institut fur Physiologie und Pathophysiologie, Philipps- indicating a direct effect. Baclofen, somatostatin, and DAMGO, agonists of € þ Universitat Marburg, Marburg, Germany. Gi coupled receptors, inhibited Ca2 signals induced by pregnenolone sulphate TRPM3 is a member of the melastatin subfamily of transient receptor potential and CIM0216 in dorsal root ganglion (DRG) neurons. The GABAB receptor (TRP) channels. It is expressed in various cell types as one of numerous isoforms, agonist baclofen also inhibited CIM0216-induced currents in DRG neurons, many of which form calcium-permeable ion channels. In somatosensory neu- and nocifensive responses elicited by this TRPM3 agonist in vivo. Our data rons, TRPM3 contributes to sensation of noxious heat and inflammatory hyper- show that Gbg inhibits TRPM3 channels upon Gi-coupled receptor activation. algesia. Furthermore, it is expressed in pancreatic beta cells and has been implicated in the regulation of glucose-induced insulin release. In the eye, mu- 3182-Pos Board B390 tations in TRPM3 are linked to inherited forms of cataract and glaucoma. TRPM7 Current Inactivation: Evidence for Inside-Out Signaling Recently, it was shown that binding of Gbg subunits of Gi/o proteins to Tetyana Zhelay, J. Ashot Kozak. TRPM3 inhibits the channel. Gi/o proteins act downstream of GPCRs like the Wright State University, Dayton, OH, USA. þ þ þ þ m-opioid receptor, whose activation by morphine leads to TRPM3 inhibition. In- TRPM7 channels conduct metal cations such as Na ,K ,Ca2 and Mg2 .In þ þ hibition of TRPM3 itself might therefore be a new therapeutic principle for a the presence of external Ca2 /Mg2 TRPM7 has a steeply outwardly rectifying þ þ novel class of analgesic drugs. By overexpressing TRPM3 proteins with various current-voltage (I-V) relation. In the absence of Ca2 /Mg2 the IV becomes deletions and Gi/o-coupled receptors in HEK293 cells, we screened TRPM3, in semi-linear. This has been explained by the removal of pore blockade by diva- 2þ 2þ order to identify regions that were dispensible for channel function, but neces- lent cations (e.g. Ca o/Mg o). TRPM7 channels are inhibited by cytoplasmic 2þ sary for the inhibition by Gbg. In these experiments, TRPM3 was activated Mg in a voltage-independent manner, primarily by a reversible reduction in with pregnenolone sulfate, the only known endogenous substance to activate the overall number of conducting channels. Here, we have examined the con- þ these channels. After identifying a stretch of 10 amino acids in the N-terminus sequences of external Ca2 removal and reintroduction on TRPM7 current ki- þ of TRPM3, we verified its importance for inhibiting TRPM3 by analyzing single netics. In whole-cell patch clamp with low internal Mg2 , we rapidly and 2þ þ point mutations. To further characterize the binding between Gbg and TRPM3 repeatedly exchanged 2 mM Ca with divalent cation free (DVF) Na or þ in vitro with a biochemical approach, we demonstrated that the stretch of Cs containing solutions to compare the time dependent changes in current. 2þ 10 amino acids of TRPM3 binds directly to Gbg. This site mediates binding to This maneuver resulted in a declining current in DVF following Ca exposure, natively prenylated Gbg, but also to an artificially soluble Gbg mutant. We which we call inactivation. This was followed by a slowly rising current when 2þ conclude that Gbg acts as an allosteric inhibitor of TRPM3 and its binding site Ca o was reintroduced. The decay of monovalent current occurred over 1-4 þ may also serve as a potential target for small molecule allosteric modulators. minutes and was monoexponential in most cases. Internal Mg2 and spermine, partially inhibiting TRPM7, revealed more pronounced decay/potentiation cy- 3180-Pos Board B388 cles than seen with Mg2þ-free internal solutions. Inactivation was observed Volatile Anaesthetics Inhibit Thermosensitive TRPM3 Ion Channels with both Naþ and Csþ as charge carriers and extracellular Mg2þ could substi- Bala´zs Kelemen1, Flo´ra Kulin1, Erika Lisztes1,Ja´nos Posta2, Thomas Voets3, tute for the potentiating Ca2þ effect. Current decay in DVF was distinct from Tama´s Bı´ro´4, Bala´zs Istva´n To´th1. TRPM7 rundown, which we previously documented in patch-clamp record- 1Department of Physiology, University of Debrecen, Faculty of Medicine, 2 ings. Inactivation persisted in perforated-patch recordings, which allow the Debrecen, Hungary, Department of Forensic Medicine, University of 2þ 3 maintenance of physiological Mg concentrations and prevent rundown. We Debrecen, Faculty of Medicine, Debrecen, Hungary, Department of Cellular have also characterized TRPM7 point mutations that abolish Ca2þ potentia- and Molecular Medicine and TRP Research Platform Leuven (TRPLe), KU 4 tion/inactivation. TRPM7 current behavior in response to switches between Leuven, Leuven, Belgium, Departments of Immunology and Physiology, divalent-containing and divalent-free solutions resembled the Ca2þ potentia- University of Debrecen, Faculty of Medicine, Debrecen, Hungary. tion/depotentiation described for calcium release activated calcium (CRAC) Volatile anaesthetics (VAs) are the most widely used compounds to maintain channels. We conclude that TRPM7 response to extracellular ions depends general anaesthesia during operation both in human therapeutic interventions on intracellular Mg2þ/spermine concentrations, presenting a novel case of and animal experiments. Although the mechanism of their action is not fully inside-out signaling. understood in details, it is generally believed, that VAs depress central nervous system functions mainly via acting on ion channels of the neuronal membrane, 3183-Pos Board B391 þ like GABA receptors, NMDA receptors or 2-pore-domain K channels. Recent PIP2 Depletion Contributes to Inhibition of TRPM8 Activity by GQ Pro- research also reported their action on nociceptive and thermosensitive TRP tein Coupled Receptors channels, TRPV1, TRPA1, and TRPM8. In our current study, we investigated Luyu Liu, Yevgen Yudin, Tibor Rohacs. the effect of VAs on TRPM3, a less characterized member of the thermosensi- Rutgers-New Jersey Medical School, Newark, NJ, USA. tive TRP channels. We investigated the effect of VAs on the activity of TRPM3 Transient Receptor Potential Melastatin 8 (TRPM8) channels detect cold tem- both in recombinant and native systems using intracellular Ca2þ measurements peratures (< 26C). They are activated by cold, depolarization, and chemical and patch clamp electrophysiology. All the investigated VAs inhibited the agents such as menthol, icilin, and WS12. Phosphatidylinositol 4, 5-bisphos- 2þ pregnenolone sulphate and CIM0216 induced Ca signals and transmembrane phate (PIP2), a minor component of the cell membrane, is required for currents in a dose dependent way in HEK293T cells overexpressing recombi- TRPM8 activity. Our lab found earlier that intracellular dialysis of PIP2 through nant TRPM3. Among the applied VAs, halothane was found to be the most the whole cell patch pipette inhibits desensitization of TRPM8. Activation of effective blocker of TRPM3 (IC50z0.5 mM). We also investigated the effect Gaq-protein coupled receptors stimulate phospholipase C (PLC), which de- of VAs on native TRPM3 channels expressed by sensory neurons of the dorsal creases cellular PIP2 levels. A recent study proposed that TRPM8 activity is in- root ganglia. We found that VAs activated certain sensory neurons indepen- hibited by G-protein coupled receptors via direct binding to Gaq to the channel. dently of TRPM3, but applied in 1 mM, they strongly inhibited the agonist In the presence of PIP2, the inhibition of TRPM8 by activated Gaq was prom- induced TRPM3 activity in a reversible way. Our results further enhance our inent in excised patches, but it is not known if a decrease in PIP2 plays a role in knowledge about VAs’ mechanism of action and might contribute to the devel- TRPM8 inhibition in a cellular context. We hypothesize that PIP2 depletion opment of novel analgesics targeting TRPM3. contributes to inhibition of TRPM8 by Gaq-protein coupled receptors. We

BPJ 8735_8739 Wednesday, February 21, 2018 643a show here that human muscarinic acetylcholine receptor 1 (hM1R) activation warm temperatures. Additionally, neutralizing an arginine residue at the center by carbachol inhibits TRPM8 in a mammalian expression system. We found of the coiled-coil (R1079A) abolishes the TRPM8 channel activation by cold. that intracellular dialysis of PIP2 alleviated the inhibition, and PIP2 was Our findings strongly support a role of the coiled-coil domain as a module high- depleted in the plasma membrane when hM1R was activated. We also found ly temperature sensitive in TRPM8 channels. that TRPM8 channels are partially inhibited by a constitutively active Gaq when PLC is not activated, confirming earlier results. Furthermore, we used 3186-Pos Board B394 an ‘‘inflammatory soup’’, which contains multiple agonists of various Gaq-pro- Identification of Clustered Phosphorylation Sites in PKD2L1: How tein coupled receptors expressed in dorsal root ganglion (DRG) neurons, to test PKD2L1 Channel Activation is Regulated by Cyclic AMP Signaling Pathway our hypothesis in neurons. We found that intracellular dialysis of PIP2 allevi- 1 1 2 1 ates the inhibition of TRPM8 by Gaq-protein coupled receptors in DRG neu- Eunice Y. Park , Misun Kwak , Kotdaji Ha , Insuk So . 1Seoul National University, Daejeon, Republic of Korea, 2University of rons. We conclude that a decrease in PIP2 level contributes to inhibition of TRPM8 channels upon the activation of Gaq-protein coupled receptors. California, San Francisco, San Francisco, CA, USA. Polycystic kidney disease 2-like-1 (PKD2L1), or polycystin-L or TRPP2, 3184-Pos Board B392 formerly TRPP3, is a transient receptor potential (TRP) superfamily member. TRPM8 Regulates Sexual Desire and Satiety It is a calcium-permeable non-selective cation channel that regulates intracel- Lusine Demirkhanyan1, Vivek Krishnan2, Swapna Asuthkar1, lular calcium concentration and thereby calcium signaling. PKD2L1 has been Brenda Alexander2, Zahir Hussain1, Padmamalini Baskaran2, reported to take part in hedgehog signaling in renal primary cilia and sour tasting Yelena Nersesyan1, Alejandro Cohen3, Evgeny Pavlov4, coupling with PKD1L3. In addition to the previous reports, PKD2L1 is recently Baskaran Thyagarajan2, Eleonora Zakharian1. found to play a crucial role in localization with b2-adrenergic receptor (b2AR) 1The University of Illinois at Chicago, Peoria, IL, USA, 2The University of on the neuronal primary cilia. The disruption of PKD2L1 leads to the loss of Wyoming, Laramie, WY, USA, 3Dalhousie University, Halifax, NS, Canada, b2AR on the primary cilia and reduction in intracellular concentration of cyclic 4New York University, New York, NY, USA. AMP (cAMP). Since the role of cAMP and PKA is frequently studied in relation Testosterone regulates dimorphic sexual behaviors in vertebrates. However, the to PKD diseases, we investigated on the mechanism of cAMP regulation in rela- mechanism by which testosterone mediates these behaviors remains elusive. tion to the function of PKD2L1 channel. In this study, we observed the activity of Here, we report that TRPM8, a potent testosterone receptor, is expressed in PKD2L1 channel increased by the downstream cascades of b2AR and found the the mammalian brain and regulates sexual and social behaviors. TRPM8 null clustered phosphorylation sites, S682, S685, and S686 that are significant in the mice exhibit increased frequency in both male-to-female and male-to-male channel regulation by phosphorylation. mountings, a delayed sexual satiety, and increased aggression compared to con- 3187-Pos Board B395 trols. These behavioral traits are associated with a concomitant increase in the Polycystin-1/Polycystin-2 Mediated Calcium Entry into Cilia during Sonic serum testosterone concentrations in TRPM8 null mice. These mice also show Hedgehog Signaling enhanced expression of aromatase, which converts testosterone to estradiol. Bryn S. Moore, Ann N. Stepanchick, Jonathan Z. Luo, Tooraj Mirshahi. The selectivity analysis of steroidal hormones to TRPM8 reveals that the chan- Molecular and Functional Genomics, Geisinger Clinic, Danville, PA, USA. nel protein is at least 10000 times more selective to testosterone over estradiol Sonic Hedgehog (SHH) signaling leads to elevation of calcium and reduction of or progesterone. Collectively, our novel findings bring to light a central role of cAMP in primary cilia. Gadolinium sensitive channels mediate the SHH effects TRPM8 in the regulation of testosterone-dependent mammalian sexual and so- on cilia calcium and cAMP. The cilia-localized calcium permeable channel has cial behaviors, as loss of TRPM8 leads to a delayed sexual satiety, altered sex- been suggested to be a member of the Trp channel superfamily: either ual preferences, and enhanced aggression. polycystin-2 (PC2), encoded by polycystic kidney disease-2 (PKD2), or poly- cystin 2-like 1. In kidney inner medullary collecting duct cells (IMCD3) and 3185-Pos Board B393 mouse embryonic fibroblasts (MEFs) we detect only PKD2 transcripts. Local- Molecular Elements for Temperature Detection in TRPM8 Channel ization of PC2 to the primary cilia is dependent on polycystin-1 (PC1). To 1 2 3 Karen Castillo , Natalia Raddatz , Melissa Alegrı´a-Arcos , determine if and how the PC1/PC2 complex regulates cilia calcium and 4 3 3 German Min˜o-Galaz , Ignacio Diaz-Franulic , Fernando Gonzalez-Nilo , cAMP levels, we used cilia targeted genetically encoded biosensors and 1 Ramon Latorre . biochemical analyses. In PKD2 knockout MEFs, SHH signaling failed to in- 1Neurociencia, Centro Interdisciplinario de Neurociencia de Valparaı´so, 2 crease calcium or reduce cAMP levels in cilia, showing that PC2 is essential Universidad de Valparaı´so, Valparaı´so, Chile, Instituto de Bioquimica for SHH signaling. Similarly, SHH did not affect cilia calcium or cAMP levels Vegetal y Fotosintesis, Consejo Superior de Investigaciones Cientificas, 3 in PKD1 knockout MEFs. Re-expressing PC2 or PC1 in PKD2 or PKD1 41092, Sevilla, Spain, Centro Interdisciplinario de Neurociencia de knockout MEFs, respectively, rescued the SHH effect of cilia calcium increase Valparaı´so, Universidad de Valparaı´so and Center for Bioinformatics and and cAMP reduction. Interestingly, we found that PC1 re-expression in PKD1 Integrative Biology, Universidad Andres Bello, Valparaı´so and Santiago, 4 knockout MEFs also increased basal cilia calcium levels. Using western blot Chile, Neurociencia, Centro Interdisciplinario de Neurociencia de Valparaı´so, and immunofluorescence, we found no difference in the expression or localiza- Universidad de Valparaı´so and Center for Bioinformatics and Integrative tion of PC2 in cilia after SHH. However, we did see increased expression of Biology, Universidad Andres Bello, Valparaı´so and Santiago, Chile. PC1 after SHH. Our data show that the PC1/PC2 complex is responsible for The TRP melastatin receptor type 8 (TRPM8) channel is the main cold trans- the SHH mediated increase in cilia calcium and decrease in cilia cAMP and ducer in vivo. TRPM8 is involved in innocuous cold sensing and in abnormal suggest that SHH-induced increase in expression of PC1 may regulate PC2 cold sensitivity during pathological conditions. Although the temperature- activity. dependent activity of TRPM8 has been well characterized, the underlying mechanism of gating by cold remains unknown. The C-terminal domain of 3188-Pos Board B396 TRPM8 has been proposed to be the temperature sensing structure, because Regulation of Ciliary Length in LLC-PK1 Renal Epithelial Cells swapping the C-terminal domain of TRPV1 with that of TRPM8, exchanges Paula L. Perez1,2, Noelia Scarinci1,2, Maria del Rocio Cantero1,2, their temperature sensitivities (Brauchi et al., 2006). Here we propose that Horacio F. Cantiello1,2. the coiled-coil domain, located at the end of C-terminus is the structural domain 1IMSaTeD Conicet, Santiago del Estero, Argentina, 2Facultad de Ciencias that defines the channel sensitivity to temperature. The progressive shortening Medicas, UNSE, Santiago del Estero, Argentina. of the TRPM8 coiled-coil results in decreased cold sensitivity, when expressed Polycystin-2 (PC2, TRPP2) is a Ca2þ permeable nonselective cation channel in Xenopus laevis oocytes, although conserving their voltage-dependent activa- whose dysfunction generates autosomal dominant polycystic kidney disease tion. Additionally, the temperature dependence is lost in the chimeric TRPM8 (ADPKD). PC2 is present in different cellular locations, including the primary channel that expresses the coiled-coil domain of TRPM7, a temperature insen- cilium of LLC-PK1 renal epithelial cells, where it may contribute to ciliary sitive ion channel. By constructing a 3D molecular model of the TRPM8 Ca2þ transport. Little is known, however, as to how renal epithelial cells con- coiled-coils, we observed that in addition to hydrophobic interactions, the cen- trol the length of the primary cilium, and in particular how PC2 function may ter of the structure also contains a cluster of polar interactions that potentially contribute to this regulation. Here, we explored the effect of maneuvers that could participate in temperature sensitivity. Using this molecular model we either control Ca2þ transport through PC2 or modulate its function, on the evaluate possible routes for thermal diffusion in the TRPM8 coiled-coil by ciliary length of LLC-PK1 cells. Cells (2-3 wk old) were labeled with a specific the anisotropic thermal diffusion (ATD) method. The role of several residues antibody against a-acetylated tubulin to identify primary cilia, and DAPI to in thermal diffusion, revealed by ATD, was experimentally tested. A single- locate cellular nuclei. Images were collected after overnight exposure of cells point mutation that substitutes a hydrophobic residue (I) by a polar one to the various experimental conditions. The length of the primary cilium was (I1103R) in the coiled-coil domain produces a channel that responds to obtained with the ImageJ software. Primary cilia length measurements did

BPJ 8735_8739 644a Wednesday, February 21, 2018 not follow a normal distribution, in any of the conditions tested. Comparisons ity and promotes Pi release and, in myocells, increases Ca2þ-sensitivity of force between groups were conducted either by group (Mann-Whitney) or in between production (Malik et al. Science 331:1439, 2011; Nagy et al. Br J Pharmacol groups (Kruskall Wallis) rendering significant differences for all conditions 172:4506, 2015). Despite the interest for OM as an activator of the myosin respect to Control (normal Ca2þ, 1.2 mM, n = 199). Cells exposed either to motor to understand motor-related myopathies and devise therapies (Spudich low Ca2þ (n = 212) or high Ca2þ (n = 331) had a decreased primary cilium Biophys J 106:1236, 2014), the details of OM effects on force generation are length by 23.8% and 14.4%, respectively (p < 0.001). Amiloride (200 mM, unclear. We used fast sarcomere-level mechanics in demembranated rabbit so- n = 287) and LiCl (10 mM, n = 92), two chemicals that reduce PC2 function, leus fibers to define the effect of OM (1 mM) on the force and stiffness of the both induced an increase in primary cilium length of 12.1% and 20.0% (p < half-sarcomere (hs) following Ca2þ-activation. The hs-stiffness was analysed 0.001), respectively. The data indicate that PC2 function controls ciliary length with a model that accounts for the contribution of the filament compliance in renal epithelial cells, suggesting a Ca2þ-mediated regulatory mechanism. and provides the estimate of the stiffness of the motors (e, which depends on Dysregulation of this mechanism may be essential in the onset of ADPKD. the number of actin-attached motors). At saturating [Ca2þ], OM depresses the isometric force by 50% by reducing the strain in the motors, without ef- 3189-Pos Board B397 fects on e. Both the force-pCa and e-pCa relations are shifted to the left in OM Functional Characterization of a Calcium-Sensing Receptor-Polycystin-2 with respect to the control (pCa50 is increased by 0.4 units) and their steepness Channel Complex in the Plasma Membrane of LLC-PK1 Cells is reduced by 50%. Thus the effect of OM on the force-pCa relation is fully ac- 1,2 1,2 1,2 Noelia Scarinci , Paula L. Perez , Maria del Rocio Cantero , counted for by that on the e-pCa relation. We conclude that OM has a dual ef- Horacio F. Cantiello1,2. 2þ 1 2 fect: it increases the Ca -sensitivity of the motor binding to actin, while it IMSaTeD Conicet, Santiago del Estero, Argentina, Facultad de Ciencias reduces the structural change associated to force generation. In the heart the in- Medicas, UNSE, Santiago del Estero, Argentina. 2þ 2þ ternal [Ca ] is below saturation and OM acts as a potentiator by increasing the Polycystin-2 (PC2, TRPP2) is a Ca -permeable nonselective cation channel number of motors. Supported by ECRF and Telethon. from the TRP superfamily. PC2 is encoded by the PKD2 gene, whose mutations are responsible for autosomal dominant polycystic kidney disease (ADPKD). 3192-Pos Board B400 Recent studies from our laboratory (Dai et al, Exp Cell Res, 2017), determined Myosin Effects on Thin Filament Activation in Slow-Twitch Human Soleus that the PC2 contribution to the whole cell conductance of wild type LLC-PK1 2þ Muscle Fibers renal epithelial cells is regulated by changes in external Ca concentration, in a Alfredo J. Lopez-Davila1, Robert Stehle2, Stefan Zittrich2, Birgit Piep1, mechanism that implicates the Calcium-Sensing Receptor (CaSR). The present 1 3 3 2þ Faramarz Matinmehr , Andras Malnasi , Anna Rauscher , study explored the effect of Ca and CaSR agonists in the regulation of PC2 Joseph Chalovich4, Theresia Kraft1, Bernhard Brenner1. from isolated LLC-PK1 plasma membranes reconstituted in a lipid bilayer sys- 1Institute for Molecular and Cell Physiology, Hannover Medical School, tem (BLM). Addition of the CaSR agonist R-568 (5 mM) to the ‘‘external’’ side Hannover, Germany, 2Institute of Vegetative Physiology, University of of the bilayer, activated single channel currents consistent with a functional PC2 Cologne, Cologne, Germany, 3Department of Biochemistry, Eo¨tvo¨s Lora´nd 5 < by 84 12% (n = 4, p 0.01). These cation-selective currents were subse- University, Budapest, Hungary, 4Department of Biochemistry & Molecular 5 < quently inhibited (91.3 1.1%, p 0.0001) by addition to the ‘‘cytosolic’’ Biology, Brody School of Medicine at East Carolina University, Greenville, side of the BLM, of a commercially available anti-PC2 C-terminal antibody NC, USA. to the ‘‘cytosolic’’ or ‘‘internal’’ side of the BLM. Interestingly, similar findings It is well known that striated muscle thin filaments are activated by calcium but were observed with the material obtained by co-immunoprecipitation of LLC- the level of activity at sub-saturating calcium is increased by myosin binding to PK1 cell lysate with anti-CaSR antibody crosslinked in agarose beads. The actin. Observations on the effects of myosin binding on activation were made data indicate the presence of a membrane locate CaSR-PC2 functional complex primarily withcardiac or fast-twitch skeletal muscle. Here we asked whether in LLC-PK1 renal epithelial cells. The presence of the receptor/channel complex 2þ similar contributions of myosin to thin filament activation occur in slow- is in agreement with the regulatory role external Ca plays in the modulation of twitch skeletal muscle fibers that contain the same myosin heavy chain isoform PC2 channel function in the plasma membrane of renal epithelial cells. This as in the human heart. receptor-channel complex may be part of a regulatory mechanism connecting 2þ We exchanged endogenous troponin of slow-twitch single human soleus fibers Ca signals and PC2 function to onset of ADPKD. for a well characterized, fast skeletal troponin complex labeled with IANBD IANBD 3190-Pos Board B398 (fsTn ). This was used as a fluorescent sensor to report the effects of cal- Activation Mechanisms Underlying Influx-Operated Calcium Entry of cium and myosin on thin filament activation. TRPP Channels Inducing maximal force development by increasing the calcium concentration from pCa 7.5 to pCa 4.5 generated a change in the emission intensity of Liu Yuxia. IANBD Medical School, Tsinghua University, Beijing, China. fsTn of on average 32% (control). Increasing the calcium concentration TRPP channels are permeable to Ca2þ, and are also regulated by Ca2þ. For in the same conditions but in presence of 50 mM of the myosin inhibitor para-amino-blebbistatin reduced force to 7% of maximum force in control con- instance, PKD2L1 (also known as TRPP3) channel can respond to the rapid in- IANBD crease of extracellular calcium, resulting in an influx-operated Ca2þ entry ditons and reduced the change in the emission intensity of fsTn to 18% (ICE) (Hu et al. 2015 Cell Reports). However, the molecular mechanism of (or to 55% of the change observed in control conditions). Similar effects ICE activation has not been elucidated. In this study, we show that both were detected at submaximal calcium concentrations. Additionally, when 2þ 2þ switching from isometric contraction to slightly-loaded shortening, changes Ca and the outer pore play dual roles: Ca can act as both charge carrier IANBD and activation ligand for channels; meanwhile, the channel pore regions can in fluorescence indicating conformational changes of fsTn had rate con- stants at least 10 times higher than those of force redevelopment. act as both the selective filter and also the activation gate. The affinity of IANBD Ca2þ in the outer pore region is highly correlated with the activation of ICE. Thus, our data with fsTn as thin filament activation sensor suggest that The negatively charged residues prefer Ca2þ over Naþ assuring channel activa- force-generating myosin heads contribute to thin filament activation at all acti- tion. Thus, less charged residues or more extracellular Naþ downregulate ICE. vation levels in human slow-twitch skeletal muscle fibers. Importantly, the rate Moreover, the actual ion-binding affinity (irrelevant of selectivity here) is high- constant of force redevelopment is not limited by the much faster equilibration ly regulated by other sites within the outer pore. Such active mechanism of among different states of the thin filament. 2þ Ca and outer pore in activation may also underlie other ion channels. 3193-Pos Board B401 The Super Relaxed State of Myosin in Human Muscle Clyde F. Wilson, Nariman Naber, Roger A. Cooke. Posters: Skeletal Muscle Mechanics, Structure, Biochem, UCSF, San Francisco, CA, USA. and Regulation II The Super Relaxed State of myosin (SRX) plays a role in maintaining the low resting metabolic rate of skeletal muscle. Pharmaceuticals that destabilize this 3191-Pos Board B399 state could increase resting muscle and whole body metabolic rate by as much An In-Situ Study of the Modulation of the Mechano-Kinetic Parameters of the as 1000 Calories/day, providing an effective treatment for obesity and type 2 dia- Slow Isoform of Muscle Myosin II by the Heart Drug Omecamtiv Mecarbil betes. Our previous work on this state in skeletal muscle has all been in animal Marco Caremani, Cristina Gallart, Valentina Percario, Gabriella Piazzesi, models. Here we extend this approach to human muscle. Biopsies from the quad- Vincenzo Lombardi, Marco Linari. riceps lateralis of both lean and obese subjects were used. Measurements of single Department of Biology, University of Florence, Florence, Italy. turnovers showed an SRX with a population of 0.27 5 0.02 and a lifetime of Omecamtiv Mecarbil (OM), a small molecule that binds to the catalytic domain 150 5 8 sec in lean subjects and a population of 0.28 5 0.02 and a lifetime of of the myosin isoforms of both cardiac and slow muscles, increases actin affin- 158 5 10 sec in obese subjects. Piperine is a small molecule that has been shown

BPJ 8735_8739 Wednesday, February 21, 2018 645a to destabilize the SRX in rabbit fast twitch fibers, with no effect on slow twitch meeting. Taken together, out data have allowed us to unravel new general prin- fibers. In human muscle, addition 100mM piperine reduced the population of ciples of molecular elasticity in muscle filament proteins and may be applicable the SRX by 34% in lean subjects and 25% in obese subjects, with little change to other filaments as well. in the lifetimes. The human quadriceps lateralis contains 50% fast twitch and References: 50% slow twitch fibers. Data obtained with piperine showed two responses [1] Pinotsis et al. (2012) PLoS Biol. 2012 Feb;10(2):e1001261. with 50% of fibers showing a strong inhibition of the SRX and the remaining [2] Obermann et al. (1996) J Cell Biol 134: 1441-1453. fibers showing very little, in both lean and obese subjects. Presumably the fibers showing strong inhibition are fast twitch, but this remains to be proven. We 3196-Pos Board B404 conclude that in human muscle the SRX and its response to piperine are similar Nebulin Stiffens the Thin Filament and Augments Crossbridge to those seen previously in rabbit muscle, with little difference between lean Interaction - An X-Ray Diffraction Study on Intact Muscle 1 1 2 1 1 and obese subjects. This shows that analogs of piperine that have higher affinity Balazs Kiss , Eun-Jeong Lee , Weikang Ma , Frank Li , Paola Tonino , 2 2 1 would provide effective treatment for metabolic diseases. Srboljub M. Mijailovich , Thomas Irving , Henk Granzier . 1Department of Cellular and Molecular Medicine, University of Arizona, 2 3194-Pos Board B402 Tucson, AZ, USA, Department of Biology, Illinois Institute of Technology, Quantifying the Titin Contribution to Muscle Force Generation using a Chicago, IL, USA. Novel Method to Specifically Cleave the Titin Springs in Situ Nebulin, the giant sarcomeric protein is thought to play a role in thin filament Yong Li1, Andreas Unger1, Marion von Frieling-Salewsky1, length regulation but little is known about nebulin’s influence on the thin fila- Jaime Andres Rivas Pardo2, Julio M. Fernandez2, Wolfgang A. Linke1. ment structure in skeletal muscle. In the present work we compared the ultra- 1Institute of Physiology II, University of Muenster, Muenster, Germany, structure of relaxed and contracting intact soleus muscle dissected from 2Department of Biological Sciences, Columbia University, New York, NY, conditional nebulin-knockout (cKO) and control (Ctrl) mice by using small- angle X-ray diffraction. Although the spacing of the actin subunit repeats USA. ˚ The giant protein titin contributes to muscle force generation. However, titin’s (27.3 A meridional reflection) was equal in both groups in relaxed muscles, dur- mechanical role in muscle is still incompletely understood because of previous ing contraction the normalized percent change is more than the double in cKO inability to specifically cleave the titin spring. To overcome this problem, we (0.0054%/kPa) compared to that of the control (0.0020%/kPa). The myofibrillar used a mouse model in which both a tobacco etch virus (TEV) protease- area was determined by a detailed cross-sectional ultrastructural analysis recognition site and a HaloTag were cloned into elastic I-band titin. The thereby making the stiffness calculation of single thin filaments possible. The Halo-TEV cassette allowed for in-situ imaging of titin, specific proteolysis dur- stiffness for 1-mm-long actin was calculated to be 38.7 pN/nm in Ctrl muscle ing myofiber mechanics, and visualization of successful cleavage on protein gels and 14.8 pN/nm in cKO soleus. Furthermore, significantly shorter left-handed thin filament helical pitch (59 A˚ meridional reflection) was found in the absence or tissue sections. Using permeabilized skeletal myofiber bundles dissected from ˚ ˚ mice homozygous for Halo-TEV-titin, we measured passive force over the of nebulin in passive muscles (58.88 A in cKO vs. 59.14 A in Ctrl); the shorter þ ˚ sarcomere-length (SL) range 2.2-3.4 mm and maximum Ca2 -triggered force 59 A helical pitch was maintained during contraction. Through the equatorial (pCa5) at 2.6 mm SL, in the absence or presence of TEV-enzyme. Wildtype my- 1,0 and 1,1 intensities we found significantly less mass attached to the thin fil- ofibers treated with TEV-protease served as additional controls. We found that aments in passive cKO muscles and inefficient myosin mass transfer during their contraction. According to our findings, nebulin plays a role in the passive state incubation of myofibers with TEV-protease at room temperature cleaved genet- ˚ ically modified titin specifically and completely in <1 hour, but had no effect on of muscle by fine-adjusting the 59 A helical pitch of the thin filaments thereby wildtype titin. Titin cleavage barely affected myofiber ultrastructure in the modulating the binding sites of regulatory proteins along the long pitch groove absence of a stretch or Ca2þ-activation; however, mechanical stressing caused of actin. Further, nebulin stiffens the thin filament which is critically important sarcomere destabilization and myosin-filament disarray. In both psoas and so- for skeletal muscle force development, explaining why the absence of the neb- leus myofiber bundles, passive forces dropped with titin cleavage by 50%, ulin in nemaline myopathy patients leads to muscle weakness. the remaining forces presumably being attributable to extracellular-matrix pro- 3197-Pos Board B405 teins. Subsequent treatment of the samples with high-salt buffer (0.6 M KCl/ Myosin Orientation in a Functioning Muscle Fiber with High Angular 1.0 M KI) to dissolve the titin anchorage caused a further 25% drop in passive Resolution force, suggesting this frequently-used extraction method consistently overesti- Yahor Savich, Benjamin P. Binder, Peter D. Martin, Andrew R. Thompson, mates the titin-contribution to passive tension. Active force development was David D. Thomas. reduced, on average by 50%, in titin-cleaved psoas or soleus fibers, compared University of Minnesota, Minneapolis, MN, USA. to controls. Our approach thus allows for a direct and reliable quantitation of We have measured the orie--ntation of myosin heads in a functioning muscle fiber titin’s contribution to passive and active forces in muscle. using electron paramagnetic resonance (EPR) and a bifunctional spin label (BSL), with angular resolution of a few degrees. Despite advances in cryo-EM, fluores- 3195-Pos Board B403 cence, and small-angle X-ray diffraction, these techniques do not provide high- Deciphering the Molecular Mechanism of Myomesin Elasticity resolution structural information about myosin heads in vitro under functional Matthias Wilmanns, Spyros D. Chatziefthimiou. conditions. A pair of (i,iþ4) Cys residues were engineered on an alpha-helix in Hamburg Unit, European Molecular Biology Laboratory, Hamburg, the skeletal muscle isoform of the regulatory light chain (skRLC) on the N and Germany. C lobes. By exchanging endogenous RLC with mutant skRLC labeled with In striated and cardiac vertebrate muscles, the sarcomeric M-band ensures the BSL on oriented muscle fibers, we were able to resolve sharp angular distributions uniform distribution of the tension across the myosin filaments during contrac- in several biochemical states due to the stereospecific attachment of BSL’s two di- tion. Myomesin, a prominent filament component of the M-band, is thought to sulfide bonds. In this setup, the accurate determination of BSL’s angular coordi- act as strain absorber keeping the thick filaments in register during muscle nates allowed us to determine the orientation of individual structural elements contraction. In previous work, we discovered a hitherto unprecedented molec- with respect to the muscle fiber axis. Addition of ATP in the absence of Ca, relax- ular elasticity mechanism for the C-terminal segment of the dimeric myomesin ing the muscle, shifted the orientational distribution to a much more disordered filament including domains 9-13/13’-9’, using a hybrid structural approach distribution. A subsequent temperature increase induced orientational order, including X-ray crystallography, electron microscopy, small angle X-ray Scat- consistent with a transition to the super-relaxed state (SRX). Based on the pro- tering and atomic force microscopy [1]. We demonstrated that highly exposed posed therapeutic importance of SRX in heart failure and obesity, this system helical linker peptide segments, connecting an five-fold repeated array of could become a useful biosensor for structural analysis and therapeutic design immunoglobulin domains can be reversibly unfolded when exposed to low in muscle. This work was supported by NIH R01AR032961 and R37AG26160 < external forces of 30 pN, leading to an estimated 2,5-fold extension in length to DDT. YS was supported by NIH T32AG29796. of the filament. Here were present additional structures of myomesin multi- domain constructs including domains 7 and 8. The connections between these 3198-Pos Board B406 domains 7-9 are completely different from those observed above and are void Cryoelectron Tomography of Isolated, Relaxed Thick Filaments from the reversible unfolding pattern. Hence our data suggest that myomesin elastic- Lethocerus Indicus Flight Muscle ity is confined to the C-terminal segment 9-13. We included these additional Nadia Daneshparvar. domains into an extended composite model 7-13/13’-7’, which we validated Florida State University, Tallahassee, FL, USA. with the same hybrid structural biology approach. The model generated is 42 The recent high resolution structure of isolated, relaxed thick filaments from nm in length, approaching the previously found M4-M4’ distance of 44 nm, Lethocerus indicus flight muscle [Hu et al., Sci. Adv. 2, e1600058 (2016)] re- which is thought to mark the distal boundaries of myomesin [2]. In vitro and vealed unprecedented detail on the arrangement of myosin rods within the thick in vivo validation experiments are ongoing and will be presented at the filament backbone and a novel orientation of the myosin heads arranged as an

BPJ 8735_8739 646a Wednesday, February 21, 2018 interacting heads motif (IHM). In addition, the blocked head component of the tors almost completely suppress the contractile force, but still substantial changes IHM of Lethocerus thick filaments appears to be a dynamic structure whose were observed in the diffraction patterns upon calcium activation. (1) The 2nd motions might be trapped by rapid freezing. However, the high resolution struc- actin layer line reflection was enhanced normally, indicating that calcium binding ture, which was determined using iterative helical real space reconstruction, re- to troponin and the subsequent movement of tropomyosin are not inhibited, (2) vealed considerably less detail for the paramyosin core and nothing about the the myosin layer line reflections became much weaker, and (3) the 1,1/1,0 inten- bare zone and the tapered end. Since the bare zone and tapered end are non- sity ratio of the equatorial reflections was increased. The observations (2) and (3) helical structures, complex structures, and little is known of the paramyosin indicate that, even in the presence of the inhibitors at a high concentration, myosin arrangement, we have resorted to cryoelectron tomography (cryoET) and sub- heads leave the helix on the myosin filaments and approach the actin filaments. volume averaging to obtain preliminary structures that might be improved upon Interestingly, the d1,0 spacing of the filament lattice remains unchanged upon by single particle 3-D image reconstruction. Subvolume averaging can also activation of inhibited fibers, in contrast to the case of normal activation in which elucidate the dynamics of the blocked-head. Supported by NIH. the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, 3199-Pos Board B407 the pull is suppressed, and as a result, the heads simply bind to actin without ex- Electron Tomography of Relaxed Lethocerus Flight Muscle Reveals Thin erting any force. The results support the idea that the inhibitors do not block the Filament Binding of Disordered ‘‘Blocked’’ Heads myosin binding to actin, but block the step of force-producing transition of the Hamidreza Rahmani. bound actomyosin complex. Florida State University, Tallahassee, FL, USA. When relaxed flight muscle fibers from Lethocerus indicus are visualized by 3202-Pos Board B410 thin section electron microscopy, they typically show pronounced shelves of ˚ Cooperative Activation of Striated Muscle Thick Filaments by S2 Binding density spaced at 145 A intervals, dubbed crowns, with occasional contacts be- Dua’a Quedan, Andrea Bernardino-Schaefer, Rohit Singh, tween what appear to be myosin heads and the troponin complex preferentially, Christopher Thang, Mithilesh Bhaskaruni, Riti Srivastava, Douglas D. Root. but not exclusively. Recent cryoEM studies on the structure of relaxed, isolated Biological Sciences, University of North Texas, Denton, TX, USA. thick filaments from Lethocerus flight muscle indicates that the myosin heads Myosin binding protein C (MyBPC) is present at a very low ratio relative to are arranged in an interacting heads motif (IHM) with a novel orientation with myosin in muscle yet has a significant impact on contractility in cardiomyop- respect to the filament axis. The IHM free head is positioned tangential to the athies. One explanation is that MyBPC cooperatively modulates the binding backbone surface with the blocked head extending to high radius and appar- of the S1 to the S2 coiled coil. To test this hypothesis, the MF30 monoclonal ently more dynamic than the free head. As such, the blocked head might be antibody is used to compete with MyBPC for binding S2. As a control, the the myosin head seen contacting the thin filaments in EMs of relaxed muscle MF20 monoclonal antibody which binds to light meromyosin does not affect fibers. The stretch activation response in Lethocerus flight muscle occurs at a muscle contraction. Expansion microscopy of myofibrils reveals that MF30 þ submaximal [Ca2 ] concentration requiring some other structure to produce but not MF20 has reduced labeling in the region of MyBPC binding consistent complete activation. Here we have used electron tomography and subvolume with competition between MF30 and MyBPC. Designed antiS2 synthetic pep- averaging to investigate the distribution of myosin head-thin filament attach- tides that modulate myosin S2 stability demonstrate cooperativity similar to ments in sections of relaxed fibers to determine their thick filament origin ˚ MF30 with higher measured Kd values than their EC50 or IC50 on myofibril and thin filament destination. Class averages of thick filament crowns (145 A contractility. Furthermore, fluorescent labeling of the antiS2 peptides reduces axial repeats) show a mixture of different conformations of the IHM. Class av- their binding affinity but not the extent of their effect on myofibril contractility. erages of thin filaments resolve actin subunits and troponin well. In this study, The antiS2 peptide that destabilizes the coiled coil had a greater maximum we are trying to investigate the statistics of different IHM conformations in a enhancement of myofibril contractility than MF30 which suggests that the pep- quantitative way as well as the in situ details of the free and blocked head con- tide may block the binding of S1 to S2 while MF30 may simply act by sterically formations in relaxed and partially relaxed IHMs. Supported by NIH. blocking MyBPC. Furthermore, MF30 has no effect on in vitro motility assays 3200-Pos Board B408 of purified actin and myosin; while the destabilizing peptide slightly enhanced Role of Myosin CaATPase in Muscle Contraction actin sliding velocities. These results support the view that a cooperative tran- Jinghua Ge1, Akhil Gargey Iragavarapu2, Yuri E. Nesmelov1. sition occurs between less active and more active conformations of myosin in 1Physics, University of North Carolina, Charlotte, NC, USA, 2Biology, the thick filament that can be modulated by a substoichiometric amount of University of North Carolina, Charlotte, NC, USA. effector. Calcium and magnesium play an essential role in muscle contraction. The role of calcium is to regulate actomyosin interaction by binding troponin of a thin 3203-Pos Board B411 filament. The role of magnesium is to form an MgATP complex to bind to a Topology of Interactions between Titin Molecules and Myosin Thick myosin active site, thus promoting actomyosin dissociation and subsequent Filaments myosin conformational change. The concentration of magnesium in cells is Miklos Kellermayer, Dominik Sziklai, Zsombor Papp, Brennan Decker, virtually uniform, whereas the calcium concentration is tightly regulated. There Eszter Lakatos, Zsolt Martonfalvi. are several buffers for calcium and magnesium in muscle. Since magnesium Semmelweis University, Budapest, Hungary. concentration in cells is high (i.e., in a millimolar range), the availability of Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. In MgATP is determined by the availability of ATP. Calcium buffers play an the A-band titin is associated with the myosin thick filament. It has been specu- essential role in the formation of a transient calcium wave in muscle upon mus- lated that titin may serve as a ruler for thick-filament formation due to the super- cle activation, and the kinetics of calcium binding to and dissociation from repeat structure of its A-band domains. Accordingly, titin might provide a buffers is important for muscle contraction. One such calcium buffer in muscle template that determines the length and structural periodicity of the thick fila- is ATP. It has been shown that the concentration of CaATP reaches 60uM in a ment. However, the accessibility of A-band anti-titin antibody epitopes and struc- sarcomere, which is about 1% of all ATP available. Myosin is not a specific tural reconstructions from electron-microscopic images suggest that titin runs on ATPase, and, therefore, both the Mg and Ca support myosin ATPase activity. the thick filament surface, raising important questions about how the two fila- Considering that about 300 myosin molecules form a thick filament, one can ments determine or affect each other’s structural arrangement. Here we tested estimate that about three heads per filament can have CaATP bound. Does the titin ruler hypothesis by mixing titin and myosin at in situ stoichiometric ra- CaATP binding to myosin and hydrolysis affect the force production by a tios (approximately 300 myosins per 12 titins) and dialyzing the sample against sarcomere? In this work we determine kinetics of actomyosin CaATPase to buffers of different filament-forming ionic strength (KCl concentration range conclude whether CaATP affects the force production in muscle. 50-150 mM). The topology of the filamentous complexes were investigated with atomic force microscopy. We found that the samples contained distinct, 3201-Pos Board B409 separate populations of titin molecules and myosin thick filaments. We were un- Effects of Myosin Inhibitors on the X-Ray Diffraction Patterns of Relaxed able to identify complexes in which myosin molecules were regularly associated and Calcium-Activated Rabbit Skeletal Muscle Fibers to either mono- or oligomeric titin in either conformationally relaxed or stretched Hiroyuki Iwamoto. states of the titin filaments. Occasionally we observed myosin thick filaments Research & Utilization Div., SPring-8, JASRI, Sayo-gun, Hyogo, Japan. with titin oligomers attached to their surface. Thus, the electrostatically driven Two potent myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS) and bleb- self-association is stronger in either myosin and titin than their binding to each bistatin, suppress the contractile force of skeletal muscle fibers at micromolar other, and it is unlikely that titin functions as a geometrical template for thick- concentrations. To gain insights into the mechanism of inhibition by these chem- filament formation. Conceivably, associated proteins, such as myosin-binding icals, we studied their effects on the X-ray diffraction patterns from rabbit skeletal protein C, and additional mechanisms are required to modulate and regulate muscle fibers under relaxing and activating conditions. At 100 mM, both inhibi- the in situ interactions between titin and the myosin thick filament.

BPJ 8735_8739 Wednesday, February 21, 2018 647a

Posters: Kinesins, Dyneins, and Other 3207-Pos Board B415 High-Speed Atomic Force Microscopic Observations on Demembranated Microtubule-based Motors II Chlamydomonas Axonemes and Dynein Arms Misaki Shiraga1, Yuka Matsuda2, Junya Kirima2, Kazuhiro Oiwa3. 3204-Pos Board B412 1Graduate School of Life Science, University of Hyogo, Harima, Japan, Using Brownian Dynamics Simulations to Identify Best Practices in Single 2University of Hyogo, Hyogo, Japan, 3National Institute of Information and Particle Tracking Communications Technology, Kobe, Japan. Annan S.I. Cook. In Chlamydomonas axoneme, nine doublet microtubules surround a pair of The Pennsylvania State University, State College, PA, USA. singlet microtubules. On each of doublet microtubules, protein motor dyneins High frequency nanoparticle tracking has emerged in recent years as a powerful are precisely aligned in two rows, outer- and inner-arm dyneins. The dynein strategy to track the nanoscale dynamics of proteins and DNA. Using dark-field arms are regulated in a coordinated fashion to produce periodic flagellar or interferometric scattering microscopy techniques (iScat) in conjunction with beating. The coordination among the dynein arms under strict spatiotemporal point-spread fitting, sub-millisecond temporal resolution images of proteins can regulation makes flagella beat in an organized manner. To understand the co- be achieved with nm spatial precision. However, there remain unresolved ques- ordination mechanism, we have performed video imaging of demembranated tions regarding the best practices for site-specific tagging of proteins. We here flagellar axonemes and dynein arms under the aqueous environment using employ Brownian dynamics simulations to investigate the influence of tether the high-speed atomic force microscope. By using this technique, unstained, length, tether stiffness, and particle size on imaging precision and protein unfixed axonemes in their natural physiological surroundings was imaged. Ax- fluctuation dynamics. We use kinesin motor proteins as a model system to onemes of the wild-type and the outer-dynein arm-less mutant oda1 were de- investigate temporal changes in position and analyze the fundamental contribu- membranated and attached on poly-L-lysine coated mica surface. To tions of each experimentally controllable parameter to results. We identify a visualize the axonemes, we used a tapping-mode high speed-AFM. AFM im- hard limit at which increasing frame rate surprisingly results in a noisier signal ages clearly highlighted many details of axonemes and difference between even when the particle saturates the image. We further observe that there is a wild-type and oda1 axonemes. In the wild-type axoneme topography, the tradeoff between tether length and collision frequency. It is therefore prudent doublet microtubules were recognized but the furrows between the doublets to consider the tradeoffs between increased tracking fidelity and perturbation were not clearly seen because of the presence of bulky blobs between the dou- of the natural dynamics of the protein system in question. blets. In contrast, the oda1-axoneme topography showed clearly the furrows and individual doublets. The difference between two topography was probably 3205-Pos Board B413 derived from the presence of outer dynein arms in wild-type axonemes. Phase Substrate Mobility Produces Velocity Time Dependence in Microtubule imaging of wild-type axonemes revealed the detail of the bulky blobs since it Gliding provides information about surface stiffness variations. The structural repeat of Joseph D. Lopes1, David Quint2, Dail Chapman3, Ajay Gopinathan1, 24nm was confirmed in the phase images. The high-speed AFM images thus Linda Hirst1, Jing Xu1. defined a detailed map of axonemal components under the aqueous environ- 1Physics, UC Merced, Merced, CA, USA, 2Center for Cellular and ment and will provide the dynamics of these components. This work was sup- Biomolecular Machines, UC Merced, Merced, CA, USA, 3Physics, UC ported by Grant-in-Aids for Scientific Research (C), JSPS, 26440089 and Irvine, Irvine, CA, USA. 17K07376 to K.O., and the Takeda Science Foundation (K.O.). Molecular motor based transport is critical for all eukaryotic cell function. Motors often work in small teams to transport a cargo in-vivo, however understanding the factors that control and regulate the group function of multiple motors bound to a 3208-Pos Board B416 lipid membrane remains a challenge. Here we couple kinesin motors to a lipid Structural Insights into Complex Formation of the Axonemal Dynein Light bilayer, utilizing the microtubule gliding assay, recording and analyzing gliding Chain-1 and OADg Stalk 1,2 1,2 1 3 velocity as a function of time. We observe a constant gliding velocity on glass Akiyuki Toda , Hideaki Tanaka , Yosuke Nishikawa , Toshiki Yagi , Genji Kurisu1,2. that is characteristic of solid substrates, while gliding on membrane resulted in 1 a larger than two-fold increase in velocity. When microtubules are immobilized Institute for Protein Research, Osaka University, Suita, Osaka, Japan, 2Graduate School of Science, Osaka University, Toyonaka, Osaka, Japan, in the absence of ATP, microtubule-bound motors are observed to build up over 3 time. We propose an analytical model relating time dependent motor protein den- Faculty of Life and Environmental Science, Prefectural University of sity to microtubule velocity, giving us a motor disassociation rate and a mecha- Hiroshima, Hiroshima City, Hiroshima, Japan. nism for the observed velocity increase. Our results provide evidence that motors Dyniens are microtubule-based molecular motors that consist of heavy, inter- coupled to a fluid-like membrane exhibit significantly different gliding behavior mediate, ligh-intermediate and light chains. Intermediate and light chains than observed on rigid substrates such as glass and hypothesize that motor diffu- play a regulatory role in cargo binding or ATPase activity. Most of the dynein sion in the membrane may play a role in biological transport processes. accessary chains interact with the N-termini of dynein heavy chains. However, it was recently uncovered that axonemal dynein light chain-1 (LC1) from Chla- 3206-Pos Board B414 mydomonas reinhardtii is bound to the microtubule-binding domain (MTBD) Microfluidic Device to Measure Collective Force Dynamics of Kinesin of outer arm dynein gamma (OADg). It was the first discovery that light chain Motor Proteins interacts with MTBD. Moreover, it was also discovered that LC1 binding to the Joseph M. Cleary, William O. Hancock. MTBD decreases microtubule-binding affinity of the heavy chain. On the other Bioengineering, Pennsylvania State University, State College, PA, USA. hand, it has been reported that ATPase activity of heavy chain is increased in Molecular motors are the fundamental driving force for intracellular transport the presence of microtubules. Thus, both results imply that LC1 indirectly and other vital biological processes. These motors have been studied exten- changes ATPase activity of OADg and tunes ciliary/flagellar beating. Howev- sively at the single molecule level in vitro, but their function in vivo involve er, the molecular mechanism of tuning ATPase activity through the MTBD at the coordinated efforts of many motors working together. To investigate the the tip of dynein stalk remains unclear. cooperative activities of multi-motor assemblies, we designed a microfluidic Here, we performed mutational and structural studies of the interaction between device. The device is made by etching a diamond-shaped pattern into acrylic LC1 and stalk region and identified two important sites for the interaction. providing a geometry that supports a laminar flow field under a wide range MTBD mainly interacts with LC1 through H5 helix and a flap region which is of flow rates. Gliding assays at low motor densities were performed in these mi- a characteristic insertion of the axonemal dynein MTBD. Based on these results, crofluidic devices with flow rates between 100 nL/s to 40 mL/s modulated by a we predicted a structural model of LC1-MTBD-Tubulin complex. In this presen- syringe pump. Velocity profiles across the flow cell were simulated using tation, we will discuss about the structural basis for the LC1-MTBD complex for- COMSOL Multiphysics, and were validated using particle tracking of 250 mation that might regulate the function of axonemal dynein heavy chain. nm fluorescent particles. Studies investigated the canonical kinesin motor pro- tein, Drosophila Melanogaster kinesin-1, and a more force-sensitive kinesin 3209-Pos Board B417 motor protein, mouse kinesin-2 KIF3A. Varying flow profiles over the gliding Creating Protein-Based Molecular Motors That Move along DNA assays generated fluid forces on the microtubules that resulted in detachment of Nanotubes the microtubules from the motors with no change in gliding velocity. With sus- Ryota Ibusuki1, Kazuhiro Oiwa1,2, Hiroaki Kojima2, Ken’ya Furuta2. tained flows, microtubule paths were measurably altered to align with the flow 1University of Hyogo, Hyogo, Japan, 2National Institute of Information and profiles. Increased bending of the taxol-stabilized microtubules also become Communications Technology, Hyogo, Japan. apparent during periods of fluid flow. Current studies are being extended to in- Linear molecular motors such as myosin, kinesin, and dynein are enzymes that crease the apparent force generation on the motors and microtubules by attach- move toward one end of cytoskeletal filaments. These motors are unusual nano- ing beads to the plus-end of microtubules during gliding assays. machines because they can robustly move unidirectionally even in the storm of

BPJ 8740_8744 648a Wednesday, February 21, 2018 thermal agitation. Despite the decades of studies, however, the essential mech- points, increase cofilin severing rate, independently of filament tension. Our ex- anisms of directional motion remain unclear. For example, we do not under- periments thus reveal how ADF/cofilin, together with capping protein, control stand how the structural change of motors, and the asymmetric structures of the dynamics of actin filament barbed and pointed ends, and how mechanics the motor-filament interface contribute to directional motion. A limitation is tune cofilin severing in actin networks. that neither motors nor cytoskeletal filaments can be rationally re-designed, making it difficult to address these key questions. To overcome this limitation, 3212-Pos Board B420 we took bottom-up approaches where new molecular motors and tracks are de- Intrinsically Disordered Region of Actin Binding Protein Regulates signed and created based on protein and DNA building blocks. Through the Dynamic Actin Assembly process of creation, we will understand the essential factors for a molecular mo- He Sun, Yansong Miao. tor to move forward. Here we constructed a new hybrid motor from dynein and Nanyang Technological University, Nanyang Technological University, a DNA-binding protein. We chose DNA as a track instead of cytoskeletal fila- Singapore, Singapore. ments because DNA have a lot of advantages: it is stable, synthesizable, and Assembly of the actin cytoskeleton are dynamically regulated under various phys- can be self-organized into higher order structures. In in vitro motility assays, iological and pathological states to fine-tune the endocytosis, cytokinesis and ve- the new hybrid motors successfully translocated 10-helix DNA nanotubes at sicular transport. A particularly interesting phenomenon is that both endocytic an average velocity of 8 nm s1. Furthermore, the multiple molecules of hybrid proteins and ABPs are highly enriched of intrinsically disordered regions motors transported single DNA origami cargoes along immobilized DNA nano- (IDRs), which were recently found as a constituent of protein crowding, protein tubes. Control experiments showed that the hybrid motors recognize the spe- coacervation, and phase separation. How the actin filament assembly are regulated cific DNA sequence that is periodically incorporated along the long axis of by these intrinsically disordered regions are largely unclear, which are important the DNA nanotube. Our strategy opens the way to systematic studies on the for understudying in vivo complex regulation of actin polymerization and organi- mechanisms of motors, and to nanotechnological applications using the power- zation. The protein dynamics, conformation, inter- or intramolecular interactions ful DNA-based molecular toolbox. for intrinsically disordered proteins (IDPs) are highly regulated by the changes of surface electrostatic interactions through the post-translational modifications, such 3210-Pos Board B418 as protein phosphorylation. We recently studied several ABPs and found striking F-Actin Meditated Focusing of Vesicles at the Cell Tip is Essential for regulation of actin filament assembly through the IDR and the phospho-regulation Polarized Growth of IDR of actin binding proteins. We will present and discuss the molecular mech- Jeffrey P. Bibeau1, James L. Kingsley2, Fabienne Furt1, Erkan Tuzel€ 2, anisms by which actin assembly receives multi-signals from intracellular or extra- Luis Vidali1. cellular cues in modulating actin filament assembly. 1Department of Biology & Biotechnology, Worcester Polytechnic Institute, 2 3213-Pos Board B421 Worcester, MA, USA, Physics, Worcester Polytechnic Institute, Worcester, Large-Scale Coarse Grained Simulations of F-Actin Interacting with MA, USA. Model Membranes Filamentous actin has been shown to be essential for tip growth in an array of Carsten F.E. Schroer, Siewert J. Marrink. plant models, including Physcomitrella patens. One hypothesis is that diffusion Groningen Biomolecular Sciences and Biotechnology Institute, Univeristy of can transport secretory vesicles, while actin plays a regulatory role during Groningen, Groningen, Netherlands. secretion. Alternatively, it is possible that actin-based transport is necessary Actin is one of the most abundant proteins in eukaryotic cells. Its importance to overcome vesicle transport limitations to sustain secretion. Therefore, a lies in the ability to form polymer strands, the actin filaments, which are the quantitative analysis of diffusion, secretion kinetics and geometry is necessary main component of the cytoskeletal actomyosin cortex. The actomyosin cortex to clarify the role of actin in polarized growth. Using FRAP analysis, we first is a cross-linked network of actin fibers and myosin that is located at the inner show that secretory vesicles move toward and accumulate at the tip in an face of the cell membrane and responsible for the shape and the mechanical sta- actin-dependent manner. We then depolymerized F-actin to decouple vesicle bility of the cell. As such, actin strands are in the direct vicinity of the mem- diffusion from actin-mediated transport, and measured the diffusion coefficient brane and their interactions are determined by the interplay between the and concentration of vesicles. Using these values, we constructed a theoretical membrane composition and specific actin-binding proteins. diffusion-based model for growth, demonstrating that with fast-enough vesicle Here, we show results from coarse grained MD simulations of filamentous actin, fusion kinetics, diffusion could support normal cell growth rates. We further interacting with model membranes of different compositions. The simulations refined our model to explore how experimentally-extrapolated vesicle fusion are based on the Martini model that has been successfully applied for a great diver- kinetics and the size of the secretion zone limit diffusion-based growth. This sity of biomolecular systems. Our results display a distinct variety of interactions model predicts that diffusion-mediated growth is dependent on the size of between the polymer and the lipid bilayers, depending on the nature of the lipids. the region of exocytosis at the tip, and that diffusion-based growth would be Furthermore, we show some preliminary results of an actin binding protein, inter- significantly slower than normal cell growth. To further explore the size of acting with the membrane and the filament at the same time. This system closely the secretion zone, we used a cell wall-degradation enzyme cocktail, and deter- resembles the situation in the actomyosin cortex and can shed some light on the mined that the secretion zone is smaller than in diameter at the tip. Taken molecular detail of the coupling of the cell membrane and the cytoskeleton. together our results highlight the requirement for active transport in polarized growth and provide important insight into vesicle secretion during tip growth. 3214-Pos Board B422 Stability on the Edge: Probing the Biophysical Mechanisms of Polarity Posters: Cytoskeletal Assemblies and Dynamics Maintenance in Motile Cells Rikki M. Garner1, Elena Koslover2, Andrew J. Spakowitz3, 3211-Pos Board B419 Julie A. Theriot4. Mechanical Regulation of Actin Filament Disassembly by ADF/Cofilin 1Biophysics Program, Stanford University, Stanford, CA, USA, 2Department Antoine Jegou, Hugo Wioland, Guillaume Romet-Lemonne. of Physics, University of California San Diego, San Diego, CA, USA, Institut Jacques Monod - CNRS, Paris, France. 3Chemical Engineering, Applied Physics, Materials Science and Actin-depolymerizing factor (ADF)/cofilins contribute to cytoskeletal dy- Engineering, Biophysics Program, Stanford University, Stanford, CA, USA, namics by promoting rapid actin filament disassembly. Here, by monitoring 4Department of Biochemistry, Department of Microbiology and the activity of the three mammalian ADF/cofilin isoforms on individual skeletal Immunology, Howard Hughes Medical Inst., Stanford University, Stanford, muscle and cytoplasmic actin filaments, we directly quantify the reactions un- CA, USA. derpinning filament severing and depolymerization from both ends. Compared Neutrophil chemotaxis to sites of inflammation and infection is one of the first to bare filaments, we find that ADF/cofilin-saturated filaments depolymerize steps in a healthy immune response. In their surveillance of the human body, neu- faster from their pointed ends and slower from their barbed ends, resulting in trophils must undergo polarized migration over long distances through a host of similar depolymerization rates at both ends. We show that, unexpectedly, different physical environments - in the bloodstream, along blood vessel walls, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their and within many mechanically distinct tissues. The long-term goal of this barbed ends are very difficult to cap or elongate, and consequently undergo research is to understand how neutrophils regulate their actin cytoskeleton in or- depolymerization even in the presence of capping protein and actin monomers. der to maintain polarized migration through dynamic physical settings. Using Because capping induced by capping proteins allows growing ADF/cofilin do- high-speed microscopy of neutrophil-like HL-60 cells, we have shown that mains to reach the barbed ends, it thereby promotes barbed end uncapping and cell leading edge shape undergoes micron-sized fluctuations that dynamically its subsequent depolymerization. We also investigated how mechanical con- appear and equilibrate on the order of seconds. We have developed image anal- straints affect ADF/cofilin severing activity. We observe that local filament ysis tools to quantify properties of these fluctuations, which we use as a read-out curvature, as well as constraining filament torsion between two anchoring of the biochemical and physical feedback mechanisms used to maintain

BPJ 8740_8744 Wednesday, February 21, 2018 649a polarization. In addition, we have developed a minimal physical model of actin ual molecules have been extensively characterized. Here, we show in vitro that growth against a membrane to interpret these results, predict how changes in flagellar-like beating can be produced naturally and robustly in polar bundles of properties of actin growth affect leading edge dynamics, and identify candidate filaments. Using surface micro-patterns of a nucleation-promoting factor, we actin binding proteins that are likely to regulate polarity maintenance. controlled the geometry of actin polymerization to produce thin networks of par- This minimal model recapitulates many aspects of lamellipodial behavior allel actin filaments. In the presence of either myosin Va or heavy-mero myosin II in vivo, such as treadmilling and decay of actin intensity away from the leading motors added in bulk, growing actin filaments self-organized into bundles that dis- edge. In the future we will iteratively test and update this model to determine the played periodic wave-like beating resembling those observed in eukaryotic cilia minimal set of feedback interactions that can reproduce polarized cell migration and flagella. The waveform of oscillation was similar for the two types of motors. behavior. We will then use the model to predict how neutrophils employ these However, oscillations with myosin II were one order of magnitude faster than with feedback mechanisms to respond to forces in their environment - informing myosin Va. In both cases, a wave of bending deformations propagated at a uniform how neutrophil migration, and thus immune function, might vary between tis- velocity from the anchored base of the actin bundle towards the freely-moving tip. sues with different mechanical properties. As polymerization proceeded, the actin bundle elongated at a constant velocity, re- sulting in a proportional increase of the period of oscillation. Remarkably, the 3215-Pos Board B423 propagation velocity of the bending wave did not vary with the increasing bundle Ultra Fast Contractions and Emergent Dynamics in a Living Active Solid - length, indicating that thebundle lengthset the wavelength. Ourwork on a minimal Trichoplax adhaerens The Epithelium of the Primitive Animal acto-myosin system demonstrates that active flagellar-like beating emerges as an Shahaf Armon, Manu Prakash. intrinsic property of polar bundles of filaments in interaction with molecular mo- Bio Engineering, Stanford University, Stanford, CA, USA. tors. Structural control over the self-assembly process provides key information to Apical contractions in epithelial sheets are often associated with embryogenesis. clarify the underlying physical principles of flagellar-like beating. Conventionally, these contractions are slow and precisely controlled in space and time, patterning the shape and form of a developing embryo. In this work 3218-Pos Board B426 we report the discovery of ultra-fast epithelial contractions (50% cell area in 1 Characterization of Actin Modulating Proteins in the Cytokinetic Ring second, at least an order of magnitude faster) in a ‘‘simple’’ primitive marine Machinery of Yeast using a Minimal In-Vitro System invertebrate, Trichoplax adherence, lacking neurons or muscles. Using theoret- Saravanan Palani, Paola Zambon, Anton Kamnev, Tomoyuki Hatano, ical calculations, we demonstrate that this speed can still be explained by existing Mohan K. Balasubramanian, Darius V. Ko¨ster. models of acto-myosin contractility and load reduction. We show that tissue ar- Centre for Mechanochemical Cell Biology, University of Warwick, chitecture is reducing the load on the molecular motors at work. Furthermore, Coventry, United Kingdom. live imaging of the whole animal in vivo reveals emerging contraction patterns, The mechanism of cell division is conserved in many eukaryotes, ranging from including propagating radial and axial waves. We hypothesize a new role of yeast to man. A contractile ring of filamentous actin and myosin II motors gen- cellular contractions in epithelium - keeping tissue integrity and enabling resil- erates the force to bisect a mother cell into two daughters (Balasubramanian, Bi ience to rupture via ‘‘active cohesion’’. Studying this primitive epithelium high- and Glotzer, Curr. Biol., 2004). The cytokinetic actomyosin ring is among the lights a novel unstudied realm in active soft matter. most complex cellular machines, comprising over 150 proteins. Understanding how these proteins organize themselves into a functional ring with appropriate 3216-Pos Board B424 contractile properties remains one of the great challenges in cell biology. Using Maturation of the Human Motile Cilia Waveform in Airway Cells supported lipid bilayers as a two-dimensional fluid scaffold and total internal Alina Oltean1,2, Philip V. Bayly1, Steven L. Brody2. reflection fluorescence microscopy, we test and characterize the function of iso- 1 Mechanical Engineering and Materials Science, Washington University in lated, actin binding proteins involved in cytokinetic ring formation. We identify 2 St louis, Saint Louis, MO, USA, Department of Medicine, Washington two actin binding domains in the membrane protein Mid1, that is important for University School of Medicine, Saint Louis, MO, USA. the correct positioning of the cytokinetic ring. One actin binding domain was ex- Motile cilia protrude from the apical surface of cells tomove fluid or particlesinthe pected but the second domain for actin binding is a new finding. More interest- brain, airways, and oviduct. Cilia are composed of paired microtubules in a 9þ2 ingly, we observed that the actin binding domain of Rng2 leads to the formation configuration that bend due to the activation of dynein motor proteins. In the of actin rings with curvatures of 0.3-1 mm1 on a planar lipid bilayer. In addition, airway, cilia are essential for clearance of mucus and particles which depends decoration of actin filaments with the tropomyosin Cdc8 leads to a more consis- on an effective back and forth waveform. Cilia motion can be evaluated in ex tent ring formation with a tighter bending of 1 mm1. Taken together, these pre- vivo samples or in vitro cultures as part of the diagnosis of diseases such as primary liminary findings reveal a minimal set of proteins that lead already to the ciliary dyskinesia. However, the characteristics of a healthy cilia waveform and the formation of tightly bound actin rings that would be key for the successful forma- relationship between cilia assembly and motion are not well characterized. To tion of a cytokinetic ring. This shows the potential of the supported lipid bilayer quantify ciliary dynamics in human samples, we analyzed the motion of cilia assay to decouple the complex, multicomponent protein-protein interactions and from donated airway samples of non-diseased donors. We compared cilia motion to follow their spatio-temporal dynamics with high resolution. from high-speed video capture of cells directly sampled from the airway to quan- tify the variation within donor samples and across donors. The cilia of ex vivo sam- 3219-Pos Board B427 ples were also compared to cells differentiated at air-liquid interface by Micro-Manipulating the Spindle to Study Chromosome Segregation in quantifying parameters such as curvature and the estimated forces exerted by Anaphase each beat cycle. To correlate cilia assembly to changes in cilia motion, we exam- Jun Takagi1, Takeshi Itabashi2, Shin’ichi Ishiwata2, Yuta Shimamoto1. 1 2 inedouterdyneinarm localization with fluorescence microscopy atdifferent stages National Institute of Genetics, Shizuoka, Japan, Waseda University, Tokyo, of ciliogenesis. Quantitative assessment of waveform parameters showed large Japan. variations in waveform shape that were dependent on cilia length and stage of dif- During anaphase of cell division, sister chromatids are separated to the opposite ferentiation. Our findings suggest that normal cilia have a wide variation in wave- ends of a cell by a pulling force generated by the microtubule-based mitotic spin- form and other motion parameters that may correspond to analogous changes in dle. The sister chromatids are topologically entangled at the onset of anaphase. dynein localization. By comparing in vitro and ex vivo samples, we suggest objec- This entanglement has to be resolved in a timely manner as it opposes the spindle tive parameters to describe cilia motion, to ultimately improve the characterization force and may create a significant strain on the DNA. Cell-based assays suggest of cells as healthy or diseased using high-speed video microscopy. that failures in relieving the strain lead to severe DNA damage. However, it is largely unknown how chromosomes cope with the spindle force while resolving 3217-Pos Board B425 the sister chromatid adhesion. In this study, we combined a microneedle-based Self Organized Wave Like Beating of Actin Bundles in a Minimal micromanipulation setup with Xenopus cell-free egg extracts to study the effect Acto-Myosin System of Controlled Architecture of force on sister chromatid motion and deformation in anaphase. Using a pair of Marie Pochitaloff1, Mathieu Richard1, Takagi Yasuharu2, force-calibrated glass microneedles, the chromatids were stretched by pulling Enrique De La Cruz3, Jim Sellers2, Jean-Franc¸ois Joanny1, Frank Julicher€ 4, apart the poles of the anaphase spindle. We found that the speed of chromatid Laurent Blanchoin5, Pascal Martin1. separation depends on the magnitude of poleward pulling force. Molecular 1Laboratoire PCC (UMR168), Institut Curie, Paris, France, 2Laboratory of perturbation suggested that the force-dependency is linked to the activity of Molecular Physiology, NHLBI - NIH, Bethesda, MD, USA, 3Molecular type II topoisomerase, a key enzyme responsible for resolving the topological Biophysics and Biochemistry, Yale University, New Haven, CT, USA, problem of DNA. Furthermore, a relatively fast pulling led to excessive stretch- 4Biological Physics, Max Planck Institute for the Physics of Complex ing of chromatid fibers, while the adhesion of sister chromatids remained per- Systems, Dresden, Germany, 5iRTSV, CEA, Grenoble, France. sisted. Together with a simple mechanical model, our data suggest a The emergent active behaviors of systems comprising large numbers of molecular mechanism of how anaphase chromosomes transmit spindle force for resolving motors and cytoskeletal filaments remain poorly understood, even though individ- their adhesion while maintaining overall integrity.

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3220-Pos Board B428 microtubule bundles and orientationally ordered phases. Additionally, steady- Minimal Ingredients for Coupled Spindle Assembly and Chromosome state structures that resemble ring-shaped spools of single or multiple bundled Bi-orientation in a Computational Model of Fission Yeast Mitosis filaments appear, which arise due to the flexibility of the filaments. These Christopher Edelmaier1, Adam Lamson1, Zach Gergely2, spools have also been observed in motility assays with actin and myosin, but J. Richard McIntosh2, Matthew A. Glaser1, Meredith D. Betterton1. their genesis and stability are poorly understood. In order to better understand 1Physics, University of Colorado Boulder, Boulder, CO, USA, 2Molecular, the behavior of these systems, we have developed simulations of gliding fila- Cellular, and Developmental Biology, University of Colorado Boulder, ments in motility assays by modeling the filaments as wormlike chains that un- Boulder, CO, USA. dergo steric interactions and experience an orientation-dependent force per unit Mitosis ensures the proper segregation of chromosomes into daughter cells, length. We characterize the phase transitions between the isotropic flocking, which is accomplished by the mitotic spindle. During fission-yeast mitosis, nematic laning, and spooling phases as a function of filament density, persis- chromosomes establish bi-orientation as the bipolar spindle assembles, mean- tence length, and driving. These simulations make predictions for the behavior ing that sister kinetochores become attached to microtubules whose growth of motility assays and can provide additional insights into self organization of was initiated by sister poles. This process must also correct erroneous attach- the cytoskeleton. ments made by the kinetochores during the attachment process. Our goal is to build a 3D computational model of spindle assembly based on a realistic 3224-Pos Board B432 description of microtubule, kinetochore, and chromosome dynamics, in order A Novel Kinase Activity of Calponin to interrogate the role of specific mechanisms in these chromosome bi- Nicholas W. Diloreto. orientation and error correction processes. We have added chromosomes to Physiology, Wayne State School of Medicine, Grosse Pointe Farms, MI, our previous computational model of spindle assembly, which included mi- USA. crotubules, a spherical nuclear envelope, motor proteins, crosslinking pro- Calponin is an actin filament-associated regulatory protein expressed in both teins, and spindle pole bodies (centrosomes). In the new model, each smooth muscle and many types of non-muscle cells. An established function chromosome is represented by a pair of sister chromatids, and sister kineto- of calponin is an inhibitory regulator of myosin ATPase in modulating smooth chores are represented as chromatid-associated polygons. In preliminary muscle contractility and cell motility (Liu and Jin, Gene 585:143-53, 2016). work, we have explored the mechanical properties of kinetochores and their Three isoforms of calponin are present in vertebrates encoded by homologous interactions with microtubules that achieve amphitelic spindle attachments at genes CNN1, CNN2, and CNN3 with conserved structures but diverged in the high frequency. A plausible set of minimal assumptions yields simulations C-terminal region. We recently reported that a monoclonal antibody (mAb) that generate chromosome attachment errors, but resolve them, much as CP3 raised against calponin 1 has a specific cross reaction to carbonate anhy- normal chromosomes do. drase 3 (Feng and Jin, Front Physiol 7:597, 2016), implicating a structural sim- ilarity between calponin and metabolic enzymes. In the present study, we 3221-Pos Board B429 developed and characterized another mAb, 1H8, raised by immunization of cal- Nonequilibrium Dissipation in Living Oocytes ponin 2. Western blotting and mass spectrometry identification demonstrated Wylie Ahmed. that 1H8 cross-reacts to creatine kinases M and B. Analysis of calponin 2 using Physics, California State University Fullerton, Fullerton, CA, USA. a commercial creatine kinase assay kit demonstrated that calponin 2 has a Living organisms are inherently out-of-equilibrium systems. We employ recent kinase-like activity. The sub-molecular location of the 1H8 epitope is mapped developments in stochastic energetics and rely on a minimal microscopic using engineered calponin isoforms and fragments. Two-dimensional gel elec- model to predict the amount of mechanical energy dissipated during active trophoresis, Western blotting and mass spectrometry identification are applied vesicle motion in mouse oocytes. Our model includes complex rheological ef- to study the presence of 1H8 epitope-like structures in other metabolic en- fects and nonequilibrium stochastic forces due to molecular motors. By per- zymes. The results are analyzed together with sequence database search to forming active microrheology and tracking micron-sized vesicles in the identify the molecular structures that give calponin the kinase-like properties. cytoplasm of living oocytes, we quantify the spectrum of dissipated energy. Functional significance of the kinase-like structures and activity of calponin We show that our model is consistent with the experimental data, and we use is being investigated using calponin knockout smooth muscle tissues and fibro- it to offer predictions for the injection and dissipation energy scales involved blasts. While extensive research has been done for the functions of calponin in in active fluctuations in the oocyte cytoplasm. regulating contractility and cell motility, the novel kinase-like structures and activities of calponin opens a new direction to understand its physiological 3222-Pos Board B430 and pathological functions. Investigating Quality of Mixing of a Biological Active Nematic Amanda J. Tan, Eric Roberts, Kevin A. Mitchell, Linda S. Hirst. Posters: Cell Mechanics, Mechanosensing, and UC Merced, Merced, CA, USA. Swarming birds, and swimming bacteria are forms of active matter that have Motility III unique properties of collective motion. Active matter consists of individual en- ergy consuming particles that move collectively in bulk. We use biopolymers 3225-Pos Board B433 Euglena gracilis (microtubules), and molecular motors (kinesin) to form an active matter Biophysics of Collective Phototaxis of network that is a self-mixing system. We investigate the fluid dynamic proper- Alan C.H. Tsang, Amy T. Lam, Ingmar H. Riedel-Kruse. ties of this biological self-mixer. When confined in 2D in an oil-water interface, Bioengineering, Stanford University, Stanford, CA, USA. the network is a self-mixing active nematic that exhibits unique fluid dynamics. High-density suspensions of actively swimming cells in fluids often self- We are interested in quantifying the mixing dynamics of this 2D system. Sys- organize into complex dynamic patterns due to the interplay between tems that exhibit good mixing show exponential stretching in their material microscopic force generation and macroscopic hydrodynamics. Though lines. We bound beads to the microtubules in the active network as a way of various biophysical models have been developed to describe the dynamics tracking filament bundles mixing in the network. Using these trajectories, we of these systems, experimental verification has been difficult to obtain. This find bead pairs that are initially close together, and measure their separation dis- is due to the complex interaction rules which govern the swimmer behavior, tance as a function of time to measure the stretching rate of the material. The and the lack of tools for delivering controlled perturbations to a defined system shows exponential stretching, which may imply that is a good mixer. subset of cells inside such a suspension. Here, we consider concentrated We also change the mixing velocity by altering ATP concentration, and suspensions of the unicellular, photoresponsive algae, Euglena gracilis. observe how the velocity affects the stretching rate. We propose an integrated experimental and theoretical platform for predict- ing the collective behavior of Euglena under light stimulation. By varying 3223-Pos Board B431 the spatiotemporal light patterns, we are able to finely adjust cell densities Organization and Dynamics of Gliding Flexible Filaments and achieve arbitrary non-homogeneous distributions, including compres- Jeffrey M. Moore, Tyler N. Thompson, Matthew A. Glaser, sion into high-density aggregates of varying geometries. Our models Meredith D. Betterton. were able to account for the light-induced aggregation that result from a Department of Physics, University of Colorado Boulder, Boulder, CO, USA. complex interplay between negative phototaxis, hydrodynamic interactions, Interactions between the microtubule cytoskeleton and its associated proteins and shading effects between the cells. This work will deepen our general give rise to complex behavior that is essential to many cellular processes. understanding of how relatively simple mechanical feedback interactions Experiments to observe microtubule organization have used surface-tethered can generate multi-cell behaviors in microswimmer suspensions, and how kinesin-1 motors to generate directed forces along microtubules in filament- to bridge such understanding over different length and time scales. Further- gliding motility assays. As microtubule density and ATP concentration are more, it may also aid the engineering of microfluidic devices and advance varied, these active systems exhibit different behaviors, including flocking concepts in active matter physics.

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3226-Pos Board B434 nuclear lamin A/C-mediated formation of the perinuclear apical actin cables pro- Frustrated Phagocytic Spreading of Human Neutrophils on Different tects the nuclear structural integrity from extracellular physical disturbances. Our Densities of Surface-Immobilized IgG findings highlight the role of the physical interactions between the cytoskeletal Zhiyu Xiao, Emmet A. Francis, Volkmar Heinrich. network and the nucleus in cellular mechanical homeostasis. Biomedical Engineering, University of California, Davis, Davis, CA, USA. Although phagocytosis by immune cells has been studied for more than a century, 3229-Pos Board B437 quantitative insights into phagocytic protrusion dynamics remain scarce, mainly Deformable Microparticles as Reporters for Probing Cellular Forces in due to the difficulty of analyzing the irregular, three-dimensional phagocyte Phagocytosis shapes. Here, we address this difficulty by examining antibody-mediated, frus- Daan Vorselen, Julie Theriot. trated phagocytic spreading of individual human neutrophils on flat coverslips Stanford University, Stanford, CA, USA. that were coated with four different densities of rabbit IgG. We use reflection inter- Macrophages are able to phagocytose targets of vastly different rigidities, ranging ference contrast microscopy (RICM) tomeasure the growing cell-substrate contact from bacteria during immune response, to apoptotic cell fragments in tissue ho- area with high precision. Using neutrophils that were preloaded with the fluores- meostasis. At the same time, experiments have shown that phagocytosis of similar cent calcium indicator Fluo-4, we concurrently monitor the cytosolic free calcium targets can be strongly affected by minor differences in target rigidity. It is concentration, which enables us to assess the timing of prominent calcium- currently poorly understood how phagocytes handle targets with such disparate signaling surges. We find that the overall speed of the contact-area growth is physical properties, or how physical cues of the target feed back into the process largely independent of the IgG density, confirming that this type of cell spreading of phagocytosis. Studying these processes is hampered by lack of tools to probe is governed by the ‘‘protrusive zipper’’ rather than the adhesion-driven ‘‘Brownian the mechanical interaction (i.e. the forces) between phagocyte and target. We zipper’’ mechanism. On the other hand, neutrophils spreading on lower IgG den- developed monodisperse (CV < 0.1) spherical hydrogel particles with tunable sities tend to exhibit less round contact regions than those on higher IgG densities, size (4-20 mm) and elastic properties. Furthermore, they can be readily conjugated and their maximum contact area often remains smaller. Regardless of IgG density, with a variety of ligands and fluorescent dyes. We show that IgG-functionalized we observe a global burst in intracellular calcium concentration in most of the microparticles are taken up by J774.1 macrophage-like cells, in a rigidity depen- actively spreading cells. However, whereas cells spreading on high IgG densities dent manner. Moreover, soft particles ( kPa) are strongly deformed during exhibit calcium bursts almost immediately after contact with the substrate, such phagocytosis, implying highly localized cellular forces (nN). Our work currently bursts tendtooccur later inneutrophilsspreading on lower densities ofIgG, usually focuses on the quantification of these forces. Such a method is expected to find only when the contact area has already reached a moderate value (50-100 mm2). broad applications in the study of in vitro and in vivo cellular forces. This result indicates that the timing of calcium bursts is governed by a threshold of 3230-Pos Board B438 the number of engaged Fcg-receptors of the spreading neutrophil. Matrix Stiffness Regulates the Fate of Breast Cancer Cells 3227-Pos Board B435 Deep Parikh, Mary Stack, Hongjun Wang. Coordination of Morphogenetic Growth and Cellular Mechanics across BCB, Stevens Institute of Technology, Hoboken, NJ, USA. Multiple Cell Layers to Shape the Drosophila Wing Disc Breast cancer metastasis is regulated by the physicochemical characteristics of Ali Nematbakhsh. their extracellular matrix (ECM). Sufficient studies have been done to demon- University of California at Riverside, Riverside, CA, USA. strate the effects of compositional changes in the ECM on breast cancer metas- Epithelia serve as barriers between the environment and internal structures of tasis, but little is known about the effects of physical changes on breast cancer organs. Epithelial morphogenesis must be carefully controlled through coordi- metastasis. Previous studies of 24-h monitoring have implied that increase in nation of cellular properties. This is critical because uncontrolled epithelial stiffness could lead to the increase of cell spreading, migration, invasion and growth is the underlying cause of more than ninety percent of tumors. However, metastatic capabilities of breast cancer. However, these model systems typi- how mechanical properties are patterned across multiple cell layers to define cally fail to recapitulate the dimensional and morphologic characteristics of overall organ shape during development is poorly understood. Additionally, natural ECM along with the negligence of the fact that the stiffness of healthy how cells regulate their heights and remodel the extracellular matrix during or- breast tissue is about 167 Pa while the malignant one is 4000-6000 Pa. To gan growth is still largely unknown. Here, we define the contributions of mul- address the challenges, a gel/fiber hybrid system is accordingly designed to tiple epithelial cell types (squamous, cuboidal and columnar) to the shaping of combine ECM-mimic fibers with agar gel that exhibits tunable stiffness the Drosophila wing imaginal disc. The wing disc has long served as a para- (157-5117 Pa) proportional to agar concentration. With the assistance of digm model system to study epithelial morphogenesis due to the ability to such a system, we observed that the cell spreading area of breast cancer cells selectively manipulate cell mechanics within the tissue. The wing disc becomes (MDA-MB-231, MCF-7) increased over the stiffness increase within 24 h after progressively more folded and buckled as the organ grows to its final size. seeding, but this trend reversed afterwards and remained comparable for the Coupled computational simulations and experiments demonstrate that overall rest of the period. Interestingly, a complete halt of cell proliferation was noticed organ shape depends on several factors: differential regulation of growth rates with a stiffness of 5117 Pa after 72 h. Meanwhile, a maximum cellular migra- between cell types, spatial patterning of mechanical properties across the tissue, tion was also observed at 5117 Pa. Cells on softer surfaces preferred to form and regulation of the underlying extracellular matrix surrounding the organ. colony, while on stiffer surfaces they appeared to be round and individual. Dif- The computational model couples a novel subcellular element (SCE) model ferential expression of metastasis-associated proteins like e-cadherin, vinculin, representing cell mechanics and a discrete sub-model describing how chemical vimentin and focal adhesion kinases was observed. Thus, we conclude that ma- signaling patterns the subcellular mechanical properties of cells. Computational trix stiffness closely and temporally regulates the phenotype of breast cancer predictions are validated through both pharmacological and genetic perturba- cells and targeting ECM may offer another avenue for cancer therapy. tions of known regulators of epithelial morphogenesis, including the bone morphogenetic protein Decapentaplegic (DPP) and regulators of actomyosin 3231-Pos Board B439 Local Epithelial Fracture and Healing Mechanics Dictate Morphogenesis contractility. This work describe a powerful computational platform for evalu- Trichoplax adhaerens ating mechanisms of three-dimensional epithelial morphogenesis through feed- and Asexual Reproduction in 1 2 1 back between multi-scale computational modeling and experimentation. Vivek N. Prakash , Arjun Bhargava , Manu Prakash . 1Department of Bioengineering, Stanford University, Stanford, CA, USA, 3228-Pos Board B436 2Department of Applied Physics, Stanford University, Stanford, CA, USA. Lamin A/C Guided Nuclear Mechanotransduction Epithelial tissues play a crucial role in determining shape and form in all adult Jeong-Ki Kim, Dong-Hwee Kim. animals by providing robust support to organs and organ systems. However, in KU-KIST Graduate School of Converging Science and Technology, Korea some situations, epithelia are also known to be dynamic structures that display a University, Seoul, Republic of Korea. fluid-like behavior with local cell rearrangements (intercalation events) regu- The distinct spatial architecture of the apical actin cables (or actin cap) facilitates lated by genetic programs during embryo morphogenesis. Here, we have rapid biophysical signaling between extracellular mechanical stimuli and intra- discovered a novel case of non-canonical morphogenesis in an early diverging cellular responses, including nuclear shaping, cytoskeletal remodeling, and the animal - the Trichoplax adhaerens. It is one of the simplest known multicellular mechanotransduction of external forces into biochemical signals. These functions animals with a flat, pancake-shaped body plan. Although the animal lacks neu- are abrogated in lamin A/C-deficient mouse embryonic fibroblasts that recapitu- rons and muscles, it exhibits complex behavioral traits reminiscent of higher late the defective nuclear organization of laminopathies, featuring disruption of animals. Using live microscopy, we reveal that adult animals are capable of the actin cap. However, how nuclear lamin A/C mediates the ability of the real-time (seconds) extreme ‘amoeboid’ shape changes exhibiting both actin cap to regulate nuclear morphology remains unclear. Here, we show that solid-like (elastic) and liquid-like (plastic) tissue behavior. To quantitatively lamin A/C present cells could form the actin cap to resist nuclear deformation study this phenomenon, we developed a novel technique for long duration in response to physiological mechanical stresses. This study reveals how the (hours) and large-scale (mm) morphogenetic tracking of thousands of cells

BPJ 8740_8744 652a Wednesday, February 21, 2018 using micro-bead tags. Single cell tracking analysis of these organism-scale Without turnover, a catastrophic intrinsic contractile instability occurs. Small flow fields has revealed a surprising fluid-like motion - with characteristic fea- density fluctuations are amplified and fracture the ring. Restoring turnover tures such as vortices and shear regions, accompanied by local non-affine reduced instabilities as turnover is faster than aggregation. (disordered) cell rearrangements. We show that these disordered cell move- ments arise from local micro-fractures in the ventral epithelium. Remarkably, 3234-Pos Board B442 it turns out that these micro-fractures are generated physiologically, and are Motile Hair Cells Distinguish Mechanical Signals from Noise Best When regulated by global mechanical forces generated by the animal’s natural move- They Operate on the Brink of Spontaneous Oscillation Daibhid O Maoileidigh1, Joshua Salvi2, A.J. Hudspeth3. ments. These micro-fractures can enlarge to form macro-fractures that may heal 1 over time and lead to permanent organismal shape change, thus demonstrating Otolaryngology-Head and Neck Surgery, Stanford University, Palo Alto, CA, USA, 2Laboratory of Sensory Neuroscience, Rockefeller University, a direct link between micro-scale fractures and macro organism-scale morpho- 3 genesis. Finally, we show that epithelial fracture and healing mechanics govern New York, NY, USA, Laboratory of Sensory Neuroscience, Rockefeller two key biologically important phenomena observed in this animal - asexual University and HHMI, New York, NY, USA. reproduction by fission, and multi-scale tissue mixing. Hair bundles are the sensory organelles of hair cells that transduce mechanical stimuli into electrical signals in the auditory and balance organs of vertebrates. 3232-Pos Board B440 These sensors are substantially more sensitive to periodic stimulation when Coherent Timescales and Mechanical Structure of Multi-Cellular they are mechanically active than when they are passive. We developed a Aggregates model of active hair-bundle mechanics that predicts that a bundle’s behavior Miao Yu1, Aria Mahtabfar2, Paul Beelen2, Yasir Demiryurek1, and function may be controlled by the bundle’s mechanical load. The model David I. Shreiber3, Jeffrey D. Zahn3, Ramsey Foty2, Liping Liu4, Hao Lin1. relies only on the existence of mechanotransduction channels and an active 1Department of Mechanical and Aerospace Engineering, Rutgers University, adaptation mechanism which recloses the channels. A noisy hair bundle’s Piscataway, NJ, USA, 2Department of Surgery, Rutgers Robert Wood phase-locked amplitude and degree of entrainment are maximized when the Johnson Medical School, New Brunswick, NJ, USA, 3Department of bundle exhibits low-amplitude spontaneous oscillations. Additionally, these Biomedical Engineering, Rutgers University, Piscataway, NJ, USA, responsiveness measures are predicted to peak as functions of the noise level. 4Department of Mechanical and Aerospace Engineering and Department of We confirmed these predictions experimentally by employing a feedback sys- Mathematics, Rutgers University, Piscataway, NJ, USA. tem to change the mechanical load of individual hair bundles. By periodically 3D cellular aggregates are a broadly studied model system for biological pro- forcing hair bundles held at different operating points, we determined the range cesses including morphogenesis, carcinogenesis, malignant invasion, wound of parameters for which the signal is detected best. A hair bundle’s active pro- healing, and tissue engineering. Tissue-like aggregates are well known to cess amplifies the stimulus preferentially over the noise, allowing a self- exhibit liquid-like behavior in the long-times (hours), particularly manifesting oscillatory bundle to detect periodic forces less than 1 pN in amplitude. We a ‘‘surface tension’’ analogous to liquids. On the other hand, the mechanical also determined that the addition of noise can improve a bundle’s ability to behavior in the short times (seconds to minutes) is very different and has detect the stimulus under similar conditions. Overall, this work indicates that received less attention. The current work uses shape relaxation experiments auditory systems rely on active elements, composed of hair cells and their me- to investigate the structural characteristics of aggregates in the short times. chanical environment, that operate on the brink of self-oscillation. Two coherent timescales are observed, one on the order of seconds; the other, 3235-Pos Board B443 tens of seconds. Both are found to be persistent across all examined cell types Molecular Mechanisms for Distinct Functions of Talin Isoforms (GBM, 3T3, Rat2, L-cells) despite drastic changes in other properties such as Krishna Chinthalapudi1, Tina Izard2. tissue surface tension (by two orders of magnitude) and adhesion strength. A 1Department of Integrative Structural and Computational Biology, Scripps precise mathematical theory previously developed is used to extract the visco- Research Institute, Jupiter, FL, USA, 2Department of Integrative Structural elastic properties of aggregates from the measured timescales. The analysis and computational Biology, Scripps Research Institute, Jupiter, FL, USA. suggests that aggregates are a composite of an unusually ‘‘soft’’ interior Multicellular organisms have well-defined and tightly regulated mechanisms for (weak bulk elastic modulus) enclosed by a relatively stiffer envelope. Addition- cell adhesion where the heterodimeric ab integrin receptors play central roles and ally, aggregate surface tension, elastic modulus, and viscosity observe propor- regulate processes for normal cell functions such as signaling, cell migration, and tionality. This result suggests that the underlying cellular properties are adhesion. By regulating cell adhesion, integrins take part in several biological regulated in a precise manner. The root-cause for multi-cellular aggregation processes such as development, angiogenesis, immune response, tumor progres- to preserve such timescales requires further investigation. sion, and hemostasis through proper control of the integrin activation mecha- nisms that involve conformational changes of these receptors. By responding 3233-Pos Board B441 to extra- and intracellular stimuli, integrins connect the extra cellular matrix to A Node Organization Generates Tension and Promotes Stability in the the cytoskeleton, and transduce signals across the plasma membrane in both di- Fission Yeast Contractile Ring rections, termed outside-in and inside-out, respectively. During inside-out Sathish Thiyagarajan1, Shuyuan Wang1, Ben O’Shaughnessy2. 1 2 signaling, isoforms of the cytoskeletal protein talin play key roles in regulating Physics, Columbia University, New York, NY, USA, Chemical the affinity of integrin for extracellular matrix ligands whereby the talin head Engineering, Columbia University, New York, NY, USA. domain activates integrin by binding to the cytoplasmic tail of b-integrin and During cytokinesis in fungi, amoeba, and animals, cells divide into two using the acidic membrane phospholipids. So far, it has been assumed that the talin iso- tension generated by a contractile actomyosin ring. Tension is generated by forms (1 and 2) function redundantly and they have similar structural arrange- myosin-II that pulls on actin filaments, but how these forces are marshalled to ments that modulate integrin activity because of their high sequence identity. generate tension is unclear. Recently super-resolution microscopy (FPALM) However, a recent study specifically showed that talin 2 binds integrin much showed that myosin-II and formin-capped barbed-ends of actin filaments colocal- more tightly, which is required for generating traction forces that drives ize in membrane-anchored protein complexes called nodes in fission yeast rings invadopodium-mediated matrix degradation, which is essential for cancer cell in- (Laplante et al., 2016). Using this information, a mathematical model showed there vasion. Until now, there are no studies that explain what mechanical or structural exists two contra-rotating families of nodes. Actomyosin forces operate both within differences in the talin isoforms dictate their distinct functions. Here, we show and between families, and both contribute equally to ring tension. Anchoring en- structural basis of the talin 2 high affinity towards the integrin. Furthermore, sures that nodes aggregate slowly such that turnover can prevent instabilities. confocal microscopy and integrin activation assays show that the interaction be- We incorporated this nodes ultrastructure in a mathematical model of the tween talin 1 and the cell membrane is essential for focal adhesion formation and fission yeast ring that uses a coarse-grained, continuous representation of the integrin activation. Thus, the unique structural features in talin 1 and 2 isoforms nodes, and is constrained by the ample experimental data about the fission yeast dictate their integrin recognition and activation mechanisms. ring. We find there are two classes of node, stochastically determined by the polarity of the actin filaments associated with the node: nodes that move clock- 3236-Pos Board B444 wise, or nodes that move counterclockwise around the ring, due to myosin-II LIM Kinase 1 and 2 Regulate Motility and Invasion in Glioblastoma pulling forces. Thus, two node families contra-rotate with respect to one Joseph Chen, Badriprasad Ananthanarayanan, Kelsey Springer, another. This agrees with the bidirectional motion measured in FPALM. Sanjay Kumar. We find that opposite-polarity nodes are pulled towards one another, and this Bioengineering, UC Berkeley, Berkeley, CA, USA. sliding filament mechanism generates 50% of ring tension. Pulling forces be- Glioblastoma is one of the most aggressive human cancers characterized by high- tween same-polarity nodes contributes another 50% and involves no relative ly invasive cells that rapidly infiltrate the surrounding normal brain tissue. The motion. The predicted ring tension agrees with experiment (390 pN), with a disruption of tumor cell motility represents a promising therapeutic strategy to best-fit force per Myo2 head of 1 pN, close to previous measurements. reduce invasive potential and improve outcomes; however, the specific

BPJ 8740_8744 Wednesday, February 21, 2018 653a mechanisms regulating aggressive motility of glioblastoma cells are not well un- of the cell to achieve various phototaxis tasks effectively is not fully under- derstood. The members of the LIM kinase (LIMK) family, LIMK1 and LIMK2, stood. We develop a biophysical model that describes the change of the cell’s are important modulators of actin polymerization and depolymerization, essential pitch axis orientation and strength in dependence of the light intensity, which cellular mechanisms associated with cell polarization, migration, and invasion. leads to a unified account of various phototactic behavioral states of these cells. Importantly, LIMK1/2 are overexpressed in aggressive cancers including glio- We experimentally test this model using different spatiotemporal light stimuli. blastoma. In this study, we investigated the role of LIMK1/2 in regulating cell We find that the frequencies of rolling and helical swimming are coupled; we polarization and motility in glioblastoma tumor cells (U373) and glioblastoma identify a new escape strategy from strongly lit regions perpendicular to the tumor-initiating cells (TICs). Using shRNA strategies, we knocked down both light direction; and we show how a simple monotonous dependence of the pitch LIMK1 and LIMK2 and observed a strong effect on invasive phenotype in axis orientation on light intensity can naturally evoke the appropriate taxis both U373s and TICs. LIMK1/2 knockdown induced a dramatic loss of persistent behavior given lighting conditions. This work has implication for taxis strate- polarization and a decrease in 2D cell motility in U373 cells. Transwell invasion gies of other natural and synthetic chiral micro-swimmers. assays revealed a two-fold decrease in TIC invasion through 8 mmporesin LIMK1/2 knockdown cells. Similarly, migration of LIMK1/2 knockdown cells 3239-Pos Board B447 through transwells with 3 mm pores led to a more than four-fold decrease in in- Direct Measurement of the Magnitude and Dynamics of Mechanical vasion suggesting a role of LIMK1/2 in TIC migration through confined spaces Forces Exerted by Single Integrins in Living Cells reminiscent of those found within brain parenchyma. We then investigated the in Steven Tan, Alice Chang, Cayla Miller, Sarang Nath, Alexander Dunn. vivo effect of LIMK1/2 knockdown by xenografting luciferase-expressing con- Stanford University, Stanford, CA, USA. trol and LIMK1/2 knockdown TICs into the brains of immunocompromised Integrins mediate cell adhesion to the extracellular matrix and enable the construc- mice and monitored the growth of the tumors via bioluminescence imaging. tion of complex, multicellular organisms, yet fundamental aspects of integrin- LIMK1/2 knockdown TICs displayed substantially smaller tumors with lower based adhesion remain poorly understood. In particular, few experiments directly bioluminescent signal over time. These data provide evidence that LIMK1/2 report on the magnitude and dynamics of the forces experienced by individual in- play important roles in the migratory and tumorigenic capacity of glioblastoma, tegrins, due principally tolimitations inherent to existing techniques. Using Fo¨rster suggesting that these molecules might be potential therapeutic targets. resonance energy transfer (FRET)-based molecular tension sensors (MTSs), we measured the distribution of forces exerted by single integrin heterodimers in living cells. We found that the load exerted by individual integrins fell into subpop- 3237-Pos Board B445 < > Repair Factor Loss and Genome Variation in Cancer Cell Invasion ulations of approximately 3 pN, 4-8 pN, and 10 pN, suggestive of distinct func- Jerome Irianto1, Yuntao Xia1, Charlotte R. Pfeifer1, Avathamsa Athirasala1, tional roles. Disruption of the actin cytoskeleton with cytochalasin D blocked the > Jiazheng Ji1, Cory M. Alvey1, Manu Tewari1, Rachel R. Bennett2, formation of integrin-ligand bonds bearing 3 pN, while mouse embryonic fibro- Shane M. Harding3, Andrea J. Liu2, Roger A. Greenberg3, blast (MEF) cells lacking endogenous vinculin could primarily only form bonds Dennis E. Discher1. bearing up to 6 pN. In separate experiments, we found that the forces exerted by 1SEAS, University of Pennsylvania, Philadelphia, PA, USA, 2Graduate a5b1 and av-class integrin heterodimers were broadly similar, indicating that the Group, Department of Physics and Astronomy, University of Pennsylvania, force per integrin was not, in this system, primarily determined by integrin class. Philadelphia, PA, USA, 3Cancer Biology, Abramson Family Cancer Additionally, we used single-molecule MTS measurements to quantify the dy- Research Institute, Perelman School of Medicine, University of namics of mechanical load transmitted by individual integrins in MEFs and pri- Pennsylvania, Philadelphia, PA, USA. mary human dermal fibroblasts. Surprisingly, we found that the force per Migration through micron-size constrictions has been seen to rupture the nucleus, integrin in both cell types was largely constant, with a rare subpopulation of release nuclear-localized GFP, and cause localized accumulations of ectopic integrin-binding interactions yielding changes in FRET efficiency consistent 53BP1 - a DNA repair protein. Here, constricted migration of two human cancer with dynamic loading or unloading. In total, our data support a model in which cell types and primary mesenchymal stem cells (MSC) increases DNA breaks the force per integrin is set by the molecular connectivity to the actin cytoskeleton throughout the nucleoplasm as assessed by endogenous damage markers and rather than the integrin class. In addition, our findings suggest that, in some cell by electrophoretic ‘comet’ measurements. Migration also causes multiple DNA types, cytoskeletal force transmission may emerge from a compositionally dy- repair proteins to segregate away from DNA, with cytoplasmic mis- namic, yet mechanically stable, linkage at the integrin-cytoskeleton interface. localization sustained for many hours as is relevant to DNA repair times. Partial 3240-Pos Board B448 knockdown of repair factors that are known to also regulate chromosome copy Modeling Insights into the Mechanical Coordination in the Collective numbers is seen to increase DNA breaks in U2OS osteosarcoma cells, with nu- Locomotion of Heart Progenitor Cells celoplasmic patterns similar to migration, and knockdown likewise increases Calina Copos1, Yelena Bernadskaya2, Lionel Christiaen2, Alex Mogilner1. aberrant levels of DNA without affecting migration. Migration-induced nuclear 1Courant Institute, New York University, New York, NY, USA, 2Biology, damage is nonetheless reversible for wild-type and sub-cloned U2OS lines, New York University, New York, NY, USA. whereas DNA arrays and sequencing reveal lasting genomic differences. Gains During embryonic development, cells often migrate in groups. The heart progen- and losses of hundreds of megabases in many chromosomes are typical of the itor cells of the ascidian Ciona provide one of the simplest examples of collective changes and heterogeneity in bone cancer. Phenotypic differences that arise migration whereby just two cells migrate with defined leader-trailer polarity. The from constricted migration of U2OS cells are further illustrated by a clone cells are also capable of migrating individually, albeit by a shorter distance, with with a highly elongated and stable MSC-like shape that depends on microtubule imperfect directionality, and with altered morphology. Thus, maintaining the assembly downstream of the transcription factor GATA4. Such changes seem leader-trailer polarity is important for directed migration to the destination. To un- consistent with reversion to a more stem-like state upstream of cancerous osteo- derstand the mechanics of this collective migration phenomenon, we develop a blastic cells. Migration-induced genomic instability can thus associate with her- computational model to study the interplay of actomyosin contractility, cell- itable changes. Such migration-induced genomic changes were also observed in matrix adhesion, and the resulting leader-trailer polarity maintained for collective another lung cancer line, A549. To test the migration-induced genomic aberra- migration and combine modeling with imaging of the migrating cells. Two tions in vivo, clonal A549s were used to establish a metastatic mouse model fol- competing hypothesis are tested to understand the mechanical coupling and coor- lowed by genomic comparisons between the cells derived from primary and dination between leader-trailer cells and the cells in the extracellular tissues: (1) metastatic sites. Indeed, genomic variations were highest at the distant metastatic cells act as a single unit in which the leader cell generates actin-driven protrusions sites like lung, suggesting an invasion related genomic aberrations. while actin polymerization is down-regulated in the contractile trailer cell; (2) alternatively, contraction at the rear of the trailer cell leads to higher osmotic pres- 3238-Pos Board B446 sure pushing on leader cell. We present insights from modeling explorations of the Euglena Versatile Phototactic Behaviors of the Chiral Microswimmer mechanochemical coordination in this model system for collective locomotion. gracilis Alan Tsang, Amy Lam, Ingmar H. Riedel-Kruse. 3241-Pos Board B449 Bioengineering, Stanford, Stanford, CA, USA. Cell Cycle Inhibition by Constricted Migration The various taxis strategies of microorganisms regarding external stimuli high- Charlotte R. Pfeifer1, Victor M. Morales Garcia2, Leeza M. Santiago light interesting solutions to control-feedback tasks bounded by biophysical Millan3, Brandon Niese4, Jerome Irianto1, Dennis E. Discher1. constraints. Here we study the versatile phototactic behaviors of the micro- 1University of Pennsylvania, Philadelphia, PA, USA, 2University of Puerto swimmer Euglena gracilis. These cells scan the surrounding light by rolling Rico at Mayaguez, Mayaguez, Puerto Rico, 3University of Puerto Rico at around their long axes while swimming along helical paths, thereby pointing Humacao, Humacao, Puerto Rico, 4Ohio University, Athens, OH, USA. their single directional photoreceptor periodically into various directions. As a cancer cell invades adjacent tissue, penetrates a basement membrane bar- How this detected time-varying light signal is coupled to active reorientation rier, or squeezes into the smallest blood capillaries, its nucleus can be highly

BPJ 8740_8744 654a Wednesday, February 21, 2018 constricted. Such nuclear stress has recently been shown to enhance DNA dam- 3244-Pos Board B452 age and genome variation. However, little is known about the interaction be- Two Isoforms of Myosin-II Cooperate to Organize the Fission Yeast tween constriction-induced damage and cell cycle/DNA replication, even Cytokinetic Ring for Maximal Tension Production though the latter is increasingly being appreciated as a major source of Shuyuan Wang1, Harvey Chin2, Sathish Thiyagarajan1, Erdem Karatekin3, cancerous mutations. Here, U2OS osteosarcoma cells, U251 glioblastoma cells, Thomas Dean Pollard4, Ben O’Shaughnessy2. and A549 lung carcinoma cells were seeded at three different densities (low, 1Physics, Columbia University, New York, NY, USA, 2Chemical medium, high) on 2D plastic and were also migrated through rigid 3 mm and Engineering, Columbia University, New York, NY, USA, 3Cellular and 8 mm pores. In each case, cell cycle analysis was performed using total DNA Molecular Physiology, Yale School of Medicine, New York, NY, USA, content combined with EdU incorporation, as measured by fluorescence micro- 4Molecular, Cellular, and Developmental Biology, Yale University, New scopy. For all three cell types, G2 was found to be suppressed most in 2D by York, NY, USA. high density, indicating possible contact inhibition. In 3D, G2 was suppressed An actomyosin contractile ring drives cytokinesis of animal, fungal and most among cells that migrated through 3 mm pores, even though this was the amoeboid cells. How ring components organize to generate tension is lowest density condition. These findings suggest that constricted migration in- unanswered. Here we show that the two myosin-II isoforms in the fission hibits cell cycle progression, perhaps due to intra-S-phase checkpoint activa- yeast ring not only exert force, but have equally important roles as tion by constriction-induced DNA damage. Indeed, super-resolution actin crosslinking agents that organize the ring, cooperating to boost microscopy of migrated cells reveals DNA damage throughout the nucleoplasm tension. even though nuclear lamina disruption and chromatin blebs localize to the Recent FPALM super-resolution microscopy measurements of the fission yeast poles. ring showed that myosin-II Myo2 is organized in protein complexes (‘‘nodes’’) anchored to the plasma membrane, persisting into constriction phase. A second 3242-Pos Board B450 myosin-II isoform, Myp2, may be unanchored (Laplante et al., 2015, Takaine et Disrupting Endothelial Cell Biomechanics through Connexin 43 Inhibition al., 2015). Md. Mydul Islam, Robert Steward, Jr. Here we measured 650 pN ring tension in wild-type fission yeast protoplasts, University of Central Florida, Orlando, FL, USA. 65% of this value in Dmyp2 mutants, and 40% in myo2-E1 mutants with Endothelial cells are understood to bear and transmit intercellular stresses negligible ATPase activity and reduced actin binding. We developed a molec- through their cell-cell junctions. However, identification of the specific cell- ularly explicit simulation of the ring with the above organization, which re- cell junction molecule responsible for intercellular stress generation remains vealed a clear division of labor: (1) Myo2 anchors the ring to, and transmits an unanswered question. Thus far, the ability of tight junctions and adherens tension to, the plasma membrane. (2) Myo2 only bundles 25 actin filaments junctions to transmit intercellular stresses has been actively investigated, but in the cross-section within its reach, but not the other 25. (3) Unanchored the role of gap junctions is currently unknown. Gap junctions are formed by Myp2 thickens the bundle, by bundling an additional 25 filaments and connecting two neighboring connexons, which are each made from individual doubling tension. Anchoring of these filaments to the membrane is indirect, connexins. Although endothelial cells primarily express Connexin 43, Con- via filaments shared with Myo2. nexin 40 and Connexin 37 for this study we focused on connexin43 as this is In simulated myo2-E1 rings 20% of the actin filaments peeled away from the among the most abundant gap junction proteins expressed by endothelial cells. ring and formed Myp2-dressed bridges, as observed experimentally. The orga- In this study, we seeded endothelial cells onto a soft polyacrylamide gel (stiff- nization in simulated Dmyp2 rings was highly disrupted, with 50% of the ness= 1.2 KPa) and inhibited gap junction activity with the molecule 2’,5’-di- actin filaments unbundled. hydroxychalcone, a well established Connexin43 (Cx43) inhibitor. We In summary, beyond their widely recognized job to pull actin and generate repeated our experiments under fluid flow in a parallel plate flow chamber to tension, myosin-II isoforms are vital crosslinking organizational mimic arterial blood flow at a fluid shear stress of 1Pa. Intercellular stresses elements of the ring, a role that allows maximal actomyosin interaction were measured in both static and fluid flow conditions. Our results reveal and tension. Cx43 inhibition to reduce intercellular stresses when compared to the untreated condition. The results we present here reveal for the first time the role gap junc- 3245-Pos Board B453 tions play in endothelial biomechanical force generation. Curvature Dependence of Nuclear Rupture Frequency Revealed by AFM Force Spectroscopy 3243-Pos Board B451 Irena L. Ivanovska, Yuntao Xia, Jerome Irianto, Dennis E. Discher. Mechanosensing of Solid Tumors by Cancer-Attacking Macrophages University of Pennsylvania, Philadelphia, PA, USA. Cory Alvey, Charlotte Pfeifer, Jerome Irianto, Yuntao Xia, Lucas Smith, Nuclear deformations that occur during cell migration in constricting spaces Larry Dooling, Dennis E. Discher. can lead to nuclear envelope rupture with loss of many factors including Biophysical Eng’g Labs, University of Pennsylvania, Philadelphia, PA, USA. DNA repair proteins. DNA repair is often compromised in cancer, and so fluo- The physical biology of immune cell interactions with solid tumors has many rescence imaging of cancer cells was combined with single cell AFM-force facets, from tissue and cell levels down to key molecules. Macrophages are spectroscopy to characterize in real-time any rupture and loss of repair factors. highly phagocytic, especially when derived from marrow, but whether they Deep indentations into the nucleus with AFM probes having low curvature do can also traffic into solid tumors and engulf cancer cells is questionable, given not cause rupture even when the tip dwells for minutes with forces of 10-20 nN. the well-known limitations of tumor-associated macrophages (TAMs). Here, a However, poking the nucleus with tips having high curvature causes rupture ‘self’ recognition receptor on macrophages, termed SIRPa, was inhibited to events to increase dramatically in frequency. Repair factors mis-localization block binding at a phagocytic synapse to its membrane ligand, the ‘‘marker from the nucleus is restricted to cytoplasm, with no obvious leakage out of of self’’ CD47 found on all other cells. The modified macrophages were then the cell. Softening the nucleus with Lamin- A knockdown dramatically in- systemically injected into mice with fluorescent human tumors that were also creases probability of nuclear rupture even with indentation by low curvature antibody targeted. Within days, the tumors regressed, and single-cell fluores- at lower forces. The nuclear lamina is therefore mechano-protective against cence analyses showed that the more the macrophages engulfed, the more curvature-induced rupture. they accumulated within regressing tumors. Human-marrow-derived macro- phages engorged on the human tumors, while TAMs were minimally phago- 3246-Pos Board B454 cytic, even toward CD47-knockdown tumors. Super-resolution imaging and Computational Model of Dictyostelium Migration by Chemo-, Mechano-, mathematical modeling at multiple scales deepen insight into the key pro- and Rigidity Sensing cesses. Consistent with tumor-selective engorge-and-accumulate processes Atsushi Suzuki1, Takumi Hayakawa1, Kyungtaek Lim2, Kazushi Ikeda3, in vivo, phagocytosis in vitro inhibited macrophage migration through micro- Chika Okimura4, Yoshiaki Iwadate4, Yuichi Sakumura3. pores that mimic features of dense 3D tissue. Accumulation of SIRPa-inhibited 1Aichi Prefectural University, Aichi, Japan, 2ERATO Sato Project, Kyoto, macrophages in tumors favored tumor regression for 1-2 weeks, but donor mac- Japan, 3Nara Institute of Science and Technology, Nara, Japan, 4Yamaguchi rophages quickly differentiated toward non-phagocytic, high-SIRPa TAMs. University, Yamaguchi, Japan. Analyses of macrophages on soft (like marrow) or stiff (like solid tumors) Dictyostelium has the ability to sense not only extracellular chemoattractant collagenous gels demonstrated a stiffness-driven upregulation of SIRPa and but also mechanical stimulus and rigidity of the extracellular substratum. Us- the mechanosensitive nuclear marker lamin-A. Mechanosensitive differentia- ing stretchable substratum, we previously showed that Dictyostelium mi- tion was similarly evident in vivo and likely limited the anti-tumor effects, grates in a direction where the external mechanical stimulus from the as confirmed by re-initiation of tumor regression by fresh injections of substratum is absent (Okimura et al, Cell Adhesion & Migration, 2016). In SIRPa-inhibited macrophages. Macrophage motility, phagocytosis, and differ- our preliminary observation using the substratum with static anisotropic ri- entiation in vivo are thus coupled and physically modulated. gidity, Dictyostelium tends to migrate in the direction where the rigidity is

BPJ 8740_8744 Wednesday, February 21, 2018 655a soft. There have been studies focusing on sensing mechanism of extracellular Posters: Cytoskeletal-based Intracellular chemoattractant, however, it is still unclear how Dictyostelium as an elasto- plastic object converts the mechano- and rigidity sensing to directional Transport migration based on the mechanical interactions within the cytoplasm and be- tween the cell and the substratum. In this work, we developed the computa- 3249-Pos Board B457 tional model of Dictyostelium to understand the signal transductions from Molecular Mechanisms of Dynein Force Persistence in Lipid Droplet mechanical stimulus and substratum rigidity to directional migration. The Transport cell was modelled as a simple elastic octagon with the center and each vertex Babu Reddy Janakaloti Narayanareddy1, Dail Chapman1, Deanna Smith2, that are mechanically coupled with each other by a spring and a damper, and Steven Gross1. the vertex was supposed to stochastically move in a direction in which the cell 1Dev& Cell biology, UC Irvine, Irvine, CA, USA, 2Department of Biology, expands. Furthermore, we introduced the myosin II characteristics; myosin II University of South Carolina, Columbia, SC, USA. inhibits formation of cell protrusion (Iwadate et al, Biophys J, 2013) and ac- Intracellular lipid droplet (LD) motion on the microtubules is bidirectional and this cumulates at the cell edge where the mechanical stimulus is applied. The transport is carried out by dynein towards the minus and kinesins drive them to plus model Dictyostelium showed an amoeba-like movement and reproduced ends, and contributes to appropriate control of metabolism. Recently we reported the three type of cell taxises; 1. chemotaxis in the presence of extracellular that lipid droplet motion in COS1 cells undergoes a surprising phenomena of dy- chemoattractant gradient, 2. directional migration in the direction of weak namic adaptation of forces, where in response to opposition to motion, force pro- mechanical stimulus, and 3. directional migration in the direction where duction in the dynein direction is improved. We have now identified molecules the substratum is soft. involved in regulation of this force adaptation, which I will discuss in detail.

3247-Pos Board B455 3250-Pos Board B458 Structural and Biomechanical Changes During Platelet-Driven Clot Size-Dependent Organelle Transport during Phagocytosis Contraction Steve Keller, Konrad Berghoff, Holger Kress. Oleg Kim. University of Bayreuth, Bayreuth, Germany. Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Phagocytosis of bacteria and other pathogens by macrophages is a key process of the PA, USA. mammalian immune system. The intracellular maturation of phagosomes which Blood clot contraction plays an important role in prevention of bleeding and often leads to the degradation of the internalized pathogens shows high organelle- in thrombotic disorders. We found that activated platelets bend and shorten to-organelle variations that are not clearly understood. An important part of the individual fibrin fibers via their filopodia that undergo sequential extension maturation is the phagosomal transport from the cell periphery towards the perinu- and retraction, as if pulling hand-over-hand. Platelets also induce compaction clear region. We hypothesize that the phagosome size influences the phagosomal of fibrin fibers into platelet-attached agglomerates. As a result of simulta- transport and therefore also potentially the maturation process. We tested this hy- neous pulling on multiple, closely-set fibrin fibers, platelets pull themselves pothesis by tracking phagosomes with different diameters between 1 mmand3 closer to each other and form secondary clusters larger than the initial aggre- mm inside macrophages. We show that the transport efficiency increases with gates. Contracting platelets actively remodel a fibrin network by increasing increasing phagosome size although the instantaneous velocities of the investigated its density followed by enhanced clot stiffness. Kinetic analysis of the time phagosomes are very similar to each other. In addition, we found that the bi- course of structural and mechanical transitions revealed a multiphasic directional motion as well as the transport from the nucleus back to the periphery behavior with at least three distinct phases that differ in duration and rate con- decreases with increasing phagosome size. We furthermore show that dynein is stants. All the observed changes were reduced or abrogated in the presence of significantly involved in the phagosomal transport, in particular in the persistent cen- specific inhibitors of non-muscle myosin IIA (blebbistatin) and the platelet tripetal transport of large phagosomes. In addition, we found that actin-dependent integrin aIIbbIII (abciximab), indicating that actomyosin-driven cell contrac- motion is also contributing to the transport, in particular to the transport of small tility and integrin-fibrin mediated platelet-fibrin interactions are crucial for phagosomes. Furthermore we investigated the spatial distribution of dyneins and contraction of blood clots. Finally, blood clot contraction was found to be a microtubules, and found that density differences between the nucleus-facing side spatially non-uniform process with faster compression of the clot edge and of phagosomes and the opposite side can explain part of the observed transport char- a delayed deformation of the clot interior. Altogether, the results provide a acteristics. Our findings suggest that a basic size-dependent cellular sorting mecha- quantitative structural basis for the mechanobiology of clot contraction at nism might exist that supports inward transport of large phagocytosed pathogens for various spatial scales from a single cell/single fiber level up to the network facilitating their digestion and that simultaneously supports outward transport of and macroscopic levels. Our results obtained on platelet-induced contraction small pathogen fragments for example for antigen presentation. of the filamentous fibrin network are of fundamental importance because they provide a foundation for understanding dynamic and complex biomechanical 3251-Pos Board B459 interplay between non-muscle cells and fibrous extracellular matrices of Controlled Disturbance of Intraflagellar Transport in C. elegans Chemo- various compositions. sensory Cilia Results in Changes of Ciliary Structure Mijalkovic Jona, Felix Oswald, Jules Girard, Jasmijn van Loo, 3248-Pos Board B456 Erwin J.G. Peterman. Probing the Physical and Molecular Basis of the Mammalian Mitotic Physics and Astronomy, Vrije Universiteit, Amsterdam, Netherlands. Spindle’s Response to Force Primary cilia act as cellular antennae to detect and transmit signals from the Pooja Suresh1, Alexandra F. Long2, Sophie Dumont3. extracellular environment. They are built and maintained by continuous cycles 1Biophysics Graduate Program, Cell and Tissue Biology, University of of intraflagellar transport (IFT), which transports ciliary proteins from base to California- San Francisco, San Francisco, CA, USA, 2TETRAD Graduate tip and back again. It is well known that defects in the IFT machinery can disrupt Program, Cell and Tissue Biology, University of California- San Francisco, ciliary structure and function, but the effects of more controlled disturbances of San Francisco, CA, USA, 3Cell and Tissue Biology, University of California- the IFT machinery are not well understood. Here we study how IFT in the chemo- San Francisco, San Francisco, CA, USA. sensory cilia of C. elegans is affected by chemical inhibition and femtosecond The mitotic spindle is the dynamic self-organizing structure that segregates laser ablation of the dendritic input. In C. elegans, anterograde IFT is driven chromosomes at cell division. To perform its function, the spindle must by two cooperating kinesins, OSM-3 and kinesin-II, and retrograde IFT by IFT generate and respond to mechanical force, and remodel itself in response to dynein. Using fluorescence microscopy, we visualize and quantify the real- mechanical and biochemical cues. How the spindle responds to diverse forces time response of ciliary proteins to both disturbances. We find that laser ablation to perform its function - and the mechanisms that drive its response - are not of the dendrite results in a three-stage response: (i) IFT motors moving at their understood. To address these questions, we are developing an approach to normal velocity redistribute, followin a sudden surge of retrograde transport trig- exert controlled, local forces on the mitotic spindle in molecularly tractable gered by an unknown signalling process; (ii) the axoneme shortens and motor ve- cells. We use a glass microneedle to manipulate spindle structures inside locities decrease; and (iii) motors leave the cilium. We propose that such a multi- mammalian cells, and monitor the real-time response via fluorescent markers. step response enables the cilium to adapt to outside changes. Early results suggest that we can reliably micromanipulate the spindle In another set of experiments, we applied the small-molecule cytoplasmic dynein without causing cell death or impeding cell cycle progression, and that there antagonists, ciliobrevin A. Acute, low-concentration ciliobrevin treatment results is poor mechanical coupling between both metaphase spindle halves. Using in shortening of cilia and reduction of transport velocity in both retrograde and this approach, we aim to determine how the spindle propagates and responds anterograde directions. Longer exposure to ciliobrevin leads to concentration- to forces across space and time, and the molecular mechanics that underlie dependent motor accumulations and axonemal deformations. We find a strong this response. correlation between IFT-dynein velocity and ciliary length. These experiments

BPJ 8740_8744 656a Wednesday, February 21, 2018 show that, in C. elegans chemosensory cilia, changes in IFT efficiency directly trapping to build single 3D microtubule intersections in vitro with relevant affect ciliary structure, highlighting that the cilium is a highly dynamic organelle nanoscale precision. We then used these fully suspended microtubule structures connecting inside and outside of the cell. to perform motility assays on kinesin-1 coated cargos. We find that some inter- section geometries influence cargos to pass along their current microtubule, 3252-Pos Board B460 while other geometries influence them to switch to the intersecting one. To un- Cytoskeleton-Mediated Dynamic Organization of Lysosomes Promotes derstand how, we use a 3D Brownian dynamics simulation of cargo transport to Their Interactions with Endosomes investigate the mechanisms which give rise to the observed switching probabil- Qinle Ba1, Guruprasad Raghavan1, Kirill Kiselyov2, Ge Yang1,3. 1 ities across separation distances and angles. Using these stochastic simulations, Department of Biomedical Engineering, Carnegie Mellon University, we find that switching probability is often determined by a competition between Pittsburgh, PA, USA, 2Department of Biological Sciences, University of 3 a stronger motor team on the primary microtubule and the intersecting micro- Pittsburgh, Pittsburgh, PA, USA, Department of Computational Biology, tubule sterically hindering that team’s progress. This understanding of the basic Carnegie Mellon University, Pittsburgh, PA, USA. mechanisms of switching at single intersections in 3D helps lay a foundation A basic strategy used by eukaryotic cells to organize their internal environment for understanding how the cell may regulate switching to control how cargos is to form specialized membrane-bound organelles such as lysosomes and en- navigate the MT network and ultimately their spatial organization. dosomes. Although this strategy provides important structural and functional benefits, specialized functions of the organelles must be coordinated and inte- 3255-Pos Board B463 grated for cell physiology. We now know that different organelles interact Development of Improved Microscopy and Data Analysis Tools for Under- directly and extensively through mechanisms such as membrane contact and standing Multimotor Transport membrane fusion, which depend critically on their colocalization and, there- Keith J. Mickolajczyk. fore, their spatial distributions. Currently, however, we cannot explain how Biomedical Engineering, Penn State University, University Park, PA, USA. these organelle interactions are mediated and regulated at the systems level Intracellular transport of vesicles, organelles, and other cargoes is driven by within the heterogeneous intracellular space. To address this deficiency, we teams of kinesin, dynein, and myosin molecular motors. Frequently there are focus on the lysosome, an organelle that plays essential roles in important multiple motors of each type and directionality on each cargo, but how teams cellular functions such as degrading macromolecules and controlling cellular of motors coordinate their activity in order to give rise to the emergent ‘‘tug of metabolism. We investigate whether and, if so, how individual lysosomes are war’’ behavior observed in cells remains poorly understood. Even less understood spatially organized so that their functions can be coordinated and integrated is the observation that knocking down specific motors diminishes transport in to meet changing needs of cells. To this end, we analyze the collective behavior both directions, not only in the direction of the inhibited motor. In vitro and intra- of lysosomes in cultured cells using spatial statistical techniques. We find that cellular cargo tracking experiments seeking to understand the molecular mecha- in single cells, lysosomes maintain nonrandom, stable, yet distinct spatial dis- nisms underlying bidirectional transport behavior have thus far been limited by tributions, which are mediated by the coordinated effects of the cytoskeleton two key factors: (1) the spatiotemporal resolution of existing microscopy tech- and lysosomal biogenesis on different lysosomal subpopulations. Furthermore, niques, without which short back-and-forth motions of the cargo blur together we find that throughout the intracellular space, lysosomes form dynamic clus- and average out, and (2) data analysis tools capable of ascribing the dynamics ters that substantially increase their interactions with endosomes. We determine of position-over-time traces to the behaviors of the individual motors taking that formation of the clusters is mediated primarily by lysosomes undergoing part in multi-motor transport. In the current work, we seek to address both of directed movement along with those undergoing constrained diffusion. these limitations. We first introduce high-speed, super-resolution scattering mi- Together, our findings reveal cytoskeleton-mediated spatial organization of ly- croscopy techniques to track cargoes with 1-nm and 1-ms resolution. We next sosomes at the whole-cell scale and provide new insights into how organelle create a stochastic differential equation model to represent coupled motor and interactions are mediated and regulated over the entire intracellular space. cargo positions, and apply it to our data using a combination of particle filtering and a stochastic expectation-maximization algorithm. Overall, we work towards 3253-Pos Board B461 quantitatively describing how each motor on the cargo dynamically interacts with Characteristic Rotational Behaviors of Rod-Shaped Cargo Revealed by the track and with the other motors in order to drive bidirectional transport. Automated Five-Dimensional Single Particle Tracking Ning Fang, Kuangcai Chen, Xiaodong Cheng. 3256-Pos Board B464 Department of Chemistry, Georgia State University, Atlanta, GA, USA. How Multiple Kinesin Motors Transport the Cargo We report an automated single particle tracking technique fortracking the x, y, z co- Saurabh Shukla1, Marco Tjioe2, Paul R. Selvin3. ordinates, azimuthal and elevation angles of anisotropic plasmonic gold nanorod 1Department of Chemical and Biomolecular Engineering, University of probes in live cells. These five spatial coordinates are collectively referred to as Illinois at Urbana-Champaign, Champaign, IL, USA, 2Department of 5D. This method overcomes a long-standing challenge in distinguishing rotational Biophysics, University of Illinois at Urbana-Champaign, Champaign, IL, motions from translational motions in the z-axis in differential interference contrast USA, 3Department of Physics, University of Illinois at Urbana-Champaign, microscopy to result in full disclosure of nanoscale motions with high accuracy. Champaign, IL, USA. Transferrin-coated endocytic gold nanorod cargoes initially undergo active rota- Multiple molecular motors work together to transport cargos to their destination tional diffusion and display characteristic rotational motions on the membrane. in vivo. These motors also need to avoid myriads of roadblocks such as Then as the cargoes being enclosed in clathrin-coated pits, they slow down the active microtubule-associated proteins (MAPs) to sustain the motion of the cargo. rotation and experience a quiet period before they restore active rotational diffusion Here, we report an in vitro microtubule gliding assay that allows direct visuali- after fission and eventually being transported away from the original entry spots. zation of multiple kinesin motors working together to transport the cargo or mi- Finally, the 3D trajectories and the accompanying rotational motions of the cargoes crotubules. Our assay also allows distinction of kinesins that actively drive the are resolved accurately to render the intracellular transport process in live cells. cargo from the inactive or unbound kinesins. Only a subset of kinesins among the group actively drive the cargo at a given moment of time. Kinesins switch 3254-Pos Board B462 roles from inactive to active and vice versa to maintain the motility of the cargo. Geometry Matters for Cargos Navigating 3D Microtubule Intersections A slight modification of our assay also allows us to directly observe kinesin mol- 1 2 3 2 Matthew J. Bovyn , Jared Bergman , Florence Doval , Manasa Gudheti , ecules avoiding roadblocks. Data suggests that kinesins avoid the roadblocks by Steven Gross4, Jun Allard5, Michael Vershinin6. 1 temporarily unbinding or by switching the protofilaments on the microtubule. We Physics and Astronomy, University of California - Irvine, Irvine, CA, USA, believe that cargos moving in cells follow the similar mechanisms. 2Biology, University of Utah, Salt Lake City, UT, USA, 3Physics and Astronomy, University of Utah, Salt Lake City, UT, USA, 4Developmental 3257-Pos Board B465 and Cell Biology, University of California - Irvine, Irvine, CA, USA, Obstacle Avoidance of Microtubule Motor Proteins 5Mathematics, University of California - Irvine, Irvine, CA, USA, 6Physics, Luke Ferro. University of Utah, Salt Lake City, UT, USA. UC Berkeley, Berkeley, CA, USA. Eukaryotic cells transport organelles and other cargos along microtubules to Kinesin and dynein transport cellular cargoes towards the plus- and minus-end of control their distribution within the cell and deliver them to distant locations. microtubules, respectively. In many cases, intracellular cargoes simultaneously While we understand how molecular motors can transport cargos along individ- recruit both types of motors and the direction of travel is dictated by the relative ual microtubules, the cell’s microtubules are usually arranged in a complex 3D activity of the two motors. How this type of bidirectional transport is regulated is network. While traversing this network, cargos need to navigate intersections an open question. In the cell, microtubules are heavily decorated with the MT- where microtubules cross at a wide variety of separation distances and angles. associated proteins (MAPs) and surrounding medium is crowded with various To gain insight into how cargos navigate these intersections, we have used a sizes of obstacles. It remains unclear how these roadblocks and obstacles affect recently established 3D construction technique based on holographic optical bidirectional cargo transport along MTs. In vitro assays suggest that kinesin-1 is

BPJ 8740_8744 Wednesday, February 21, 2018 657a less capable than dynein of maneuvering past MT-bound obstacles, consistent 6457%, respectively. In contrast IRþcATR exhibited no difference from IR with differences in their stepping behaviors. Yet, kinesin-driven cargoes are (p>0.5) with NaPyr or NaSucc, 5557%, 7156%. BKA induces the ‘‘m’’ able to navigate past obstacles in vivo. We reconstituted the ability of kinesin- conformation of ANT (only ADP/ATP transport), while cATR induces the 1 to avoid synthetic obstacles in vitro. We found that, in contrast to single- ‘‘c’’ conformation of ANT (ADP/ATPþnonspecific transport), so the differential molecules, multi-motor cargoes of kinesin-1 are able to avoid synthetic obstacles effect of BKA on CRC suggests at least a partial role for modulation by ANT in in a manner comparable to dynein. This suggests that kinesin may work in teams the ‘‘m’’ conformation when CSA was given to delay mPTP opening. These re- to navigate past obstacles present in its path in the cell. sults indicate enhanced efficiency of CSA to stall mPTP opening in IR injured mitochondria based on the contention that BKA promotes ANT to ‘‘m’’ being Posters: Mitochondria in Cell Life and Death favorable over the ‘‘c’’ conformation induced by cATR or by IR injury.

3258-Pos Board B466 3260-Pos Board B468 Mitochondrial Ca2D Influx Contributes to Arrhythmic Risk in Nonische- CLIC4 and CLIC5, Mitochondrial Chloride Channel Proteins Mediate mic Cardiomyopathy Cardioprotection Against Ischemia Reperfusion Injury An Xie1, Zhen Song2, Hong Liu1, Anyu Zhou1, Guangbin Shi3, Lai-Hua Xie4, Devasena Ponnalagu1, Neel J. Patel2, Ankur Chaudhury2, Erhe Gao3, Zhilin Qu2, Samuel C. Dudley1. Walter J. Koch3, Andrew R. Kohut2, Harpreet Singh1. 1University of Minnesota, Minneapolis, MN, USA, 2University of California, 1Pharmacology and Physiology, Drexel University College of Medicine, Los Angeles, Los Angeles, CA, USA, 3Rhode Island Hospital, Providence, Philadelphia, PA, USA, 2Department of Medicine, Drexel University College RI, USA, 4The State University of New Jersey, Newark, NJ, USA. of Medicine, Philadelphia, PA, USA, 3Center for Translational Medicine, Introduction: It had been reported that mitochondrial Ca2þ was overloaded in Temple University, Philadelphia, PA, USA. ischemia-reperfusion injury. Inhibition of mitochondrial Ca2þ uniporter (MCU) Cellvolumedysregulation plays a predominant role inthe originofischemic injury prevented cell death. In this study, pharmacological block or genetic knockdown in heart as well as other organs. Chloride channels maintain cell volume as well as MCU inhibited mitochondrial Ca2þ uptake, depressed early afterdepolarizations ionic homeostasis, thereby maintaining cell integrity. Chloride intracellular chan- (EADs) and reduced ventricular fibrillation in nonischemic heart failure (NI- nel (CLIC) are unique class of dimorphic ion channel proteins. Amongst the six HF) mice. Methods: A model of NI-HF was induced in CD1 mice by hyperten- mammalian paralogs (CLIC1-CLIC6), CLIC4 and CLIC5 are present in the car- sion. Echocardiography, patch-clamp (and Rhod-2 fluorescence density recorded diac mitochondria of rats. CLIC4, an outer mitochondrial membrane protein synchronously), telemetry monitoring, real-time PCR and Western blotting were modulated the calcium retention capacity (CRC) of the mitochondria whereas employed in this study. Computer simulations were carried out. Results: Echocar- CLIC5, present in the inner mitochondrial membrane maintains the reactive oxy- diography showed an impairment of systolic function as the average EF value gen species (ROS) generation. Inhibition of CLICs using pharmacological inhib- decreased from 63 5 2%to51 5 3%inNI-HF mice (p<0.05).Isolated myopathic itor (IAA-94), increased myocardial infarction (MI) due to ischemia-reperfusion myocytes showeddecreasedcytoplasmic Ca2þ transients, increasedmitochondrial (IR) injury but the mechanism of cardioprotection by CLICs was unknown. This Ca2þ transients, and increased action potential duration at 90% (APD90) repolar- intrigued us towards understanding the role of cardiac CLICs in cardioprotection ization. The alteration of APD90 was consistent with in vivo QTc prolongation and from IR injury. In this study, we performed left main descending coronary artery could be explained by augmented L-type Ca2þ currents, increased NCX and (LCA) occlusion to induce IR injury in clic-/- and wild type (Wt) mice. Mice were decreased total Kþ currents. Sixty-six percent of myopathic ventricular myocytes subjected to 40 min of ischemia and 24 h of reperfusion. Clic4-/-and Clic5-/- mice showed EADs compared with 17% of sham myocytes (p<0.05). Intracellular showed increased myocardial infarction (60.695 6.07%, n=4; 5259.5%, n=5, application of 1 mM Ru360 could reduce mitochondrial Ca2þ transients, decrease respectively) as compared to wild type mice (41.4752.63%, n=5 CD1Wt; APD90 and ameliorate EADs. Furthermore, genetic knockdown of MCU inhibited 38.6154.85%, n=3, C3H/HeJWt respectively). In addition, clic5-/- mice showed mitochondrial Ca2þ uptake, reduced NCX, decreased APD90, depressed EADs, a significant decrease in ejection fraction (25% vs 34%, p<0.05) in comparison to and reduced ventricular fibrillation in NI-HF mice. Inhibiting mitochondrial wild type mice. Clic1-/-mice showed similar myocardial infarction as wild type NCX slowed mitochondrial Ca2þ uptake, decreased APD90, and depressed mice ruling out the role of CLIC1 in cardioprotection from IR injury. Thus, our re- EADs. Computer simulations showed that EADs were promoted by heart failure sults demonstrate that CLIC4 and CLIC5 are involved in cardioprotection from in remodeling, which were abolished by blocking either the MCU or the L-type vivo IR injury probably by maintaining the calcium homeostasis and ROS gener- Ca2þ current. Simulations revealed that EADs were promoted via a positive feed- ation by the mitochondria, respectively. back loop among APD, SR Ca2þ, and mitochondrial Ca2þ, resulting in an all-or- þ none behavior. Conclusions: Mitochondrial Ca2 handling plays an important 3261-Pos Board B469 role in EADs seen with chronic and moderate nonischemic cardiomyopathy. Role of Calcium and ADP Infusion Rates in Cardiac Mitochondrial Fuel Selection 3259-Pos Board B467 Sunil M. Kandel1,2, Santosh Dasika2, Ranjan K. Dash1, Daniel A. Beard2. The ‘‘M’’ Conformation of Adenine Nucleotide Translocase Enhances 1Department of Biomedical Engineering, Medical College of Wisconsin, D Cyclosporine A-Induced Delay of Mitochondrial Ca2 Uptake after Milwaukee, WI, USA, 2Department of Molecular and Integrated Physiology, Cardiac Ischemia/Reperfusion Injury University of Michigan, Ann Arbor, MI, USA. Mark A. Goss1, James S. Heisner1, Wai-Meng Kwok2, Calcium ion concentration modulates the function of several mitochondrial en- Amadou K.S. Camara3, David F. Stowe4,5. zymes. Specifically, the kinetic operations of the decarboxylating dehydroge- 1Anesthesiology Research, Medical College of Wisconsin, Milwaukee, WI, nases pyruvate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate USA, 2Anesthesiology Research/Pharmacology and Toxicology, Medical dehydrogenase are all affected by [Ca2þ]. Previous studies have shown that, College of Wisconsin, Milwaukee, WI, USA, 3Anesthesiology/Physiology, despite its ability to affect the function of specific dehydrogenases, [Ca2þ] Medical College of Wisconsin, Milwaukee, WI, USA, 4Anesthesiology/ does not substantially alter mitochondrial ATP synthesis in vitro or in vivo Physiology/Biomedical Engineering, Medical College of Wisconsin, in the heart. We hypothesize that, rather than contributing to respiratory con- Milwaukee, WI, USA, 5Biomedical Engineering, Marquette University, trol, [Ca2þ] plays a role in contributing to fuel selection. Specifically, cardiac Milwaukee, WI, USA. mitochondria are able to use different primary carbon substrates (carbohy- þ Mitochondria can take up large amounts of Ca2 during IR injury. How excess drates, fatty acids, and ketones) to synthesize ATP aerobically in the living þ mitochondrial Ca2 leads to formation/opening of the mitochondrial perme- cells. To determine if and how [Ca2þ] affects the relative use of carbohydrates ability transition pore (mPTP) is poorly understood. Adenine nucleotide translo- versus fatty acids in vitro we measured oxygen consumption and TCA cycle case (ANT), while not a mPTP component, may regulate mPTP opening. Our intermediate concentrations in suspensions of cardiac mitochondria with aim was to assess if ANT has a role in regulating mPTP after IR injury of isolated different combinations of pyruvate and palmitoyl-L-carnitine in the media þ guinea pig hearts. We measured Ca2 retention capacity (CRC), a marker of bio- and at various calcium concentrations and ADP infusion rates. energetic activity, in mitochondria isolated after no IR (time control, TC) or after Stoichiometric analysis of the data reveals that when both fatty acid and carbo- IR injury. Cyclophilin D (CypD) interacts with mitochondrial proteins, including hydrate substrates are available, fuel selection is sensitive to both the rate of ANT, to initiate mPTP opening. Cyclosporine A (CSA) blocks CypD, thus delay- ADP infusion and to the calcium concentration. Under low-flux (leak state) þ ing mPTP opening induced by Ca2 overload if CSA is given during initiation of conditions and with zero added Ca2þ, b-oxidation provides roughly 70% of þ Ca2 efflux. Therefore, we examined if ANT inhibitors carboxyatractyloside acetyl-CoA for the citrate synthase reaction, with the rest coming from the py- (cATR) or bonkrekic acid (BKA) alter CSA-induced delay of mPTP opening. ruvate dehydrogenase reaction. With both increasing rates of oxidative ATP þ We found that CSA-induced delay of mPTP opening (assessed by Ca2 release) synthesis and with increasing [Ca2þ], the fuel utilization ratio shifts to was greater for IRþBKA vs. IR only; all data vs. TC (100%): For substrates Na- increased fractional consumption of pyruvate. The effects of ATP synthesis Pyr and NaSucc: IRþBKA, 88510%, 97510% (p<.05) vs. IR, 6157%, load and [Ca2þ] are shown to be interdependent.

BPJ 8740_8744 658a Wednesday, February 21, 2018

3262-Pos Board B470 other proapoptotic proteins into the cytosol. The latter events trigger activation Matrix Calcium Efflux via the Putative Mitochondrial Calcium-Hydrogen of caspases and apoptosis. Despite the vital importance of mPTP in controlling Exchanger: Role in mPTP Opening cell life and death pathways, the molecular structure and identity of mPTP is Lyall Glait, Jyotsna Mishra, James S. Heisner, David F. Stowe, not yet known. We have gathered evidence that F1Fo-ATP synthase c-subunit Amadou K.S. Camara, Wai-Meng Kwok. ring forms a large conductance ion channel by using different molecular Anesthesiology, Medical College of Wisconsin, Wauwatosa, WI, USA. biology, electrophysiology, and cell biology techniques. We have shown the þ Mitochondria play a key role in regulating and buffering intracellular [Ca2 ]. critical role of the c-subunit leak channel in neuronal death and survival in pri- þ Ca2 transport across the mitochondrial membrane occurs through the mitochon- mary hippocampal cultures and its regulation by different pharmacological þ þ þ drial Ca2 uniporter (MCU), ryanodine receptor (mRyR), Na /Ca2 exchanger agents. We are currently studying the role of c-subunit leak channel in mPT þ þ þ (mNCE), and putative Ca2 /H exchanger (mCHE). Exposure to excess Ca2 by using ATP synthase c-subunit CRISPR knockdown and knockout mouse leads to opening of the mitochondrial permeability transition pore (mPTP), embryonic stem cells. We find that in wild-type mitochondria ATP synthase in- þ releasing Ca2 and triggering apoptosis. The molecular components of mPTP hibitors prevent ciclosporin A (CsA, a well-known inhibitor of mPTP) sensi- are currently unknown, but cyclosporine A (CsA) and ADP both delay its open- tivity during mitochondrial swelling and calcium retention capacity assays. ing. In this study, we isolated mitochondria from rat hearts, energized them with However, CsA is still effective in the presence of the adenine nucleotide trans- þ þ Na -free K succinate (to block contribution of mNCE), and measured extra- locase (ANT) inhibitors. This suggests that the location of CsA sensitivity is in 2þ 2þ matrix [Ca ] with spectrophotometry to monitor matrix Ca uptake. By block- the F1 portion of the ATP synthase. We also study the ATP synthase structure þ ing Ca2 uptake through the MCU with Ru360, we uncovered a persistent efflux and conformational changes necessary for c-subunit gating by using cryo- þ þ of Ca2 from mitochondria at [Ca2 ] lower than those that induced mPTP open- electron microscopy technique. These findings will provide us with the þ ing. The rate of Ca2 efflux was proportional to the concentration of added increased understanding of the molecular composition and structure of mPTP. CaCl2. There was no significant difference in the rate of efflux between Ru360 and ruthenium red. Since we eliminated the contributions of MCU, mRyR, and 3265-Pos Board B473 mNCE, this efflux is likely through mCHE. ADP greatly delayed mPTP opening Genipin Lacks the Specificity for UCP2 Inhibition and significantly (p=0.016) slowed the rate of efflux. CsA delayed opening of Jurgen€ Kreiter1, Anne Rupprecht1, Lars Zimmermann1, Maria Fedorova2, mPTP and marginally slowed the rate of efflux. Thus, the rate of Ca2þ efflux ap- Michael Moschinger1, Tatyana I. Rokitskaya3, Lars Gille1, peared to depend on the matrix Ca2þ binding capacity, as it slowed slightly in Yuri N. Antonenko3, Elena E. Pohl1. response to CsA and significantly in response to ADP. Our results suggest that 1Department of Biomedical Science, University of Veterinary Medicine mCHE plays a pivotal role in Ca2þ efflux. Under conditions of increased Vienna, Vienna, Austria, 2Center of Biotechnology-Biomedicine, Leipzig, Ca2þ, mCHE may facilitate mPTP opening or possibly be a component of mPTP. Germany, 3Belozersky Institute of Physio-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation. 3263-Pos Board B471 Genipin is a natural cross-linker found in Gardenia jasminoides Ellis and is often Molecular Assembly of the Mitochondrial Permeability Transition Pore used as a specific inhibitor of uncoupling protein 2 (UCP2). UCP2 belongs to the Giuseppe F. Amodeo1, Nelli Mnatsakanyan2, Maria E. Solesio1, mitochondrial carrier superfamily, is abundant in rapidly proliferating cells1 and Magdalena Klim3, Piotr Kurcok3, Eleonora Zakharian4, Elizabeth A. Jonas2, mediates a regulated proton leak through the inner mitochondrial membrane. Evgeny V. Pavlov1. Here we tested the hypothesis that the action of genipin in cells is not only confined 1Department of Basic Science, New York University, New York, NY, USA, to UCP2. For that, we investigated (i) the activity of several recombinant proteins 2Department of Internal Medicine, Yale University, New Haven, CT, USA, reconstituted in artificial bilayers2 in the presence/absence of genipin (ii) mitochon- 3Centre of Polymer and Carbon Materials, Polish Academy of Science, drial membrane potential in neuroblastoma cells after addition of genipin using Zabrze, Poland, 4Department of Cancer Biology and Pharmacology, confocal microscopy and (iii) genipin-mediated modifications of proteins and lipids University of Illiniois, Peoria, IL, USA. using mass spectrometry. Our findings outline that genipin, in addition to UCP2, Mitochondria are considered the power plants of cells as they produce ATP as a inhibits its homologs UCP1 and UCP3 and complex III of the respiratory chain, result of oxidative phosphorylation. Oxidative stress and elevated cytosolic Ca2þ but not the non-mitochondrial protein a-hemolysin. Based on competitive inhibi- lead to mitochondrial damage under acute pathological conditions. The mito- tion experiments and on implementation of mutant proteins, we propose the molec- chondrial dysfunction is caused by the loss of the electrochemical potential across ular mechanism which may be responsible for the multiple action of genipin. the inner mitochondrial membrane (IMM) due to the opening of the so-called 1Rupprecht et al. (2014), PLOS ONE, Volume 9(2) mitochondrial permeability transition pore (mPTP). The molecular composition 2Beck et al. (2006), BBA - Bioenergetics, Volume 1757, 474-479 of the pore of mPTP is not well understood and is a subject of hot debate. Our work is aimed at establishing the complete chemical composition of mPTP, 3266-Pos Board B474 which is a critical step towards understanding the mechanism of its formation/ Loss of Mitochondrial Phosphate Carrier in Skeletal Muscle: Dissociation activation. In our work we purify mPTP complexes from calcium stimulated of Muscle Dysfunction from Lower ADP Phosphorylating Potential rat liver mitochondria by chloroform extraction. This is followed by chemical Erin Seifert, Lauren Anderson-Pullinger, Yana Sharpadskaya. and functional characterization of the complex. We confirmed the channel activ- MitoCare Center, Thomas Jefferson University, Philadelphia, PA, USA. ity of the purified complex by planar lipid bilayer recordings. Immunoblots using Many mutations in the mitochondrial genome and in nuclear genes that encode antibodies against C-subunit of the ATP-synthase (Csub) resulted in the detection mitochondrial proteins hamper oxidative phosphorylation (oxphos) and lead to of a band at 50 kDa suggesting that Csub oligomers are integral parts of the mPTP tissue dysfunction. Yet, whether an energy deficit is necessarily a direct cause complex. A complementary MALDI approach demonstrated the presence of a of tissue dysfunction is not clear. We addressed this question in mice in which polymer with an average molecular weight of 2,000 Da, which is likely associ- the mitochondrial phosphate carrier, PiC, was depleted from skeletal muscle ated with the Csub. Although Csub was not detected with MALDI in the chloro- (skm) by tamoxifen-induced gene excision (Creþ). Three weeks after tamox- form extract, suggesting that it is unavailable to the gas phase of the ifen, PiC protein in skm was <5% of levels in tamoxifen-treated wild-type chromatography, in contrast, when Csub is immuno-purified as a monomer it (Ctrl) mice. Oxphos, measured in mitochondria isolated from skm, was greatly is detected by MALDI. Combined, these data support the hypothesis that at least suppressed to 35% or 50% of Ctrl, depending on substrate; the residual oxphos in wild type organisms the native mPTP channel requires the presence of the may be driven by alternate Pi uptake on the dicarboxylate carrier. Residual ox- Csub and polymer parts. Whether in the absence of Csub mPTP can be replaced phos was similar at 3 and 9 wks of PiC depletion, suggesting that the oxphos by other membrane protein (s) remains to be established. defect was stable for this interval. Furthermore, subunits of the oxphos machin- ery were robustly upregulated (50-70% rise) at both time points. Nonetheless, 3264-Pos Board B472 treadmill running capacity worsened during this time. Time-to-exhaustion was Molecular Composition, Structure and Regulation of the Mitochondrial 50 mins in Creþ mice at 3 wks after PiC loss, but 25 mins when PiC was Permeability Transition Pore absent for 9 wks. Ctrl mice ran for the scheduled 80 mins. Oxphos defects have Nelli Mnatsakanyan, Han-A Park, Jing Wu, Paige Miranda, been associated with stress signaling via transcription factor ATF4, elevated Elizabeth A. Jonas. MTORC1 signaling and FGF21 synthesis. Investigating these pathways, we Internal Medicine, Yale University, New Haven, CT, USA. found evidence for FGF21 production and stronger MTORC1 signaling after Mitochondrial permeability transition (mPT) is the main cause of necrotic and 9 but not 3 wks of PiC loss. Overall, these observations suggest that skm apoptotic cell death during neurodegenerative diseases and stroke. The opening dysfunction is not related to an oxphos deficit in a simple way. Rather, the ox- of the mitochondrial permeability transition pore (mPTP) leads to mitochon- phos deficit may drive adaptive mechanisms that either become exhausted or drial inner membrane permeabilization and dissipation of membrane potential, turn maladaptive when ongoing over a longer term, or trigger additional pro- which is followed by outer membrane rupture and release of cytochrome c and cesses that are deleterious for muscle function.

BPJ 8740_8744 Wednesday, February 21, 2018 659a

3267-Pos Board B475 (mPTP). Cyclosporin A (CsA), a specific inhibitor of the mPTP, is thought to Complex I Inhibition Enhances Mitochondrial Calcium Uniporter Current suppress pore opening by binding to cyclophilin D (CypD) and inhibiting its Enrique Balderas-Angeles1, Salah Sommmakia1, Sadiki Deane1, peptidyl-prolyl cis-trans isomerase (PPIase) activity. This process facilitates Dipayan Chaudhuri2. high matrix free Ca2þ-induced conformational change of the pore. In the pre- 1Cardiovascular Research and Training Institute, The University of Utah, Salt sent study, we tested a novel hypothesis that CsA impacts matrix calcium buff- Lake City, UT, USA, 2Cardiovascular Research and Training Institute, ering and, consequently modulates mPTP opening. To test this hypothesis, we Cardiology Division, Department of Internal Medicine, The University of determined the effect of CsA on matrix Ca2þ buffering and Ca2þ-mediated Utah, Salt Lake City, UT, USA. mPTP opening by monitoring mitochondrial Ca2þ retention capacity (CRC) 2þ Ca in the mitochondrial matrix regulates several physiological processes, from during boluses of CaCl2 challenges in isolated energized cardiac mitochondria. 2þ ATP synthesis to cell death. Most mitochondrial Ca uptake occurs through the In addition, we measured redox state (NADH) and membrane potential (aJm). þ mitochondrial Ca2 uniporter, a highly-selective ion channel embedded in the in- During extra and intra matrix Ca2þ measurements, addition of CsA increased þ ner membrane. We recently demonstrated that both Ca2 uptake and uniporter the driving force for Ca2þ uptake, possibly by sequestering the added Ca2þ channel activity are increased in a mouse model of mitochondrial cardiomyopa- as indicated by maintenance of matrix [Ca2þ] constant. In the absence of thies. In these diseases, which primarily present in infants and children, charac- CsA, a gradual increase in free matrix [Ca2þ] led to gradual depolarization teristic deficits in oxidative phosphorylation produce a signal that boosts of aJm and oxidation of NADH. In contrast, the CsA mediated increase in 2þ 2þ mitochondrial Ca levels. Here we investigated the mechanism for such Ca uptake was associated with preserved aJm and NADH. In other experi- enhancement. By selective pharmacological inhibition of individual electron ments, CsA added just when pore was about to open restored free matrix [Ca2þ] transport chain complexes, we found that rotenone-induced complex I dysfunc- to basal levels while increasing Ca2þ uptake. Our data suggest that CsA may þ þ tion increases mitochondrial Ca2 uptake. Since Ca2 transport is governed by have two modes of preventing mPTP opening: by directly modulating the mito- both uniporter activity and the transmembrane voltage gradient (DJ), we chondrial calcium buffering system, and by inhibiting CypD-PPIase activity. measured uniporter activity directly using whole-mitoplast patch-clamp. We propose that CsA-mediates enhanced matrix Ca2þ buffering and so de- 2þ HEK293T cells were incubated with 0.1, 0.3 and 1 mM rotenone for 72 hr to creases matrix free [Ca ] and preserves aJm and NADH; this in turn contrib- chronically inhibit complex I. After such inhibition, we found that uniporter cur- utes to more Ca2þ uptake and buffering, and a delay mPTP opening. rent density increased from 72 5 6 pA/pF for control mitoplasts (0 nM rote- none) to 90 5 6 pA/pF in 0.3 mMrotenoneand130 5 20 pA/pF in 1 mM 3270-Pos Board B478 rotenone. Such an increase was not due to acute effects of rotenone on the uni- The Spatial Distribution of the NaD/Ca2D Exchanger in Cardiac porter channel itself, as it did not affect or only mildly inhibited current densities Mitochondria Enhances the Efficincy of the Mitochondrial Ca2D Signal at these concentrations. Our data indicate that chronic inhibition of complex I Generation produces a signal that increases the activity of MCU. Sergio De la Fuente1, Celia Fernadez-Sanz2, Jonathan P. Lambert3, John W. Elrod3, Shey-Shing Sheu2, Gyorgy Csordas1. 3268-Pos Board B476 1MitoCare Center, Pathology, Anatomy and Cell Biology, Thomas Jefferson Arsenic Targets Local ROS and Calcium Homeostasis at the University, Philadelphia, PA, USA, 2Center for Translational Medicine, Mitochondria-ER Interface Thomas Jefferson University, Philadelphia, PA, USA, 3Center for Rafaela Bagur1, Arnaldo Souza1, Georgia Gunther€ 2, Raymond Reif2, Translational Medicine, Temple University School of Medicine, Peter Va´rnai3, Gyo¨rgy Csorda´s1, Gyo¨rgy Hajno´czky1. Philadelphia, PA, USA. 1Pathology, Anatomy and Cell Biology, MitoCare Center for Mitochondrial In cardiac muscle, mitochondria take up Ca2þ through the Ca2þ uniporter Imaging Research and Diagnostics, Thomas Jefferson University, (mtCU), while Ca2þ is extruded predominantly via the 3Naþ/Ca2þ exchanger Philadelphia, PA, USA, 2Leibniz-Research Centre for Working Environment (NCLX). Both transports are electrogenic, thus consume mitochondrial mem- and Human Factors, Technical University Dortmund, Germany, 3Physiology, brane potential (dPsim). We have reported that mtCU is strategically positioned Faculty of Medicine, Semmelweis University, Budapest, Hungary. in cardiac muscle at the mitochondria-junctional SR (Mito-jSR) association to Environmental agents are associated with altered ROS production, dysregulation support efficient local Ca2þ signaling. Last year we presented data suggesting þ of Ca2 homeostasis and mitochondrial cell death. However, the causative path- that NCLX-mediated Ca2þ extrusion is largely excluded from Mito-jSR asso- ways remain largely unknown partly because the role of mitochondria in cell sur- ciation areas. We developed fractionation-based assay comparing mitochondria vival is mediated through local communication between mitochondria and other in the mitochondrial fraction (8,500*g, less Mito-jSR associations) to mito- organelles, which have been difficult to directly monitor. Our aim was to study chondria in the jSR (40,000*g, more jSR associations) fraction. WB analysis þ the effect of environmental agents such as arsenic (As) on Ca2 and ROS homeo- found most NCLX in the mitochondrial fraction and 45Ca2þ flux assays stasis in hepatocytes, paying special attention to the local mitochondria- showed more efficient Naþ-dependent Ca2þ extrusion in the mitochondrial endoplasmic reticulum (ER) communication. In vivo liver imaging of mice treated fraction. Now, we have validated these assays using NCLX cKO mice. Mito- with 100ppm As in the drinking water revealed suppression of the fast cytoplasmic chondrial fraction from NCLX cKO showed both, diminished NCLX levels þ þ [Ca2 ] rise triggered by glucagon and increase in the mitochondrial Ca2 uptake. and Naþ-dependent Ca2þ extrusion, confirming that the Ca2þ extrusion is To study the local mitochondria-ER communication, we targeted fluorescent indeed mediated by NCLX. Furthermore, transgenic NCLX overexpression re- protein-based sensors to the organellar interface in isolated hepatocytes. These sulted in significant increase in Naþ-dependent Ca2þ extrusion in the mito- studies showed an As-induced dose and time dependent increase in ROS exposure, chondrial but not the jSR fraction, underscoring the efficacy of NCLX which was accompanied by suppression of IP3-induced Ca2þ signaling. The exclusion from the dyad area (overexpression cannot override it). Finally, we changes were stronger at the mitochondria-ER interface than in the bulk cyto- wanted to test the concept whether increasing NCLX in the same domain as þ plasm. Antioxidant pre-treatment abolished the As-induced Ca2 dysregulation, mtCU increased the energy (dPsim) expense to attain a given [Ca2þ]m signal. suggesting that the IP3R desensitization was induced by ROS. Despite the Thus, we performed dPsim and [Ca2þ]m measurements in permebilized H9c2 þ decreased IP3-linked Ca2 mobilization, As (3 mM) exposure stimulated mito- cells overexpressing NCLX (transiently transfected with NCKX6-FLAG). þ þ chondrial Ca2 uptake without any change in the expression of the Ca2 uniporter Upon 10mMCa2þ addition, by the time of the [Ca2þ]m peak the associated components. This might be caused by the increased tightness of the mitochondria- dPsim drop was larger in the NCLX-overexpressing than control cells. Collec- ER contacts observed after As exposure. In conclusion, our results suggest that the tively, mitochondrial Ca2þ uptake and Naþ-dependent extrusion are recipro- As-induced liver injury might be mediated by increased ROS, which causes cally polarized, likely to optimize the energy-efficiency of [Ca2þ]m signals þ impaired local mitochondria-ER Ca2 communication. arisen mostly from Ca2þ entry at the dyadic interface.

3269-Pos Board B477 3271-Pos Board B479 Cyclosporin A: New Insights into its Potential Role in Mitochondrial Mgr2 and the Channel Activity of TIM23, a Gateway for Mitochondrial Calcium Buffering Protein Import Jyotsna Mishra1, Ariea J. Davani1, David F. Stowe2, Wai-Meng Kwok3, Oygul Mirzalieva1, Layla Drwesh2, Abdussalam Azem2, Cory Dunn3, Amadou K.S. Camara2. Pablo Peixoto1. 1Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, USA, 1Baruch College, CUNY, New York, NY, USA, 2Tel Aviv University, Tel 2Anesthesiology/Physiology, Medical College of Wisconsin, Milwaukee, Aviv, Israel, 3University of Helsinki, Helsinki, Finland. WI, USA, 3Anesthesiology/Pharmacology/Toxicology, Medical College of Mitochondrial biogenesis relies on the correct sorting of nuclear-encoded pro- Wisconsin, Milwaukee, WI, USA. teins into the inner membrane or the matrix thought the import complex þ Mitochondria Ca2 handling is important to cellular function. However, excess TIM23. A newly discovered subunit of TIM23, Mgr2, seems to regulate the þ matrix Ca2 induces opening of the mitochondrial permeability transition pore sorting process and has been coined ‘‘the gateway for lateral release’’ of inner

BPJ 8740_8744 660a Wednesday, February 21, 2018 membrane proteins. This work examines how Mgr2 impacts the previously However, direct experimental evidence of E73 involvement in VDAC1 gating characterized channel activity of TIM23. Our preliminary analysis shows is missing. Using an established protocol of assessing voltage gating of VDACs that Mgr2 contains a conserved amino acid sequence (GXXXG) in domains reconstituted into planar lipid membranes, we show that VDAC1 gating prop- spanning across the mitochondrial inner membrane. These sequences were erties do not change when E73 is replaced by either neutral (E73Q) or hydro- also found on two subunits (Tim23 and Tim17), which form the pore of phobic (E73A) residues. We conclude that residue E73 is not involved in the TIM23. This led to our hypothesis that Mgr2 depletion would affect pore struc- voltage-dependent closure of the channel. We also show that cholesterol ture and functioning. For this purpose, we tested examined TIM23 activity does not affect gating characteristics of mammalian VDAC1 wild type and Mgr2 mutant yeast strains by patch clamp electrophysiology. Our preliminary the mutants. In comparison, we found a pronounced effect of the phospholipid results suggest that depletion of Mgr2 affected both the basal membrane poten- head group charge and type on channel gating. Our data shed light on poorly- tial as well as the response to depolarizing and hyperpolarizing agents. Interest- understood role of E73 in VDAC functioning: we can now rule out its involve- ingly, the patch clamp analysis suggested a decrease in pore size and gating ment in mammalian VDAC1 gating. Consequently, previously identified natu- properties. In future studies we will investigate the import of fluorescent pep- ral modulators, such as cholesterol, do not affect channel gating through tides and how GXXXG amino acid sequences are involved in Mgr2 function. interaction with this residue. However, their established binding to E73 may modulate mitochondria functioning in vivo. 3272-Pos Board B480 Phosphorylation of Cardiac Mitochondrial VDAC1 at S215 Facilitates Cell 3274-Pos Board B482 Death Inorganic Polyphosphate (polyP) Promotes Protein Aggregation to Protect Meiying Yang1, Michael Grzybowski2, Qunli Cheng1, David F. Stowe1, Mitochondria Against Stress 1 1 Aron Geurts2, Po-Chao Wen3, Nandan Haloi3, Emad Tajkhorshid3, M. de la Encarnacio´n Solesio Torregrosa , G. Federico Amodeo , 2 3 1 1 1 Pia Elustondo , Alejandro Cohen , Evgeny V. Pavlov . Amadou K.S. Camara , Wai-Meng Kwok . 1 2 1 Basic Sciences, NYU, New York, NY, USA, Physiology and Biophysics, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, 3 WI, USA, 2Department of Physiology, Medical College of Wisconsin, Dalhousie University, Halifax, NS, Canada, Proteomics and Metabolomics Milwaukee, WI, USA, 3Biochemistry, University of Illinois at Urbana- Core Facility, Dalhousie University, Halifax, NS, Canada. Champaign, Urbana, IL, USA. Inorganic polyphosphate (polyP) is a ubiquitous biological polymer, - Post-translational modifications of the mitochondrial VDAC1 have been impli- well conserved throughout the evolution and various species. Isoenergetic cated in a myriad of diseases. In particular, phosphorylation of VDAC1 has with ATP, polyP is composed of multiple subunits of orthophosphate, linked been involved in neurological and cardiac diseases. However, the functional together by phosphoanhydride bonds. We previously showed that this poly- consequences of VDAC1 phosphorylation at specific residues are not well mer highly localizes in mammalian mitochondria, where it plays different defined. This is compounded by the dual functional roles of VDAC1 as a chan- roles. In fact, cells with reduced amounts of mitochondrial polyP showed nel and as a receptor for pro- and anti-apoptotic proteins. Using mass spectrom- higher levels of cell death in response to calcium dyshomeostasis. Here etry, we have identified a phosphorylated S215 on mitochondrial VDAC1 we show that the chaperone effect of polyP is mainly mitochondrial. More- following ischemia-reperfusion of isolated rodent hearts. To investigate the over, we also show that decreased amount of polyP also decreases cells - physiological relevance of S215 phosphorylation, we used CRISPR/Cas9 to resistance to heat shock induced cytotoxicity. In the absence of polyP, cell - generate knockin of S215E, a phosphomimetic variant, into the rat VDAC1 death is also increased after the addition of aggregated a synuclein. Inter- gene and nucleofected into H9c2 cells. Following hypoxia-reoxygenation, estingly, in brains from mice showing amyloid plaques, polyP levels were LDH release in S215E knockin H9c2 cells was significantly greater than in increased. Our data also shows that the chaperoning activity of polyP is in- the wild-type cells, indicative of S215E-facilitated cell death. Since S215 is dependent on any transcriptional changes. These findings are in agreement a putative GSK3b phosphorylation site, it potentially modulates the binding with the recently proposed role of polyP as a molecular chaperone, involved of the anti-apoptotic protein hexokinase II (HKII) to VDAC1, a process that in cell stress response. We hypothesize that polyP is crucial for the mito- is regulated by GSK3b activity. GSK3b is active when dephosphorylated and chondrial formation of fibrils, preventing protein misfolding toxicity. control experiments in H9c2 cells confirmed that hypoxia resulted in decreased 3275-Pos Board B483 phosphorylation of GSK3b at S9. Molecular dynamics simulations were con- Optogenetic Regulation of Mitochondrial ROS Emission In Vivo ducted to investigate the consequence of S215 phosphorylation on the biophys- Stephen Madamba1, Nicomedes Rivera2, Brian Nguyen2, Pablo Peixoto1. ical properties of VDAC1. Simulations did not reveal any direct effects of S215 1Baruch College and Graduate Center, CUNY, New York, NY, USA, phosphorylation on the ionic current (at least for small ions). This was corrob- 2Baruch College, CUNY, New York, NY, USA. orated additionally by recording VDAC1 activity using an artificial planar Amounting research in the past decade is establishing emerging signaling roles bilayer system. The S215E variant retained the characteristic voltage- of chemically reactive oxygen species (ROS) emitted from mitochondria, the dependence of the wild-type VDAC1. Our results have identified the phosphor- power plants of eukaryotic life. However, the chemically reactive nature of ylation of S215 on VDAC1 as a potential key step in modulating HKII binding ROS imposes a spatiotemporal constraint for induction and measurement. As and, consequently, cell death. a consequence, most studies rely on methods for induction and measurement of saturated emission irrespective of cell type or intracellular source. To 3273-Pos Board B481 circumvent this issue, we generated the first fly model for non invasive and Assessing the Role of Residue E73 in VDAC1 Voltage Gating mitochondria-specific induction of ROS emission using optogenetics. Using 1 2 2,3 1 Marı´a Queralt-Martı´n , Lucie Bergdoll , Jeff Abramson , Daniel Jacobs , this model, we present a preliminary study of the effect of specific ROS 1 1 Sergey M. Bezrukov , Tatiana K. Rostovtseva . signaling on motor function. 1Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 3276-Pos Board B484 Bethesda, MD, USA, 2Department of Physiology, David Geffen School of Mitochondrial Dysfunction due to Intracellular Beta Amyloid Oligomers Medicine, University of California Los Angeles, Los Angeles, CA, USA, Patrick T. Toglia1, Angelo Demuro2, Ian Parker2, Ghanim Ullah1. 3Institute for Stem Cell Biology and Regenerative Medicine (inStem), 1Physics, University of South Florida, Tampa, FL, USA, 2Neurobiology and National Centre for Biological Sciences–Tata Institute of Fundamental Behavior, University of California Irvine, Irvine, CA, USA. Research, Bellary Road, Bangalore, Karnataka, India. Intracellular accumulation of b amyloid (Ab) oligomers are now believed to The voltage-dependent anion channel (VDAC) regulates transport of ions and play a major role in the early phase of Alzheimer’s disease (AD) as their rise metabolites such as ATP and ADP across the outer mitochondrial membrane, correlates well with the early symptoms of the disease. However, little is known thus governing mitochondrial respiration. Under applied potential, VDAC re- about the pathways targeted by intracellular Ab oligomers. In this work, we use constituted into planar lipid bilayers characteristically switches from a high- multiscale modeling in conjunction with patch-clamp electrophysiology of sin- 2þ conductive, open state, to a variety of low-conducting, closed states, through gle inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca channel on the endo- which metabolite transport is diminished. This process involves structural rear- plasmic reticulum (ER) and total internal reflection fluorescence (TIRF) 2þ rangements, although the exact mechanism behind it remains elusive. Struc- microscopy of whole-cell Ca signaling to show that intracellular Ab42 olig- tural studies ascribed residue E73 to participate in the channel closure due to omers inflict cytotoxicity by impairing mitochondrial function. Specifically, we its involvement in protein instability. Importantly, VDAC1 crystal structure first develop a single channel model for the kinetics of IP3R based on patch- shows that this charged residue faces the hydrophobic lipid environment. clamp data, on which a model for whole-cell Ca2þ signals is built. The þ E73 has also been associated with hexokinase-I and Ca2 binding, pointing whole-cell model is then fitted to fluorescence signals from TIRF microscopy 2þ to an enigmatic role of this residue in VDAC1 function. Moreover, there is to quantify the overall Ca -release from the ER due to Ab42 oligomers through a strong support that E73 constitutes a specific binding site for cholesterol. G-protein-mediated stimulation of IP3 production. This allows us to show that

BPJ 8740_8744 Wednesday, February 21, 2018 661a

Ab42 impairs mitochondrial function due to pathological Ca2þ uptake and 3279-Pos Board B487 diminished mitochondrial inner membrane potential resulting in overall lower Docosahexaenoic Acid Remodels the Cardiac Mitochondrial Phospholipi- ATP availability to the cell and increased production of reactive oxygen species dome and Impairs Respiratory Enzymatic Activity by Disrupting Lipid (ROS) and [H2O2]. We further show that mitochondrial function is restored by Domain Formation and Lipid-Protein Binding 2þ 1,2 1,2 3 Ca buffer EGTA, which could explain the abrogation of Ab42 cytotoxicity by Edward R. Pennington , E. Madison Sullivan , Genevieve C. Sparagna , EGTA. This work is supported by NIH grant RO1 AG053988. James Washington1,2, Ethan J. Anderson4, Tonya N. Zeczycki1,2, David A. Brown5, Saame Raza Shaikh1,6. 3277-Pos Board B485 1Department of Biochemistry & Molecular Biology, Brody School of Enhanced Respiratory Reserve Sustained by Lipid Oxidation and Auto- Medicine, East Carolina University, Greenville, NC, USA, 2East Carolina phagy Underlie Extended Lifespan in High- Compared to Low-Running Diabetes & Obesity Institute, Greenville, NC, USA, 3Department of Capacity Rats Medicine, Division of Cardiology, University of Colorado Denver Anschutz Sonia Cortassa1, Miguel A. Aon1, Magdalena Juhaszova1, Medical Campus, Aurora, CO, USA, 4Department of Pharmaceutical Science Jose A. Gonzalez-Reyes2, Miguel Calvo-Rubio2, Jose M. Villalba2, and Experimental Therapeutics, Fraternal Order of Eagles Diabetes Research Bruce Ziman1, Sarah J. Mitchell3, Irene Alfaras3, Khalid Chakir1, Center, University of Iowa, Iowa City, IA, USA, 5Department of Human Jessie E.C. Axsom1, Kelsey Bullock3, Edward Lakatta1, Steven J. Sollott1. Nutrition, Foods, and Exercise, Virginia Tech Corporate Research Center, 1Laboratory of Cardiovascular Sciences, NIH/NIA/IRP, Baltimore, MD, Virginia Tech, Blacksburg, VA, USA, 6Department of Nutrition, Gillings USA, 2Departamento de Biologia Celular, Fisiologia e Inmunologia, School of Public Health and School of Medicine, University of North Universidad de Cordoba, Campus de Excelencia Internacional Carolina at Chapel Hill, Chapel Hill, NC, USA. Agroalimentario, ceiA3, Cordoba, Spain, 3Translational Gerontology Phospholipids of the inner mitochondrial membrane regulate various mitochon- Branch, NIH/NIA/IRP, Baltimore, MD, USA. drial processes including apoptosis and oxidative phosphorylation. In a range of Mitochondrial network function might be involved in healthspan by playing a cardiovascular diseases, mitochondrial inner membrane phospholipids undergo central role in slowing the rate of damage accumulation and senescence. significant acyl chain remodeling during the course of the disease. One unique Although a direct cause-and-effect relationship between mitochondrial remodeling event that occurs in the cardiac mitochondrial phospholipidome is dysfunction and disease is well established, their specific roles in life- an increase in the levels of docosahexaenoic acid (DHA), an n-3 polyunsatu- expectancy and -quality remains uncertain. We hypothesized that respiratory rated fatty acid. Here, we used diet as a tool to investigate how DHA impacted reserve and maximal respiration of cardiac muscle is affected by aging- cardiac mitochondrial phospholipid composition and thereby mitochondrial related deficits in mitochondrial turnover resulting in a progressively damaged respiratory enzyme activities. Mice were administered DHA in the context of mitochondrial population that may shorten lifespan. This was tested in a cross- a western diet. Phospholipidomics with mass spectrometry analysis revealed sectional study of 6, 17, and 24 months aged, low- (LCR) and high-running ca- that mice on a western diet enriched with DHA dramatically remodeled the pacity (HCR) female rats, in which HCR exhibit a 40% increased longevity and acyl chain composition of cardiac mitochondrial cardiolipin, phosphatidylcho- an intrinsic 4-fold higher distance-running capacity. We found that, across ages line, and phosphatidylethanolamine. These changes were associated with tested, isolated HCR cardiomyocytes display higher respiratory reserve and decreased complex I, IV, V, and IþIII enzymatic activities. Mechanistically, maximal respiratory capacity than LCR, in the presence of the fatty acid palmi- lowered enzymatic activity was not driven by modifications to mitochondrial tate (palm) with or without glucose (gluc). For example, in glucþpalm respira- respiratory protein abundance or supercomplex formation. Instead, the replace- tory reserve differences between HCR vs. LCR were significantly higher in ment of tetralinoleoyl-CL [(18:2)4CL] with tetradocosahexaenoyl-CL 4 young ([in nmol O2/min/10 cells]: 4.250.3 vs. 1.850.2; n=47, 6 experiments, [(22:6)4CL] in biomimetic membranes prohibited the formation of lipid micro- p<0.0001) than in old rats (5.750.9 vs. 4.850.7; n=24, 4 experiments). HCR domains, which was driven by DHA’s influence on thermodynamics of phos- cardiomyocytes showed significantly higher autophagy/mitophagy than LCR pholipid mixing. Furthermore, (22:6)4CL modified CL-protein binding as a function of age, especially at middle-age (>36% HCR vs. LCR, n=47, 2 kinetics relative to (18:2)4CL. The reintroduction of linoleic acid, via fusion experiments, p<0.0001), as measured by quantitative electron microscopy, of (18:2)4CL, to mitochondria isolated from mice consuming DHA, prevented which correlated with 64% (p<0.001) lower intracellular concentration of the major losses in the mitochondrial phospholipidome and rescued complexes, acetyl-CoA in HCR in the presence of glucþpalm. These data show that greater I, IV, and V activities. Altogether, these results suggest that an increase in mito- mitochondrial turnover underlies higher cardiomyocyte energetic performance chondrial membrane DHA disrupts membrane organization and enzyme activ- correlated with preferential selection of lipid oxidation with improved control ities, which has major implications for the ongoing debate concerning of intracellular acetyl-CoA. Although aging is correlated with declines in all polyunsaturated fatty acids and cardiac health. these positive indexes, they are better preserved in HCR vs. LCR, consistent with healthier aging and extended lifespan. 3280-Pos Board B488 Mitochondrial Quality Control in Aging and Heart Failure: Influence of 3278-Pos Board B486 Ketone Bodies Lipids Catalyze Mitochondrial Fission via Geometric Instability Charles Miller, Maura Ferrero, Donald M. Bers, Elena N. Dedkova. Ehsan Irajizad1, Rajesh Ramachandran2, Ashutosh Agrawal1. Pharmacology, UC Davis, Davis, CA, USA. 1University of Houston, Houston, TX, USA, 2Case Western Reserve In heart failure (HF) ketone body (beta-hydroxybutyrate and acetoacetate) University, Cleveland, OH, USA. levels are significantly elevated, but the cardiovascular consequences of this In eukaryotic cells, tubular mitochondria form intricate networks and undergo elevation are poorly understood. We monitored Parkin accumulation and incessant fission and fusion. While balanced mitochondrial dynamics is LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 believed to be essential for apoptosis, disrupted dynamics is linked to lung can- months), aged (2.5 years) and rabbits with HF (2.5 years) induced by combined cer, cardiac dysfunction and neurogenerative disorders. Pioneering experi- aortic insufficiency and stenosis. mCherry-Parkin and GFP-LC3 were ex- mental studies have provided insights into the molecular machinery that pressed via adenovirus and assessed via confocal microscopy and biochemistry. executes mitochondrial constriction and fission. The fission pathway is charac- Young rabbits exhibited low levels of mitochondrial Parkin accumulation terized by three key steps: i) the initial constriction carried out by actin poly- (average puncta size 1 mm2), but a 4-fold increase in Parkin puncta was detected merization and actomyosin contraction, ii) the intermediate constriction in the aging heart, which correlated with an increase in LC3-mediated autopha- executed by Drp1 (dynamin-related protein 1), and iii) the final fission carried gosome formation. In HF the number of Parkin and LC3 puncta was signifi- out by dynamin. cantly lower than in similar aged hearts, but very large Parkin-rich regions While the fission proteins play an inarguably critical role, a growing body of were observed. These Parkin-rich areas in HF myocyte also exhibited evidence reveals that conical lipids regulate mitochondrial morphology and completely depolarized mitochondria. Western blot analysis failed to detect fission. But how conical lipids contribute to fission remains an open question. Parkin levels in control and aging hearts, however Parkin was clearly detectable Here, we computationally model tubular mitochondria to reveal a new buckling in HF. Also, levels of p62 and LC3I (precursor for LC3 II) declined during instability-based mechanism for achieving a stable geometry conducive aging, but were not significantly altered in HF. This suggests that Parkin for fission. Employing membrane physics and differential geometry, the degradation and mitophagic flux were impaired in HF. Beta-hydroxybutyrate study reveals that buckling instabilities, triggered synergistically by cylindrical (2 mM) increased mitophagic flux in the aging heart but not in HF hearts. curvatures from proteins and spherical curvatures from conical lipids, help Several enzymes in ketone metabolism were impaired in HF leading to beta- achieve superconstrictions for fission. We validate the role of conical lipids hydroxybutyrate polymerization and poly-beta-hydroxybutyrate (PHB) forma- by an in vitro study in which membrane tubules with reduced concentration tion. PHB formation in HF was associated with enhanced opening of the mito- of conical lipids (PE) fail to undergo necking despite the presence of Drp1 pro- chondrial permeability transition pore and enhanced mitochondrial teins. depolarization. We conclude that elevated ketone body levels may be beneficial

BPJ 8740_8744 662a Wednesday, February 21, 2018 for mitochondrial repair in the aging heart; however impaired ketone body Airy-scan detector. Over 80% of Drp1 is located within 200nm radius of metabolism in HF limited this benefit and was associated with reduced Parkin RyR2. Similar results were observed in preparations by using immunogold- degradation and impaired mitophagic flux. TEM. Drp1 distribution along the dyads was studied by the localization of Drp1 along the transversal side of the mitochondria(TOM20). A significantly 3281-Pos Board B489 increased translocation of Drp1 to the transversal sides was observed upon High Intrinsic Aerobic Endurance Capacity Preserves Cardiomyocyte 15min-2Hz electric-field stimulation in cardiomyocytes superfused with iso- Quality Control, Mitochondrial Fitness and Lifespan proterenolþCa2þ(1mM) in comparison to cardiomyocytes incubated with 1 1 1 Magdalena Juhaszova , Sonia Cortassa , Miguel A. Aon , quasi-Ca2þ-free(2.5mM) buffer. Finally, isolated hearts form rat retrogradely 2 2 2 Jose A. Gonza´lez-Reyes , Miguel Calvo-Rubio , Jose M. Villalba , perfused (Langendorff) with Krebs buffer supplemented with isoprotere- 1 1 1 3 Dmitry B. Zorov , Evgeny Kobrinsky , Bruce D. Ziman , Lauren G. Koch , nolþCa2þ(1.8mM) showed a significantly elevated Drp1 accumulation at 3,4 1 1 Steven L. Britton , Edward G. Lakatta , Steven J. Sollott . the MAM. Conclusion: According to these results, we conclude that Drp1 is 1Laboratory of Cardiovascular Science, National Institute on Aging, NIH, 2 preferentially positioned at the mitochondria-SR associations and its recruit- Baltimore, MD, USA, Departamento de Biologı´a Celular, Fisiologı´a e ment likely involves Ca2þ signaling and/or beta-adrenergic activity. Inmunologı´a, Universidad de Co´rdoba, Campus de Excelencia Internacional Agroalimentario, ceiA3, Co´rdoba, Spain, 3Department of Anesthesiology, 3283-Pos Board B491 University of Michigan, Ann Arbor, MI, USA, 4Department of Molecular & Enhancing Mitochondrial Biogenesis with a CRISPR/ndCas9 Adenoviral Integrative Physiology, University of Michigan, Ann Arbor, MI, USA. Vector System in Cardiomyocytes Growing evidence has established cardiorespiratory fitness (CRF) as a strong Deepthi Ashok, Agnes Sidor, Brian O’Rourke. and reliable quantitative predictor of elderly mortality and cardiovascular Johns Hopkins University, Johns Hopkins University, Baltimore, MD, USA. events. To gain insight into potential mechanisms linking high CRF to Mitochondrial dysfunction contributes to heart failure and the genes involved increased healthspan and lifespan, we examined cardiomyocytes from rats in mitochondrial biogenesis and quality control, such as the peroxisome prolif- selectively bred for low and high intrinsic aerobic capacity, the so-called low erator activated receptor gamma coactivator-1a (PGC-1a) and mitochondrial capacity runners (LCR) and high capacity runners (HCR). LCR vs HCR transcription factor A (TFAM), are downregulated in a guinea pig model of have been shown to differ in numerous ageing related diseases (including the heart failure and sudden cardiac death. Here, we apply RNA-guided metabolic syndrome and cardiovascular complications) and longevity. We hy- nuclease-dead Cas9 (ndCas9) technology as an activator to target sequences up- pothesized that differences in mitochondrial function, homeostatic perfor- stream of the transcriptional start site to promote transcription of PGC-1a or mance and stress-resistance (i.e., fitness) underlie the ageing- and CRF- TFAM. To facilitate gene transfer to cardiomyocytes we developed an adeno- linked risks of developing chronic disease and multimorbidity. We demon- viral gene delivery system for expression of the CRISPR/ndCas9 activating strated that autophagy/mitophagy is significantly diminished in old compared complex and a targeting gRNA to modulate expression of any gene of interest to younger rats, and is significantly lower in LCR vs HCR compared across in differentiated cardiomyocytes, with our initial focus on activating the meta- all age groups (14%, 26% and 30% lower in LCR vs HCR at 7, 17 and 24- bolic gene program.We use ndCas9 fused to the activation domain VP64 and month-old, respectively). Accumulation of lipofuscin (the progressive buildup guide RNAs for activation of target genes. Proof-of-principle evidence was ob- of intralysosomal debris) gradually increased with age (by 5-fold at 24 vs 7 tained by activating a cyclic AMP response element (CRE)-driven luciferase months) reaching significantly higher levels in aged LCR vs HCR, in parallel reporter gene and activation of endogenous CRE-regulable genes (PCK1 and with the decline in autophagic turnover, suggesting significant alteration in PGC-1a). We have successfully upregulated a reporter gene (luciferase) and the balance between damage and repair. Indeed, impaired autophagy/mitoph- endogenous CRE-regulated genes PCK1 and PGC-1a in the same reporter agy may perturb mitochondrial quality control as manifested by a pronounced cell line with ndCas9-VP64, as evidenced by significant increases in both age-dependent decrease in the ROS-threshold for opening of the permeability mRNA and protein expression levels. Next, we constructed and delivered transition pore (50% decrease in 24-month vs 7-month-old; LCR>HCR) and ndCas9-gRNA adenoviral vectors to target TFAM in the rat genome that are hence a deterioration in fitness of the organelle, eventually, leading to a decline efficiently expressed by the host cells (NRVMs and H9c2 cells) and showed in cell function. increased expression of TFAM in H9c2 cells. Importantly, an increase in In summary, observed changes in mitochondrial repair, turnover and fitness mtDNA nucleoids was evoked by TFAM activation. For the first time, we may contribute to a decreased functionality and increased vulnerability to show that the CRISPR/ndCas9 system can be used as a powerful tool for modu- injury of the aged heart that is more pronounced in LCR vs HCR, and may un- lating transcription of native genes involved in mitochondrial biogenesis in car- derlie differences in healthspan and lifespan. diac cells, opening the door to developing novel therapeutic approaches for heart failure. 3282-Pos Board B490 Signaling Mechanisms of Drp1 Translocation to the Mitochondria-SR 3284-Pos Board B492 Associations in Adult Murine Cardiomyocytes Conserved Dynamic Characteristics of Mitochondrial Networks Celia Fernandez Sanz1, Sergio De La Fuente2, Zuzana Nichtova2, Greyson Lewis, Wallace Marshall. Sebastian Lanvermann1, Gyo¨rgy Csorda´s2, Wang Wang3, Shey-Shing Sheu1. Biochemistry and Biophysics, UC San Francisco, San Francisco, CA, USA. 1Center for Translational Medicine, Thomas Jefferson University, Mitochondria are the respiratory powerhouses of eukaryotic cells. These organ- Philadelphia, PA, USA, 2MitoCare Center, Pathology, Anatomy & Cell elles form a variety of organized physical networks in many biological con- Biology, Thomas Jefferson University, Philadelphia, PA, USA, texts, ranging from multi-cell structures in skeletal muscle tissue to highly- 3Mitochondria and Metabolism Center, University of Washington, Seattle, fused networks in single cells of S. cerevisiae. Though mitochondrial networks WA, USA. are quite common, the reason for maintaining a regular network structure is un- Mitochondrial dynamics, including fission, fusion, and movement, is a funda- clear. Here we begin to test mathematically the similarity between mitochon- mental mechanism in regulating mitochondrial function. The high level of Drp1 drial networks in S. cerevisiae and network structures that have been in the heart seems to be at odds with numerous studies showing that mitochon- optimized for a variety of tasks, such as from macromolecular transport and drial dynamics in adult ventricular myocytes occurs rather infrequently. Elec- minimal-energy rejection of low-quality components. To do this, I develop a tron micrographs show that in adult mammalian cardiac myocytes, the majority ‘‘fingerprint’’ using the mathematical concept of persistent homology, which of inter-myofibrillar mitochondria appear to be fragmented and tethered to the characterizes the scale-dependence of topological characteristics in a structure. sarcoplasmic reticulum (SR) near the dyads. We tested the hypothesis that Drp1 Preliminary data suggests that this fingerprint is quite different for graphs that is strategically accumulated at the mitochondria-SR associations (MAM) and are superficially similar, though built using distinct sets of construction rules. that during continuous excitation-contraction coupling processes (e.g. heart- By computationally simulating mitochondrial-like networks incorporating beats), the localized high Ca2þ in the SR-mitochondria junction further in- different types of construction rules, then comparing their fingerprints, I will creases translocation of cytosolic Drp1 to the MAM. Western blots analysis be able to differentiate between networks optimized for different tasks. I will shows a Drp1 presence in the SR and crude mitochondria fractions. Further- then compare these fingerprints to those of true mitochondrial networks in more, proteomic results show that Drp1 is mostly presented and enriched in S. cerevisiae. By analyzing these fingerprints over time, I will be able to deter- the Percoll purified MAM but not in the pure mitochondrial fraction. To mine conserved characteristics of true mitochondrial networks, allowing a confirm this data, the distance between SR(RyR2) and Drp1 in adult cardio- more acute approach toward understanding the properties for which these net- myocytes was quantified in nanometer scale by using confocal microscope- works are optimized.

BPJ 8740_8744 Wednesday, February 21, 2018 663a

Posters: Systems Biology and Disease safe transport of pathogens from within the United States and internationally. Unfortunately, these methods require large amounts of paperwork, specialized shipping and handling practices, and costly materials. A new way to transport 3285-Pos Board B493 TM Targeting Proteome-Scale Networks to Design and Synthesize Potent Anti- and store pathogens is by 903 Protein Saver Cards. These cards allow for 5 cancer and Cell-Specific Immunomodulatory Compounds samples, up to 100 mL per spot, to be transported safely due to the cards being Gaurav Chopra. dry and the pathogen still being viable for detection methods such as Polymer- Chemistry, Purdue University, West Lafayette, IN, USA. ase Chain Reaction (PCR). Currently, there are no high-throughput experiments to quantify all biochem- In this presentation, we demonstrate the compatibility of these cards to ical interactions of small molecules in an organism (human) that is essential to maintain the genetic material of Listeria monocytogenes and Vibrio cholerae ascertain both toxicity and efficacy of a drug. Furthermore, the synthesis of for lysing and detection applications. A spot of the protein card is cut, the bacteria is rehydrated, and then extracted from the card for cellular lysing compound/drug libraries explores diverse chemical space that is unrelated Ò to the disease with no guidance from disease-specific biological pathways using the Lyse-It technology. Following cellular lysing, genomic DNA is (targets and antitargets). Recognizing this unexplored area, we have devel- detected using PCR. The positive results indicate that genetic material oped proteome-scale target/antitarget guided lead optimization methods contained in the cell is still viable for specific identification of these (CANDESIGN) for discovery and de novo design of safe chemical libraries. pathogens. Selection of target/anti-target networks are identified iteratively using ma- Within microwave cellular lysis and literature today remains the question of chine learning on experimental data for desired cellular phenotypes in vitro how the DNA is being fragmented. Literature suggests that reactive oxygen and in vivo. We model interactions with proteomes from different organisms species, such as singlet oxygen, play a vital role in DNA fragmentation. We (currently 48,278) to infer homology of compound/drug behavior. Our present evidence of increased and/or rapid microwave-induced singlet oxygen approach has been verified for drug repurposing, design then synthesis of generation. Singlet oxygen is detected using Singlet Oxygen Sensor Green, potent non-toxic anticancer pathway-specific libraries, and compounds that where the endoperoxide increases the fluorescent intensity when excited by a alter specific cell functions in vivo. Repurposed leads are identified from 473 nm laser. This detection of singlet oxygen is indicative of its formation un- 1 billion predicted compound-proteome interactions with 35% accuracy der microwave irradiation and therefore one player in the role of DNA across nine indications (immunological, metabolic, infectious). Compound li- fragmentation. braries synthesized for specific hormone signaling pathways result in 3288-Pos Board B496 picoMolar anticancer leads to combat resistant cancers. Our leads are non- Cellular Adhesions Predict Mobility Propensities of EMT toxic in vitro and in vivo and more efficacious than current treatments on Lewis Scott, Christopher Lemmon, Seth Weinberg. patient-derived tumor xenograft mouse models. Similarly, we designed com- Virginia Commonwealth University, Richmond, VA, USA. pounds to specifically change function of Myeloid Derived Suppressor Cells Epithelial-mesenchymal transition (EMT) is an epithelial phenotype adapta- (MDSC) from upregulated gene networks of monocytic MDSCs in the cancer tion governed by biomechanical and biochemical interactions at the cellular microenvironment. Cancer cell lines (human, mouse, dog) are insensitive to interface. Intercellular and extracellular interactions induce gene regulatory our MDSC-specific library in vitro but exerts antitumor impact in vivo via im- networks that mediate cellular adhesions. A gain of migratory function is munomodulation. We observed decreased frequencies of MDSC expressing indicative of the transition to the mesenchymal phenotype, whereas epithelial suppressive functional markers (iNOS, PD-L1) and increased frequency of þ þ cells express the cell-cell adhesion protein, E-cadherin, to stabilize a cohesive IFN-g CD8 T-cells for antitumor activity. We conclude that proteome- monolayer such as for lining organs. However, EMT acts more as a cascading scale networks result in safer ‘‘chemical probes’’ to effect similar pathways rather than an all-or-none process: an intermediate phenotype retains partial across different diseases therapeutically. expression of E-cadherin and the capacity to revert to an epithelial state. 3286-Pos Board B494 Hence, adhesion switches constitute an underlying mechanism of EMT- Systems Analysis of a Combined Interactome of Lithium-Sensitive linked cellular patterning observed in developmental, physiological, and aber- Proteins rant processes. To further understand the biomechanical consequence of EMT Weihao Ge, Eric Jakobsson. in cellular patterning of epithelial sheets, we developed a model of cell-cell University of Illinois, Urbana, IL, USA. adhesion kinetics of thermodynamically-constrained cells on a finite element In outline, the biophysical bases of lithium action are fairly well-understood substrate, wherein cell-cell and cell-substrate interaction energies are defined and summarized in Jakobsson, et.al., Journal of Membrane Biology, in press. by a Hamiltonian function. Neighboring cell-occupied lattice sites form inter- In membranes, eight different protein-mediated modes of transmembrane cellular adhesions as a function of E-cadherin receptor-ligand dynamics, transport have been identified. Since none of these transport mechanisms thereby reducing the interaction energy between two cells. Hence, cells ex- are highly selective for lithium, its concentration in biological compartments pressing high levels of E-cadherin tend to form archetypical epithelial sheets is not regulated, but rather varies widely in proportion to intake. Within bio- to minimize free energy of the system: Forces across cell-cell adhesions bal- logical compartments, lithium’s major mode of action is to inhibit proteins ance substrate forces producing a stable epithelial monolayer. Future work that have magnesium cofactors. Because of the enormous variety of effects will investigate the role of cellular interaction energy on EMT state transition of these proteins, it is necessary to use a systems approach to understand by varying transforming growth factor beta 1 (TGF-b). We anticipate that the biological significance of the biophysical effects. In this presentation, TGF-b will bias free energy minimization at the cell-substrate interface by we approach the systems biology of lithium by a phylogenetic (clustering) increasing the energy cost of cell-cell adhesion. These findings would suggest analysis of interactomes of some important lithium-sensitive proteins. We that biomechanical intercellular interactions alter molecular signaling and find that some KEGG pathways are strongly enriched in gene products manifest in cellular patterning. Our findings suggest critical mechanisms un- from the interactomes of several different lithium-sensitive proteins. In broad derlying adhesion switching during EMT and delineate causes of aberrant categories, pathways implicated in carbohydrate metabolism, signal transduc- EMT in fibrosis and metastasis. tion, cancer, and neurodegenerative diseases are especially enriched. Notable enriched pathways outside these broad categories include long-term depres- 3289-Pos Board B497 Vibrant sion, Chagas disease, insulin signaling pathway, thyroid hormone signaling Chemical Fluctuation Theorem Governing Reaction Networks in pathway, dopaminergic synapse, platelet activation, amoebic dysentery, Living Cells adrenergic signaling in cardiomyocytes, long-term potentiation, and hepatitis Jaeyoung Sung. C. On the other hand, some KEGG pathway categories are extremely under- Chemistry, Chung-Ang University, Seoul, Republic of Korea. represented or even absent in the lithium-sensitive interactome, including To understand how accurately living cells can control their biological func- many biosynthetic pathways. tions, it is essential to construct a rigorous mathematical description for chemical dynamics of intracellular networks interacting with complex and 3287-Pos Board B495 hidden cell environment. In this talk, we introduce a novel model and stochas- Viable Pathogenic Organism Transportation and Recovery from a Low- tic kinetics for intracellular networks interacting with cell environment, Cost Support employing a complete description of cell state dynamics and its coupling to Tonya Santaus, Chris Geddes. the system network. For a general model of intracellular birth-death Chemistry/ Biochemistry, University of Maryland, Baltimore County - processes, we present the chemical fluctuation theorem (CFT) that relates Institute of Fluorescence, Baltimore, MD, USA. microscopic dynamics of the birth and death processes to the cell-to-cell There are two leading causes for rapid detection of pathogens: pathogenic variation in the product number. Combined with a new mathematical descrip- transport and sample preparation. A variety of regulations is in place for the tion of the gene expression network, CFT provides an excellent quantitative

BPJ 8745_8748 664a Wednesday, February 21, 2018 explanation of the cell-to-cell variation in the mRNA and protein levels for structure propensity and binding tendency, to quantify the effect of amino various recently investigated systems in a unified manner. Our analysis of acid substitutions. For this study, we develop a framework for large-scale mRNA counting statistics in Escherichia coli shows that the transcription of mapping of established linear sequence-based paralog and ortholog conserva- an active gene is a strongly non-Poisson process whose rate coefficient is a tion scores onto the tertiary structures of human proteins. This framework can dynamic stochastic variable with either oscillatory or monotonically decaying be utilized to map the spatial distribution of mutations on solved protein struc- time correlation function (TCF). The shape of the TCF of the rate coefficient tures as well as homology models. As a proof of concept, using a homology has an important consequence for the cellular control over the transcriptional model of the human Nav1.2 voltage-gated sodium channel structure, we noise. This work demonstrates a promising, new approach in quantitative observe spatial clustering in distinct domains of mutations, associated with biology, attempting to make complex dynamics of chemical reactions in Autism Spectrum Disorder (>20 variants) and Epilepsy (>100 variants), living cells and its biological consequence accessible to a rigorous mathemat- that exert opposing effects on channel function. We are currently character- ical description. izing all variants (>300k individuals) found in ClinVar, the largest disease variant database, as well as variants identified in >140k individuals from gen- 3290-Pos Board B498 eral population. The variant mapping framework and our score, informed with Stochastic Analysis of Coagulation and Fragmentation of Self-Assembly by structural information, will be useful in identifying structural motifs of pro- Solving Discrete Chemical Master Equation (dCME) with Acme teins associated with disease risk. Farid Manuchehrfar1, Wei Tian1, Tom Chou2, Jie Liang1. 1Bioengineering, UIC, Chicago, IL, USA, 2Biomathematics & Mathematics, 3292-Pos Board B500 UCLA, Los Angeles, CA, USA. Translational Reprogramming in Salmonella typhimurium Modifies Envi- Coagulation and fragmentation (CF) is a process in which particles aggregate ronmental pH to Sustain Higher Growth Rates before Entry into Station- into clusters and clusters break down into smaller clusters or particles. This ary Phase ubiquitous process plays important roles in life-threatening diseases such as Manohary Rajendram, Lillian Zhu, Kerwyn C. Huang. Alzheimer’s disease and brain shrinkage. This process often happens in Stanford University, Stanford, CA, USA. confined space with limited number of particles, and thus the behavior of the Bacterial batch cultures in rich media are punctuated by differing substrate system is highly stochastic. A fundamental approach to study CF and its sto- utilization patterns with corresponding changes in pH associated with the chasticity is through solving the corresponding Discrete Chemical Master excreted metabolites. Salmonella typhimurium is an intracellular pathogen Equation (dCME), which provides exact descriptions of the time-evolving that is capable of replicating in highly acidic conditions such as macrophages. and the steady states of the system of biochemical reaction networks. However, However, the biological mechanism which allows S.typhimurium to replicate current models of CF have limitations. For instance, stochastic simulation algo- in such hostile environments is still unclear. S.typhimurium grown in anaer- rithm has difficult in sampling rare events and its convergence is difficult to obic conditions in LB displays an intriguing pH profile and a diauxic growth determine. The error in the reconstructed steady-state probability distribution pattern, where the maximum growth rate corresponds to acidification of the is also often unknown. Recent theoretical models consider the process as one surrounding medium, followed by a period of alkalization and a secondary dimensional and do not account for attachment, detachment, synthesis and growth burst corresponding to another cycle of acidification. We performed degradation together. Here we study CF by solving the dCME exactly using a high-throughput growth screen of a library of S.typhimurium non-essential the newly developed ACME method [1][2], and investigate systematically gene deletions in anaerobic conditions and identified genes that are involved how synthesis, degradation, attachment and detachment of molecular particles in the different phases of growth of S. typhimurium. We found that S.typhimu- affect the behavior of a CF system. In addition, we examine the effects of rium follows distinct substrate utilization patterns in each phase of growth and dimensionality of clusters. We demonstrate how these factors may have pro- that mutants that are unable to perform alkalization of the growth medium found impacts on system behavior. Systematic analysis of the CF process as lack the observed secondary growth burst. We discovered that deletions of described in this study can help in further understanding their roles in biological two genes epmA and epmB, that help alleviate translational pausing at poly- problems such as amyloid-beta aggregation in neurodegenerative disease and proline stretches, are unable to perform alkalization of the growth medium actin cable formation. and hence have lower secondary growth rates. We postulate that S.typhimu- [1] Youfang Cao, Anna Terebus, and Jie Liang, SIAM Multiscale Modeling and rium’s growth advantage in low pH environments is conferred by global trans- Simulation, 2016. 14(2):923-963. [2] Youfang Cao, Anna Terebus, and Jie lational reprogramming allowing for easy switching between different Liang, Bulletin of Mathematical Biology, 2016. 78(4):617-661. metabolic states. 3291-Pos Board B499 3293-Pos Board B501 Identification and Characterization of Variant Intolerant Sites across Mechanistic Systems Modeling to Improve Understanding and Prediction Human Protein 3-Dimensional Structures of Cardiotoxicity Caused by Targeted Cancer Therapeutics Sumaiya Iqbal1,2, Jakob Berg Jespersen3,4, Eduardo Perez-Palma5, Jaehee Shim. Patrick May6, Henrike Heyne1,2, Kasper Lage3, Rikke Steensbjerre Møller7,8, Biophysics and Systems Pharmacology (BSP) Training Area, Icahn School of Florence F. Wagner1, Mark Daly1,2, Arthur J. Campbell1, Dennis Lal1,2. Medicine at Mount Sinai, NYC, NY, USA. 1Stanley Center for Psychiatric Research, Broad Institute of MIT and Tyrosine kinase inhibitors (TKIs) are a class of cancer therapeutics that have Harvard, Cambridge, MA, USA, 2Analytic and Translational Genetics Unit, been linked with serious cardiotoxicity including left ventricular dysfunction, Massachusetts General Hospital, Boston, MA, USA, 3Department of Surgery, heart failure, and QT prolongation.To elucidate mechanistic details and Massachusetts General Hospital, Boston, MA, USA, 4Department of Bio and develop therapeutic strategies to either mitigate or reverse these cardiotoxic- Health Informatics, Technical University of Denmark, Lyngby, Denmark, ities, we have performed computational analysis using in-vitro data based quan- 5Cologne Center for Genomics, University of Cologne, Cologne, Germany, titative systems pharmacology (QSP) modeling. Specifically, we have 6Luxembourg Centre for Systems Biomedicine, University of Luxembourg, integrated mRNAseq data with mathematical models descriptive of hypertro- Esch-sur-Alzette, Luxembourg, 7The Danish Epilepsy Centre, Dianalund, phy and excitation-contraction (EC) coupling, both of which have been impli- Denmark, 8Institute for Regional Health Research, University of Southern cated in TKI-induced cardiotoxicity. The mRNAseq data used in this analysis is Denmark, Odense, Denmark. an open-access data from the Drug Toxicity Signature Generation (DToxS) The functional interpretation of genetic variation in disease-associated genes Center at Mount Sinai. Using the approach described above, we have evaluated is far outpaced by data generation. Existing algorithms for prediction of the hypertrophic risk of 27 FDA approved TKIs under different physiological variant consequences do not adequately distinguish pathogenic variants conditions which includes stimuli such as isoproterenol and endothelin-1. The from benign rare variants. This lack of statistical and bioinformatics analyses, simulations predict that, as a class, the TKIs induce an increase in cellular hy- accompanied by an ever-increasing number of identified variants in biomed- pertrophic risk compared with 17 non-cardiotoxic control drugs. Interestingly, ical research and clinical applications, has become a major challenge. Estab- this augmented hypertrophic risk was especially pronounced in simulations that lished methods to predict the functional effect of genetic variation use the calculated effects of physiological stimuli such as endothelin-1 and epineph- degree of amino acid conservation across species in linear protein sequence rine, suggesting a ‘‘two hit’’ hypothesis in which cancer therapeutics make car- alignment. More recent methods include the spatial distribution pattern of diac myocytes more susceptible to environmental cues. Finally, a secondary known patient and control variants. Here, we propose to combine the linear analysis on simulation results identified nodes that are part of classical cardiac conservation and spatial constrained based scores to devise a novel score survival pathway, such as ERK1/2 and JNK, and their downstream effectors that incorporates 3-dimensional structural properties of amino acid residues, such as GATA4 and CREB, as potential convergent mechanisms for drug- such as the solvent-accessible surface area, degree of flexibility, secondary induced toxicities. Overall, this analysis integrating genome-scale expression

BPJ 8745_8748 Wednesday, February 21, 2018 665a analysis with mechanistic models generates experimentally-testable predic- Bacteria in large communities such as biofilms compete for nutrients and must tions and suggests novel mechanisms potentially underlying drug-induced therefore coordinate so that cells maintain sufficient access to resources. In cardiomyopathy. biofilms of Bacillus subtilis bacteria, such coordination is mediated by an electrical cell-to-cell signaling, with ion channels regulating the release of po- 3294-Pos Board B502 tassium, creating spatially propagating potassium waves. However, participa- Radioprotective Effects of Lactobacilli with Antagonistic Activities tion in the signaling carries a measurable cost to individual cells. As a result, Against Human Pathogens not all cells participate, and yet the wave successfully propagates from the 1,2 1 1,2 Astghik Pepoyan , Marine Balayan , Anahit Manvelyan , middle center to the edge of the biofilm. Using mathematical modeling, 2,3 2 2 Sofi Pepoyan , Lilit Malkhasyan , Tatevik Bezhanyan , fluorescence microscopy, and gene deletion strains, we investigate how 2 4 4 Ruzanna Paronikyan , Margarita Malakyan , Sergey Bajinyan , robust signal transmission occurs despite heterogeneous participation in 2 5 2,6 Vardan Tsaturyan , Shigeru Kamiya , Michael Chikindas . signaling. 1Armenian National Agrarian University, Yerevan, Armenia, 2International Association for Human and Animals Health Improvement, Yerevan, 3 4 3297-Pos Board B505 Armenia, Biophysics, Yerevan State University, Yerevan, Armenia, The A Guanine Nucleotide Exchange Modulator, GIV-GEM, Acts as a Research Centre of Radiation Medicine and Burns, Yerevan, Armenia, 5 6 Compartmental Controller for Growth Factor Signal Pathways Kyorin University School of Medicine, kyorin, Japan, Rutgers State Michael Getz1, Pradipta Ghosh2, Padmini Rangamani3. University, New Brunswick, NJ, USA. 1University of California San Diego, La Jolla, CA, USA, 2Department of During our previous research, we isolated lactobacilli strains: E5-1, M1-3 and Cellular and Molecular Medicine, University of California San Diego, La ZPZ, that have antagonistic activities against A. baumannii, S. aureus, Jolla, CA, USA, 3Mechanical and Aerospace Engineering, University of K. pneumoniae and Enterobacter gergoviae that are prevalent in Yerevan hos- California San Diego, La Jolla, CA, USA. pitals. The aim of the current investigation was to evaluate radioprotective Localization of signals within the cell are paramount for deciding cellular activities of these strains. 90 healthy adult Wistar rats in the weight range function; dysregulation of these signaling pathways leading to certain dis- 150-200g were randomly placed into the following nine groups: control, eases, including cancers. Recently, an alternative paradigm for cAMP control-placebo, irradiated, three control-lactobacilli and three irradiated- signaling has emerged, in which growth factor-receptor tyrosine kinases lactobacilli. The three irradiated-lactobacilli groups received an appropriate (RTKs; e.g., EGFR) access and modulate G-proteins via cytosolic guanine- feeding cannula for seven days prior to receiving a 4.5 Gy irradiation. A sig- nucleotide exchange modulator (GEM), GIV/Girdin; dysregulation of this nificant decrease in the blood leukocyte count (WBC) was described after the pathway is frequently encountered in cancers. We recently computationally 5 9 5 9 seventh day of rats’ irradiation ((0.80 0.07)x10 /L vs. (7.12 0.39) x10 /L modeled this process to show that GIV-GEM exerts dual control over 5 9 < and (6.84 0.77)x10 /L; P 0.05). We did not find any significant changes cAMP production through control of compartmentalisation speeds and Gi/s 5 in the rats’ blood glucose levels (BGL) adopted placebo ((5.79 0.37) proteins. This model showed cAMP modulation via mechanisms distinct 5 > mMol/L vs. (6.42 0.33) mMol/L; P 0.05), but an insignificant increase from the two most-often targeted classes of cAMP modulators, GPCRs and within the normal range was observed for the irradiated-E5-1 rats PDEs. Yet, this does not fully explain how GIV-GEM controls the prolifera- 5 5 5 ((5.79 0.37) mMol/L and ((6.43 0.33) mMol/L vs. (8.12 0.2) mMol/L; tive nature of the cell, typically seen through MAPK/ERK expression. We < P 0.05)). Parallel to this, we described a statistically confirmed decrease propose a comprehensive network-based compartmental model for GEM- 5 9 of WBC after the trial by the LactobacillusM1-3 ((5.21 0.12)x10 /L vs. dependent signaling. This comprehensive framework reveals unforeseen 5 9 5 9 < (7.12 0.39)x10 /L and (6.84 0.77)x10 /L; P 0.05). Also, the irradiated- crosstalk and network dynamics between upstream events and the various 5 E5-1 rats kept their initial BGL after the irradiation: (7.70 0.16) vs. 6.74; feedback loops that fine-tune the GEM action of GIV. We further validate < 5 P 0.05 (in comparison with irradiated rats) and (7.7 0.16) vs. our model by demonstrating its ability to capture experimentally observed 5 > (8.12 0.2); P 0.05 (in comparison with control-E5-1 rats). This group compartmental control and signal attenuation. We hypothesize that GIV- of rats were also characterized by the statistically confirmed increase of GEM’s Early Endosome Antigen 1 (EEA1) endosome maturation control 5 9 WBC in comparison with its irradiated controls ((0.80 0.07)x10 /L vs. can help isolate membrane scaffold dependant signals, i.e ERK1/2, to the 9 < 1.58x10 /L; P 0.05), and decrease of WBC in comparison with its controls plasma membrane. We show that the localisation of these membrane associ- 5 9 9 < ((6.7 0.38)x10 /L vs. 1.58x10 /L; P 0.05). These findings are of signifi- ated proteins, such as Ras and ERK, controls the timescale and response cance for the LactobacillusE5-1 application as a starter for the production strength allowing modulation of kinetics causing different phenotypic conse- of functional food with radioprotective activities. quences; from sinister to protective. Published experimental evidence shows 3295-Pos Board B503 EEA1 endosomes are major sites for proliferative signaling, and the endoso- The Role of Autophagy in Alzheimer’s Disease: Modeling and Simulations mal lifespan is controlled by Gas and GIV-GEM. We then combine the pre- > MooYoung Choi1, Kyungreem Han2, Jinwoong Kim3. viously created RTK- cAMP pathway and calcium signaling pathways to 1Physics, Seoul National University, Seoul, Republic of Korea, 2NHLBI, elucidate how GIV’s phenotypic response also controls second messenger National Institutes of Health, Bethesda, MD, USA, 3Pharmacy, Seoul signaling including cAMP and calcium. National University, Seoul, Republic of Korea. Autophagy is a key lysosomal degradative mechanism used to eliminate 3298-Pos Board B506 abnormal proteins and organelles. Neurons are particularly vulnerable to dis- Reflected Conduction Attributed to Sodium Channel Distribution Within ruptions of autophagy pathway, and thus dysregulation of autophagy causes Cardiomyocytes: A Possible Mechanism of Ventricular Fibrillation Induc- various neurological disorders, especially for age-related disorders such as Alz- tion in Brugada Syndrome heimer’s disease and Parkinson’s disease. This study focuses on the role of Kunichika Tsumoto, Yoshihisa Kurachi. autophagy in Alzheimer’s disease by means of modeling and computer simu- Graduate School of Medicine, Osaka University, Suita, Japan. lations. We develop a ’mathematical model for autophagy’ to integrate various Brugada syndrome is characterized by the ECGs with both a right bundle- individual molecular and cellular data sets into a unified framework. Simula- branch block pattern and ST-segment elevation in the right precordial leads tions focus on the identification of correlations between defects arose at (V1-V3) and by the higher incidence of sudden cardiac death due to ventric- different stages of the autophagy pathway and pathogenesis of Alzheimer’s dis- ular tachycardia/fibrillation. It is commonly believed that the lethal arrhyth- ease. Specifically, we analyze profiles of autophagy pathway such as time evo- mias are elicited by the closely coupled premature ventricular contractions lutions of autophagy fluxes, levels of autophagosomes/autolysosomes, and via the reflected conduction (i.e., phase-2 reentry) mechanism. Although corresponding cellular changes using quantitative indices measuring autophagy loss of function mutation in cardiac sodium (Na) channels have been linked activities. to Brugada syndrome, the relation between the loss of function in Na channels and the arrhythmogenic mechanism remains unclear. We have recently 3296-Pos Board B504 reported that the action potential propagation was greatly affected by the sub- Electrical Signal Transmission in a Heterogeneous Population of Bacteria cellular distribution of Na channels in a physiologically-accurate myofiber Joseph W. Larkin1, Xiaoling Zhai2, Kaito Kikuchi1, Aleksandra Walczak3, model where myocytes were electrically coupled with both gap junctions Garcia-Ojalvo Jordi4, Arthur Prindle5, Andrew Mugler2,Gurol€ Suel€ 1. and intercellular cleft conductor (electric field mechanism). Here, we 1Molecular Biology, University of California-San Diego, San Diego, CA, extended the simulation to the initiation of reflected conduction in Brugada USA, 2Physics, Purdue University, West Lafayette, IN, USA, 3Physics, Ecole syndrome. We conducted computer simulations of action potential propaga- Normale Superieure, Paris, France, 4Experimental and Health Sciences, tion in the myofiber model, and investigated the effects of subcellular Universitat Pompeu Fabra, Barcelona, Spain, 5Northwestern University, distributions of Na channels on the development of reflected conduction. Chicago, IL, USA. We found that the myofiber model with specific subcellular Na channel

BPJ 8745_8748 666a Wednesday, February 21, 2018 distribution (all Na channels were distributed only in the intercalated disks Posters: Emerging Techniques and Synthetic and there were no Na channels along the lateral side of each myocyte) resulted in early repolarization followed by reflected conduction. Subcellular Na chan- Biology nel distribution might be responsible for fibrillation induction in Brugada syndrome. 3301-Pos Board B509 A Genetically Encoded Toolbox of Orthogonal Adhesins for Bacterial Self- 3299-Pos Board B507 Assembly Examining UBE3A’s Possible Role in Dendritic Spine Morphogenesis David S. Glass, Ingmar H. Riedel-Kruse. Judy E. Bloom1,2, Carissa Sirois1,3, Michael L. Blinov2, Bioengineering, Stanford, Stanford, CA, USA. Stormy J. Chamberlain3, Leslie M. Loew2. In over a decade, synthetic biology has developed increasingly robust gene net- 1Neuroscience, University of Connecticut School of Medicine, Farmington, works within single cells, but constructed very few systems that demonstrate CT, USA, 2Richard D. Berlin Center for Cell Analysis and Modeling, multicellular spatio-temporal dynamics. In particular, to our knowledge there University of Connecticut School of Medicine, Farmington, CT, USA, exists no convenient method for engineering cell-cell adhesion. Towards filling 3Genetics and Genome Sciences, University of Connecticut School of this gap in synthetic biology’s toolbox, here we report the first 100% geneti- Medicine, Farmington, CT, USA. cally encoded self-assembly platform, based on modular cell-cell adhesion in UBE3A is a ubiquitin ligase responsible for targeting proteins to be the bacterium Escherichia coli. In this system, adhesive selectivity is provided degraded by the 26S proteasome. In neurons, UBE3A is only produced by by a library of outer membrane-displayed peptides with orthogonal intra-library the maternal allele as the paternal associated gene region is silenced. The specificities, while affinity is provided by intrinsic adhesin affinity, media con- loss of the maternal copy of UBE3A results in Angelman Syndrome (AS). ditions, and inducible expression across the entire library. We demonstrate this On the other hand, the duplication or triplication of the maternal chromo- tool by building a variety of well-defined multicellular patterns, including mul- some region containing UBE3A results in Chromosome 15q Duplication tiple cell types in cluster-, mesh-, and lattice-like arrangements and comparing syndrome (Dup15q) which is associated with autism. We used iPSC- these to a biophysical model. This orthogonal, composable adhesion system derived neurons from AS and Dup15q afflicted individuals to show that den- will enable future development of synthetic multicellular systems for use in dritic spine density is affected during neuronal development. Here we use a consortia-based metabolic engineering, in living materials, in tissue engineer- rule-based model to investigate UBE3A’s possible role in dendritic spine ing, and in controlled study of minimal multicellular systems. morphogenesis. Our model encompasses the ubiquitin proteasome pathway, 3302-Pos Board B510 co-activator of UBE3A, two UBE3A substrates (RING1B and Ephexin5) Optically Patterned Biofilms via Transcriptional Control of Adhesin and their related pathways. We believe these two substrates in our model Expression are critical for dendritic spine formation and maintenance. Ephexin5, a Xiaofan Jin, Ingmar Riedel-Kruse. RhoA GEF, remains active and prevents mature synapse formation until Bioengineering, Stanford University, Stanford, CA, USA. EphB signaling occurs. In the absence of UBE3A, Ephexin5 levels are Bacterial biofilms represent a promising opportunity for engineering of micro- increased and the dendritic spine density is reduced. RING1B is another -/- bial communities - however our ability to control spatial structure in biofilms E3 ligase that has been shown to shuttle AMPA receptors. In UBE3A remains limited. We engineer Escherichia coli to form biofilm when illumi- mice RING1B is significantly increased and the knockdown of RING1B re- nated by blue light, using a light-activated transcriptional promoter to regulate sults in an increase of dendritic spines. Using our model, we predict the adhesin gene expression. Under patterned illumination, these engineered cells number of spines that would be stable, silent, or mature given the Ephexin5 form structured biofilm on a variety of substrates with spatial resolution on the and AMPA receptor concentration. We then test how adjusting levels of order of 20 microns. We present a Monte-Carlo simulation model to explain a UBE3A may affect the spine development. (Supported by: Connecticut potential biophysical mechanism behind the achieved spatial resolution. Over- Regenerative Medicine Fund & National Institute of General Medical Sci- all, this platform provides the ability to culture structured biofilms, a key step ences (P41 GM103313)). towards the engineering of living biomaterials and bottom up approaches to mi- crobial consortia design. 3300-Pos Board B508 Tuberculosis (TB) and Noodletree: Verifying Genetic Sequence Data and 3303-Pos Board B511 Functionality of a Generally Transduced M. Tuberculosis Toxin through Droplet Based Measurements of Mechanical Forces and Material Proper- a Viral Carrier ties, In Vivo and In Vitro Britt Int-Hout, Lydia Flores. Elijah Shelton1, Adam Lucio1, Hannah Gustafson2, Alessandro Mongera1, Biotechnology, Collin College, Plano, TX, USA. Friedhelm Serwane3, Otger Campa`s1. Discovered in 2016, the genome of Mycobacteriophage NoodleTree was an- 1Mechanical Engineering, UC Santa Barbara, Santa Barbara, CA, USA, notated following isolation and amplification through host Mycobacterium 2Biology, UC Santa Barbara, Santa Barbara, CA, USA, 3Max Planck Institute smegmatis (mc2155). NoodleTree’s genome reveals interesting characteristics for Medical Research, Heidelberg, Germany. among C1 cluster phage, most notably genomic protein 8 (gp8) which is only The spatiotemporal patterning of physical stresses and mechanical properties found in nine other C1 phage. Gp8 potentially codes for a protein that has inside living tissues ultimately guide morphogenesis, both during normal devel- Mycobacterium tuberculosis activity. Specifically, gp8 may code for a tuber- opment and cancer progression. Here, we present various droplet-based tech- culosis necrotizing toxin (TNT) and its immunity factor (IFT). In M. tubercu- niques for measuring these physical quantities in biological systems, both losis, the toxin protein is secreted through the outer cell membrane and into its in vivo and in vitro. We describe the methods for generating and functionalizing host. Once inside the host cell, TNT hydrolyzes NADþ in the cytosol inhib- droplets, which can be placed in a tissue and imaged in 3D. We present an open iting its usage and leading to apoptosis. Gp8 expresses coverage for this func- source Matlab based computational tool recently made available to the commu- tional protein at 84.5238% with an 85.4% probability according to HHPRED. nity that performs automated 3D surface segmentation and curvature analysis Further analysis reveals gp8 protein residues have 100% identity with M. from a volumetric image stack with typical errors in radial and curvature mea- tuberculosis Rv3902c IFT (176/176 residues) and Rv3903c TNT (199/199 surements less than 2 and 20 percent, respectively. By placing cell-sized fluo- residues). However, preliminary BLAST data shows less than 40% identity rocarbon droplets within multicellular spheroids, we measure anisotropies in between NoodleTree’s gp8 and a known toxin (TNTþIFT) module. As there cell-generated stresses and quantify how these stresses change over time and are over 20 M. tuberculosis toxin modules along with other host carriers, space within the aggregates. Furthermore we present in situ calibrated measure- nucleotide and protein sequences need to be verified for exact coverage ments of cellular stresses inside the developing zebrafish tailbud (10-12 somite amounts through various bioinformatic sources (HHPRED, NCBI, Phag- stage) obtained with ferrofluid droplets (30-50 microns in diameter). Ferrofluid esDB). Upon confirmation of sequence alignment and protein identification, droplets were also used to measure mechanical properties using magnetic actu- future directions will include verifying the functional activity of gp8. RT- ation. In spheroids, stresses are observed to increase significantly over time PCR will be conducted using the gp8 nucleotide sequence. If toxin function- while maintaining a weak spatial dependence. In vivo studies in the zebrafish ality can be elucidated in a new host, then NoodleTree may have the ability to tailbud, however, reveal no significant spatial dependence in the magnitude transfer virulence to a host that shows no previous infectious activity. Through of cellular stresses, although patterning of the material properties is observed. generalized transduction, phage may demonstrate genetic transfer of viru- This work highlights the potential for droplet based in situ measurements to lence, likely contributing to an increased rate of infection and wider range elucidate mechanical phenomena underlying processes in developmental and of potential pathogenic hosts. cancer biology.

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3304-Pos Board B512 These results suggest that ribosomal assembly intermediates, which are esti- Giant Lipid Membranes Supported by Glass Beads for Membrane-Protein mated to occupy five-times more nuclear volume than chromosomes, dominate Interaction Studies the physical properties of the nucleus. Thus, mTORC1, a central regulator of Andrew Bogard1, Mark Smith2, Colleen Calzacorta3, Jessika Dagostino3, metabolism controls the biophysical properties of the nucleus by tuning macro- Nisha Shrestha2, Denise Wingett2, Daniel Fologea1. molecular crowding. Using nuc-GEMs in S. cerevisiae, we have discovered a 1Physics, Boise State University, Boise, ID, USA, 2Biomolecular Sciences, mechanism for tuning the properties of the nucleus that should be universal Boise State University, Boise, ID, USA, 3Biological Sciences, Boise State to all eukaryotes. University, Boise, ID, USA. Artificial membrane systems are essential elements for investigating funda- 3307-Pos Board B515 mental biophysical features such as biomolecular interactions between mem- Tuning DNA and Lipid Binding Proteins to Sense Changes in Cellular brane components and exogenous molecules that target them. While planar Geometry bilayer lipid membranes and liposomes have been extensively used for de- Clifford W. Sandlin, Matthew C. Good. cades for such studies, their poor stability and small size may seriously limit Cell and Developmental Biology, University of Pennsylvania, Philadelphia, their applicability. To overcome these roadblocks, we examined the use of PA, USA. large glass beads as support for the formation of stable membranes. Mem- Adenocarcinoma samples from the Penn Medicine BioBank show evidence of brane formation and stability was assessed in fluorescence microscopy studies oversized cells. These cells may affect cancer progression. However, testing that used lipophilic dyes for membrane imaging. The same technique was uti- whether large cells are relevant to pathology in a mouse requires a way to lized to indicate biomolecular interactions between fluorescent cholera toxin target only oversized cells. Targeting this subpopulation cannot be accom- B subunit and GM1 gangliosides embedded in the membrane as target for plished with drugs or any other method. Therefore, we are building synthetic the toxin. In addition, we succeeded to reconstitute membrane systems by us- genetic circuits to target only oversized cells. This synthesis approach has ing glass beads as support and intact red blood cell membranes as precursors. another benefit: it teaches us about the biophysical constraints upon size- The lipid partition of the resulted membrane was identified by lipophilic sensing proteins. staining, while the presence of proteins was confirmed by specific antibody To synthesize size-sensing, I am building proteins to sense cellular geometry. I staining. Membranes of either artificial or natural origin showed an describe how DNA- and membrane lipids titrate with cell volume, and simulate outstanding stability, which recommends the supported membranes for inves- binding of a protein to DNA or membranes as a function of volume. Simula- tigations on biomolecular interactions in which membrane stability is an tions of this ‘‘binding-with-volume’’ predict an increase the free activity of a essential factor. protein at larger cell sizes. This effect could be employed to activate a cell- death program. To account for nuclear transport effects on binding, I solve par- 3305-Pos Board B513 tial differential equations for nuclear binding and transport as a function of size. Active Deformations Compensate for the Excess Membrane Area during The results show that altering import and export rates of a DNA-binding protein the Adhesion of Cytoskeletal Vesicles will shift its binding curve. Overall, increasing any of the parameters of KD, Renu Vishavkarma. binding specificity, protein concentration, or nuclear export all shift size- Applied Sciences, Technical University Delft, Delft, Netherlands. response curves to smaller sizes. Every process in cell is complex and involve a number of signalling pathways I apply this theory to experiments in cell-sized droplets by showing binding vs. and feedback mechanisms. One way to understand the physical basis of a volume of HMG proteins to DNA-coated beads, and an amphipathic helix to cellular process, one at a time, is to adopt the bottom up approach. Using phospholipids. As expected, protein fluorescence in the lumen of droplets is this approach, the Giant unilaminar vesicles (GUVs) have been established depleted at smaller sizes. To show this behavior is relevant in cells, I demon- as the model system to study cellular adhesion. Number of experiments and strate the binding-with-volume effect in cell-like compartments containing theoretical models on vesicle-substrate interactions has provided the physical Xenopus laevis oocyte extracts and reconstituted sperm nucleii. Finally, I framework behind the membrane-substrate interaction during cellular adhe- describe the differences between theory and experiment and differences be- sion. So far the role of cytoskeletal to membrane interaction has not been tween the buffer and extract-based system. included in these studies. We show, how the interaction of a cytoskeletal vesicle to a rigid surface is different from a cortex free vesicle. Vesicle 3308-Pos Board B516 with cortex attached to its membrane are called the cytoskeletal vesicles. Probing Dynamics of Proteins via Self-Labeling Tags These vesicles lack excess membrane area stored in membrane undulations. Heejun Choi1, Ya-Cheng Liao1, Luke Lavis1, Yoon J Young2, Therefore, these vesicles cannot deform to gain in contact area and get Jennifer Lippincott-Schwartz1. ruptured by the lateral tension arising from the binding affinity of ligand- 1HHMI/Janelia Research Campus, Ashburn, VA, USA, 2Albert Einstein receptor pairs, in the strong adhesion regime. We show that, under the hyper- College of Medicine, Bronx, NY, USA. tonic osmotic stress, the cytoskeletal vesicles develop crumpled up shapes/de- Self-labeling protein tags such as HaloTag or SNAP-Tag utilize the covalent formations. This is because the volume is reducing while the surface area ligation of biorthogonal ligands to a tagged protein. By linking the ligands remains constant. If the vesicle has an active cortex, i.e., actin network with various synthetic fluorophores, orthogonal self-labeling protein tags with myosin motors, then these deformations can change over time. The permit the expansion of the chemical toolbox for fluorescence measurements lateral forces in the case of deswelled active vesicle act against the active de- for protein localization, as well as protein-protein interaction. With our formations, resulting in the smoothening of vesicle membrane. Therefore, the recent spectrally-tuned azetidine-containing rhodamine JF (Janelia Fluor) active deformations in the active vesicles serve the purpose as excess mem- dyes, each self-labeling tag can be designed to bind different fluorophore- brane area and helps the vesicle to survive strong adhesion without membrane conjugated ligands. Here, we demonstrate that the self-labeling system allows rupture. facile fine-tuned fluorescence resonance energy transfer (FRET) system by combining orthogonal self-labeling tags with a variety of donor-acceptor JF 3306-Pos Board B514 dye pairs. mTORC1 Controls the Physical Properties of the Nucleus Liam J. Holt. 3309-Pos Board B517 Institute for Systems Genetics, NYU Langone Medical Center, New York, Knockout Sudoku of Esoteric Microbes for Sustainable Energy NY, USA. Buz Barstow1, Oluwakemi Adesina2, Isao Anzai3, Michael Baym4, The mechanisms that determine the physical properties of the nucleus remain Lev Shaket2. largely unknown. We recently developed Genetically Encoded Multimeric 1Chemistry (to be Biological and Environmental Engienering as of 1/2/2018), (GEM) nanoparticles to study the physical properties of the cell. GEMs are ho- Princeton University (to be Cornell University as of Jan 2 2018), Princeton, momultimeric proteins that self-assemble into bright, stable fluorescent parti- NJ, USA, 2Chemistry, Princeton University, Princeton, NJ, USA, 3Molecular cles of defined size and shape. GEMs are extremely useful as tracer particles Biology, Princeton University, Princeton, NJ, USA, 4Biomedical Informatics, to probe the biophysical properties of cells. We added a nuclear localization Harvard Medical School, Boston, MA, USA. signal (NLS), to create nuc-GEMs that enabled rapid microrheology experi- Biology offers unique solutions to the problems of sustainable energy, but the ments within the nucleus. We leveraged this technology for a genetic screen genetic basis of many of these capabilities remain undiscovered. However, we and found that inhibition of mTORC1 dramatically altered the motion of lack the genetic characterization and engineering tools to take full advantage of nuc-GEMs, doubling their effective diffusion coefficient. Epistasis experiments this potential. In fact, 30-40% of the genes in any genome sequenced today are revealed that this biophysical effect was due to decreased ribosome production. of unknown function. This means that many of the genes underlying behaviors

BPJ 8745_8748 668a Wednesday, February 21, 2018 from carbon fixation and hydrocarbon synthesis to antibiotic resistance and 3312-Pos Board B520 sensitivity remain undiscovered. My team has developed Knockout Sudoku: Two-Photon Absorption Analysis of Red Fluorescent Genetically-Encoded a new technique that democratizes the creation of the most important genetic Calcium Ion Indicators characterization tool for any organism: a collection of single-gene knockout Rosana S. Molina1, Yi Shen2, Yong Qian2, Robert Campbell2, mutants for each of the thousands of non-essential genes in an organism’s Thomas E. Hughes1, Mikhail Drobizhev1. genome (a whole genome knockout collection). Knockout Sudoku uses proba- 1Cell Biology & Neuroscience, Montana State University, Bozeman, MT, bilistic inference algorithms to replace complex robotics to increase the speed USA, 2Chemistry, University of Alberta, Edmonton, AB, Canada. of constructing whole genome knockout collections by 100 and reduces the Genetically-encoded calcium ion indicators (GECIs) illuminate brain activity cost by 30 compared to the next comparable method. It brings this modern in model organisms. The basic design consists of a Ca2þ binding domain genomic tool to many more organisms at price that almost any investigator attached to a fluorescent protein in such a way that the binding of Ca2þ mod- can afford. This technique now allows my team to learn how to apply biology ulates its fluorescence. The current favorite GECI is the green GCaMP6, due to challenges across sustainable energy from enhancing the efficiency of photo- to the large increase of fluorescence upon binding Ca2þ (up to a 50-fold synthesis for biofuels to the safe use of nuclear power and enabling the wide- change). However, red fluorescence scatters less than green fluorescence, spread use of batteries. which is especially desirable for deeper imaging in tissue. Although there are red fluorescent GECIs available, they are less popular for reasons that include a smaller change in fluorescence. Three factors can contribute to Posters: Neuroscience: Experimental the change in fluorescence: 1) different quantum yields of the Ca2þ-bound and Ca2þ-free forms; 2) different extinction coefficients or cross sections, Approaches and Tools in one- or two-photon imaging, respectively; and 3) a redistribution of the neutral and anionic forms of the chromophore in the presence of Ca2þ. 3310-Pos Board B518 The third factor was previously shown to be the predominant mechanism The Position and Dynamics of Glutamate Receptors Measured by Bright- for modulating the fluorescence of GCaMP6. We present a thorough analysis ness- and Size-Equalized Small Quantum Dots of the one-photon and two-photon properties of several red GECIs and use 1,2 3 1,2 3 Sang Hak Lee , Phuong Le , Yeoan Youn , Andrew M. Smith , this data to determine the main factor(s) contributing to the increase in fluo- 1,2 Paul R. Selvin . rescence upon binding Ca2þ, particularly under two-photon excitation. In 1Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2 3 most cases we observe both the change of the maximum two-photon cross Center for Physics of Living Cells, Urbana, IL, USA, Bioengineering, section and the shift of equilibrium between the neutral and anionic forms University of Illinois at Urbana-Champaign, Urbana, IL, USA. of the chromophore upon binding Ca2þ. Quantitative analysis of the shapes Quantum dots have been widely used for biomedical imaging due to their of the two-photon absorption spectra provides us with clues regarding changes brightness and extraordinary photo-stability. Recently, they have also been in the electrostatic environment of the chromophore and can potentially direct used to study neuronal receptors. However, commercially available quantum þ mutagenesis efforts towards creating red GECIs with a larger Ca2 -depen- dots (cQD) tend to be large, 25 35 nm in diameter, which is problematic dent fluorescence increase. because steric hindrance prevents their entry into the 30 nm synaptic cleft. In this study, we develop new and small quantum dots that are brightness- 3313-Pos Board B521 and size-equalized (12 nm in hydrodynamic diameter), called BSE-QD. We Potassium Channel-Based Two Component Optogenetic Tool for Silencing developed two different-colors BSE-QDs (605 nm and 725 nm) to label and of Excitable Cells observe two different neuronal receptors, AMPARs and NMDAR, in the syn- Yinth Andrea Bernal Sierra1, Benjamin Rost2,3, Silvia Oldani2,3, apses in live neurons. They were each able to enter the synaptic cleft, allowing Franziska Schneider-Warme4, Reinhard Seifert5, Dietmar Schmitz2,3, us to observe and measure diffusion. We find that the receptors were in con- Peter Hegemann1. strained ‘‘nanodomains’’. Through cross-correlation of the two receptors, we 1Experimental Biophysics, Institute for Biology, Humboldt-Universit€at zu found that the nanodomains of AMPAR and NMDAR are colocalized in the Berlin, Berlin, Germany, 2Neuroscience Research Centre, range of 100 nm 400 nm, implying that excitatory synapses need their coop- Universit€atsmedizin Berlin, Berlin, Germany, 3German Center for erative interaction to be effective. Neurodegenerative Diseases (DZNE), Berlin, Germany, 4Institute for Experimental Cardiovascular Medicine, University Heart Centre Freiburg, 3311-Pos Board B519 Bad Krozingen, Freiburg, Germany, 5Abteilung Molekulare Neurosensorik, Diffusive Dynamics of NMDA Receptors in Live Neurons using Super- Center of Advanced European Studies and Research (Caesar), Bonn, Resolution Imaging and Tracking Germany. Chaoyi Jin1, Sang Hak Lee1, Phuong Le2, Yeoan Youn1, Pinghua Ge1, Opsin-based optogenetic tools still lack a light-gated potassium channel for Okunola B. Jeyifous3, Andrew M. Smith2, Sheldon Park4, William N. Green3, cell inhibition. Silencing of excitable cells had been achieved so far by the Paul R. Selvin5. use of light driven pumps for protons and chloride as well as light-gated chlo- 1Center for Biophysics and Quantitative Biology, Center for Physics of ride channels. Unfortunately, changes on pH or Cl- concentration could lead Living Cells, University of Illinois, Urbana-Champaign, Urbana, IL, USA, to undesirable effects on synaptic transmission limiting the use of these tools 2Department of Bioengineering, Micro and Nanotechnology Laboratory, for neuronal silencing. We have developed a potassium channel-based inhib- University of Illinois, Urbana-Champaign, Urbana, IL, USA, 3Department of itory tool combining the small cAMP-gated potassium channel SthK (from Neurobiology, University of Chicago, Chicago, IL, USA, 4Department of Spirochaeta thermophile) and a photoactivated adenylyl cyclase. From all Chemical and Biological Engineering, SUNY, Buffalo, Buffalo, NY, USA, the PACs tested we found that bPAC (from the bacterium Beggiatoa), and 5Department of Physics, Center for Physics and Living Cells, University of TpPAC (from bacterium Turneriella parva) produced the best results. Charac- Illinois, Urbana-Champaign, Urbana, IL, USA. terization of both optogenetic-tools in ND7/23 cells showed that application N-methyl-D-aspartate receptors (NMDA receptors or NMDARs) are ionotropic of short blue light pulses to cells transfected with SthK and a PAC, produced glutamate receptors (iGluRs) located on the membrane of neuronal cells. Their high-amplitude potassium outward currents that are dependent of the light in- location and dynamics are strongly associated with synaptic plasticity. Howev- tensity. The TpPAC-based construct needed 10 times more light to reach the er, we know little about the distribution between extra- and intra-synaptic loca- maximum current compared to bPAC. However, this is an advantage in bio- tions and dynamics for two important NMDAR subunits, GluN2A and logical systems where a lower production of cAMP is needed or where the GluN2B. Results have been presented using large quantum dots (20 nm in ubiquitous phosphodiesterase activity is very low. Two construct configura- diameter), making it inconclusive about their synaptic localization. Here we tions were made. In the first configuration the SthK channel and the PAC use three dimensional super-resolution fluorescent microscopy to quantify are expressed as a fused protein (PAC-SthK) allowing localized expression the diffusion properties of these two subunits by tracking them with conjugated of cAMP at the plasma membrane. In the second configuration both proteins organic dyes (4 nm in diameter) and small quantum dots (sQDs, 10 nm in are separated by a P2A site (SthK-P2A-PAC) and the PAC is expressed in the diameter). More than 70% of these two subunits were found to diffuse in con- cytoplasm. The fused constructs produced an increase on the current’s latency strained sub-synaptic regions with similar diffusion constant regardless of and a decrease on the current density. Nevertheless, both configurations were development stage of neurons. This suggests that majority of both GluN2A- capable of inhibiting neuronal action potential firing in cultures of hippocam- and GluN2B-containing NMDARs are synaptic in early and late stage of pal neurons. Cyclic nucleotide-gated potassium channel - photoactivated cyc- neuronal development, their diffusion constant is a function of location rather lese based constructs are a very promising tool for acute optogenetic than subunit type. inhibition.

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3314-Pos Board B522 3316-Pos Board B524 Improved Microbial Rhodopsins for Ultrafast Red-Shifted Optogenetics Solar Cell Nanowires as Approach for Single Cell Direct Activation Thomas Mager1, David L. de la Morena2, Vitaly Shevchenko3, Jann I. Harberts1, Aune Koitm€ae1, Robert Zierold1, Cornelius Fendler1, Verena Senn4, Phillip G. Wood1, Johannes J. Letzkus4, Valentin Gordeliy3, Irene Fernandez-Cuesta1, Gabriele Loers2, Maria Thereza Perez3, Tobias Moser2, Ernst Bamberg1. Christelle Prinz4, Gaute Otnes4, Magnus Borgstro¨m4, Heiner Linke4, 1Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt, Robert H. Blick1. Germany, 2Institute for Auditory Neuroscience and InnerEarLab, University 1Center for Hybrid Nanostructures, University of Hamburg, Hamburg, Medical Center Go¨ttingen, Go¨ttingen, Germany, 3ICS-6: Structural Germany, 2Center for Molecular Neurobiology Hamburg, University Biochemistry, Research Centre Julich,€ Julich,€ Germany, 4Neocortical Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3Division of Circuits Lab, Max Planck Institute for Brain Research, Frankfurt, Germany. Ophthalmology, Lund University, Lund, Sweden, 4Solid State Physics, Lund The discovery of the light-gated ion channels (Channelrhodopsins, Nagel et University, Lund, Sweden. al., 2002; Nagel et al., 2003), together with the well-known light driven ion Novel fabrication techniques for nanoscale solar cells and establishment of pumps, paved the way for the optogenetic control of electrically excitable hybrid junctions between semiconductors and biological systems offer innova- cells. Channelrhodopsins are generally used for neuronal photoactivation tive approaches for single cell direct activation and sensing. We use indium (Boyden et al., 2005). Light-driven ion pumps, so far, have solely been phosphide solar cell nanowires and cerebellar granule cells to investigate used for the optogenetic silencing of neurons (Zhang et al., 2007). We, for such a system. Here, vertically aligned nanowires serve as culturing substrate the first time, show light-driven spiking in neurons via a light-driven ion for the neurons. We demonstrate that a neural network is formed on the nano- pump (Shevchenko et al., 2017). This was achieved by the heterologous, wires. Scanning microscope images show that the cells and axons stay on top of neuronal expression of the light-driven, inward proton pump Xenorhodopsin the tips caused by the high nanowire density. Cross sections via focus ion beam from the nanohaloarchaeon Nanosalina. We demonstrate that the light- reveal a slight wrapping of the cell membrane around the nanowire tips. Our driven inward transport of a single proton by each NsXeR molecule is suffi- patch clamp experiments show that the electrophysical properties such as firing cient to trigger action potentials, thereby enabling minimal invasive experi- of action potentials remain similar compared to measurements on planar refer- ments. Moreover, we present a unifying concept for the construction of ence substrates. The p-n junctions are build-in vertically and thus form an elec- Channelrhodopsins with ultrafast closing kinetics. We focused our functional trical contact at the neural cell and the underlying substrate. This opens up for analysis on the red-light activated Chrimson (Klapoetke et al., 2014) variants, possibly using both pin and nip designed nanowires for both hyper- and depo- because red light minimizes light scattering and phototoxicity in tissues. larization of the cell. The novel Chrimson variant vf-Chrimson (toff = 2.7 5 0.3 ms) is at least t 5 as fast as Chronos (Klapoetke et al., 2014) ( off = 3.6 0.2 ms), which is 3317-Pos Board B525 the fastest Channelrhodopsin to date. At the same time it has the same spectral Nanoparticle-Mediated Heating of Cellular Membrane Induces Changes peak as Chrimson wt (lmax=590 nm), which is the most red-shifted Channelr- in Membrane Capacitance and Ionic Conduction hodopsin to date. The novel Chrimson variant enable neural photostimulation Bernardo I. Pinto1,2, Joa˜o L. Carvalho-de-Souza2, Francisco Bezanilla2. with high temporal precision up to the intrinsic limit of fast spiking 1Centro Interdisciplinario de Neurociencias, Santiago, Chile, 2Department of parvalbumin-positive interneurons ( 300 Hz). Moreover, we demonstrate Biochemistry and Molecular Biology, The University of Chicago, Chicago, the remote control of auditory signalling by ultrafast red-shifted optogenetics IL, USA. in vivo. The novel microbial rhodopsin variants facilitate neuroscience The ‘‘optocapacitive’’ effect is the generation of fast transient capacitive cur- research and potential medical applications of optogenetics in hearing rents due to a fast change in membrane temperature. This effect can be used restoration. to generate action potentials in dorsal root ganglia (DRG) neurons using milli- second to microseconds laser pulses of mJ to nJ energy, respectively. We used 3315-Pos Board B523 carbon nanoplatelets (CNP) as light-to-heat transducers in order to study Focused Ultrasound Activates Task Potassium Channels, Increases Mem- heating-induced currents in HEK293 cells and DRG neurons under whole brane Capacitance, and Modulates Action Potential Waveform and Firing cell voltage clamp. To that end we delivered 6 mJ (1.2 mW during 5 ms) of a Properties in Hippocampal Brain Slices 405 nm laser to CNP touching the cell of interest. In HEK293 cells in the Martin L. Prieto1, Daniel V. Madison1, Butrus T. Khuri-Yakub2, absence of permeant ions, heating induced transient inward currents at voltages Merritt Maduke1. from 100 to þ100 mV, consistent with a reversal potential of þ120 mV ex- 1Molecular and Cellular Physiology, Stanford University, Stanford, CA, pected from a pure optocapacitive effect. In neurons the heating-induced cur- USA, 2E. L. Ginzton Laboratory, Stanford University, Stanford, CA, USA. rents measured show a fast component associated with the optocapacitive Focused ultrasound is a promising technology for modulating brain activity effect and a second, slower component that follows the time course of the tem- in vivo. To guide the development of this new technology, we seek to under- perature change. The current to voltage (I-V) curve of this second component stand the physical and biophysical mechanisms underlying ultrasound’s ef- follows the I-V curve of the dominant ionic conductance. The I-V curve shows þ fects on action potential firing. We measured the effects of ultrasound at 25 a reversal potential of þ40 mV when the Na conductance is present and of þ and 43 MHz in CA1 pyramidal neurons of rat hippocampal brain slices us- 60 mV when the K conductance is present. The time course and voltage ing patch-clamp recording. Ultrasound has several effects on the electro- dependence of this light-activated current suggest that it is generated by the physiological properties of CA1 pyramidal neurons. First, ultrasound conductances change of channel’s open pore due to increased temperature, hyperpolarizes resting potential by 2-0.5 mV. This effect was not sensitive and not due to membrane damage. Our results indicate that the heating of to inhibitors of the major voltage- or calcium-dependent Kþ channels ex- the cell membrane generates an optocapacitive current followed by an increase pressed in CA1 pyramidal neurons, but was eliminated by bupivacaine, an in ionic conductances. Both mechanisms play a role in the applicability of this inhibitor of two-pore-domain Kþ channels of the TASK family. Second, technique for the stimulation of neurons. Support: NI-R01GM030376, NI- ultrasound increases membrane capacitance by several pF. The time course R21EY027101 of the capacitance change is fast relative to the membrane time constant at rest, such that there is a transient depolarization at the onset of the 3318-Pos Board B526 ultrasound stimulus, and a transient hyperpolarization at the offset. Ultra- Magnetogenetic Proteins: Mechanism and New Candidates sound also compresses the action potential waveform, decreasing its height Guillaume Duret1, Sruthi Polali1, Martin A. Bell1, and width, and increases the latency to the first spike in response to a depo- Constantine N. Tzouanas2, Jacob T. Robinson1. larizing current step. When the baseline action potential firing rate is low, 1Electrical and Computer Engineering, Rice University, Houston, TX, USA, ultrasound increases the interval between spikes. However, if action poten- 2Bioengineering, Rice University, Houston, TX, USA. þ tial frequency is increased by the application of K channel inhibitors, ul- The wireless stimulation of specific neurons using magneto-sensitive chan- trasound decreases the interval between spikes. We are currently nels will improve the exploration of neural networks, the mapping of regions investigating to what extent the effects on action potential waveform and of the brain that cannot be reached with optogenetic tools, and the stimula- firing properties can be explained by the increased capacitance and TASK tion of neurons in freely moving animals. Significant progress has been conductance, or whether effects of ultrasound on other channels are achieved by engineering proteins that couple TRP channels to the biogenic involved. Notably, the size of the capacitance change suggests that ultra- magnetic nanoparticle ferritin. Ferritin allows for a fully genetically en- sound may cause sufficient membrane tension to mechanically modulate coded magnetogenetic tool that doesn’t require additional injections of ion channel activity. magnetic nanoparticles. Channel gating occurs when these constructs are

BPJ 8745_8748 670a Wednesday, February 21, 2018 stimulated by alternating or static magnetic fields. It has been argued that 3321-Pos Board B529 the heat produced by ferritin in an alternating magnetic field is too weak Hopping-Mode Scanning Ion-Conductance Microscopy Resolution during to activate the nearby thermoreceptor and that the mechanical force between Synaptic Imaging adjacent ferritin is at least eight orders of magnitude too weak for mechan- Jake H. Rabinowitz1, Krishna Jayant2, Martin A. Edwards3, Ozgur Sahin4, ical stimulation. Recently we have proposed that a change in the magnetic Rafael Yuste4, Kenneth L. Shepard1. entropy of biogenic nanoparticles generates sufficient heat to gate 1Electrical Engineering, Columbia University, New York, NY, USA, temperature-sensitive ion channels. We believe that this magnetocaloric ef- 2Electrical Engineering & Biological Sciences, Columbia University, New fect is the underlying mechanism explaining the functional biogenic magne- York, NY, USA, 3Chemistry, University of Utah, Salt Lake City, UT, USA, togenetics proteins that respond to slowly varying magnetic fields. Using 4Biological Sciences, Columbia University, New York, NY, USA. this effect as a working hypothesis, we propose to improve the existing mag- Scanning ion conductance microscopy (SICM) is a non-contact scanning-probe netogenetic channels, and to develop new, more sensitive constructs that topography imaging technique, in which current measured between an respond to low frequency magnetic stimuli. electrolyte-filled nanopipette and an external bath electrode is monitored through closed-loop feedback to maintain a constant distance between the 3319-Pos Board B527 probe and the sample. Topography is mapped by scanning this nanopipette Magnetogenetics for Drosophila laterally across the sample in either direct-current or alternating-current Charles E. Sebesta1, Guillaume Duret2, Constantine N. Tzouanas1, mode. Both modes suffer from limitations when imaging in the presence of Jacob T. Robinson2. large morphological undulations, such as with neuronal bodies. The hopping- 1Bioengineering, Rice University, Houston, TX, USA, 2Electrical and probe scanning (HP-SICM) mode, however, is ideally suited for imaging Computer Engineering, Rice University, Houston, TX, USA. neuronal topography. In HP-SICM, the pipette current reduces upon approach- Magnetically sensitive genetically encoded channels enable remote activa- ing the sample, and height is recorded when the current reaches a pre- tion of specific neural networks deep within the brain of freely moving an- determined threshold (i.e. a fraction of the far-away value). The probe then re- imals. Recent progress coupling various TRP channels and the biogenic tracts by a distance larger than the tallest structural feature, moves laterally by a magnetic nanoparticle ferritin in mammalian cells shows significant promise pre-defined step, and reapproaches the sample. Here, using detailed finite- for creating such ‘‘magnetogenetic’’ technology, but this approach relies on element simulations incorporating the Poisson-Boltzmann and Nernst-Planck ferritin assembly by endogenously expressed subunits. This reliance on formalisms, we examine the factors that affect HP-SICM resolution when im- endogenously expressed ferritin subunits makes it difficult to study the effi- aging synapses in live hippocampal cultures. We find that currents originating cacy of ferritin formation and iron loading and prevents transfer of the tech- from the imaged membrane can affect the recorded sample height, thereby nology developed with mammalian ferritin to species like Drosophila that introducing artifacts that can range from a few nm to 100nm. This feature express divergent ferritin proteins. In addition, magnetogenetics in of HP-SICM resolution critically depends on the set threshold value, pipette Drosophila provides two key advantages for optimizing the magnetic sensi- concentration and pipette geometry but provides an opportunity to extracellu- tivity of the proteins: 1) because Drosophila ferritin is secreted and circu- larly map activity from small dendritic spines and pre-synaptic terminals lates throughout the animal within hemolymph, the particles can be more (diameter < .5um) that are practically inaccessible with conventional electro- easily isolated for characterization, 2) once isolated, we can bind these physiology. Last, we explore the current-clamp response near the membrane ferritin nanoparticles to other proteins with minimal reduction of iron and find that pipette geometry and local ion gradients affect the voltage loading. By developing magnetogenetic channels for Drosophila with an response. extracellular binding site for functionalized ferritin or superparamagnetic Funding: U. S. Army Research Laboratory and U. S. Army Research Office. nanoparticles, we can study the responses of the channels to low frequency Contract number W911NF-12-1-0594 (MURI). magnetic stimulation with fewer unknowns and create driver lines express- ing functionalized magnetogenetic ferritin nanoparticles. Drosophila also enable this technology to be rapidly tested in various cell types by taking 3322-Pos Board B530 advantage of large depositories of UAS/Gal4 drosophila lines. We believe Improvements in Action Potential Recording in Human Stem Cell-Derived developing these magnetogenetic channels and functionalized biogenic Neurons using Dynamic Clamp magnetic nanoparticles will create a testbed for behavioral studies in Mark W. Nowak1, Brian K. Panama1, Sanjot Singh1, Brandon Franks1, Glenna C.L. Bett2, Randall L. Rasmusson2. drosophila and enable researchers to rapidly test responses to stimuli 1 2 throughout the animal while gaining a deeper understanding of the physics Cytocybernetics, Buffalo, NY, USA, Jacobs School of Medicine and behind magnetogenetics. Biomedical Sciences, SUNY, Buffalo, NY, USA. Human stem-cell derived (hiPSC) neurons are a model for studying neuronal 3320-Pos Board B528 electrophysiological properties. A major limitation when recording Magnetothermal Deep Brain Neuromodulation in Awake, Freely Moving from neuronal hiPSCs is the significantly depolarized resting membrane po- tential, which results in the inability to evoke stable action potentials (APs). Mice þ Rahul Munshi, Shahnaz Qadri, Arnd Pralle. This is a result of the lack of K leak current. To correct this problem, we used dynamic clamp to electronically express, in real time, an outward recti- University at Buffalo, Buffalo, NY, USA. þ Methods to remoted activate or silence specific neuronal activity in-vivo are fying K leak current in neuronal hiPSCs to generate stable neuronal resting valuable tools for unraveling the connection between behavior and brain cir- potentials. Neuronal hiPSCs (Cellular Dynamics, WI) were cultured accord- cuits. If translatable to human application they also hold great potential as ingtothemanufacturer’sinstructionsandused4-14daysafterplating. diagnosis and treatment tools. We explore magnetothermal activation and Standard electrophysiology voltage clamp, using whole-cell rupture or Am- silencing of neuronal activities by local heating around superparamagnetic photericin B perforated patch techniques, was used. Dynamic clamp was im- nanoparticles upon exposure to external alternating magnetic fields heated. plemented via a Cybercyte System (Cytocybernetics, NY). In current clamp mode, APs were triggered by stimulating with 0.25-0.5 nA pulses for 1.5 ms The nanoparticles bound to the neuronal cell membrane heat the membrane þ sufficiently to activate TRPV1 which in turn depolarized the cell to evoke at 0.5 Hz. The outwardly rectifying K leak current was modeled using the action potentials. Other channels are used to hyperpolarize the membrane Goldman-Hodgkin-Katz equation (IK-GHK). In the absence of electronically to silence neurons. The approach is tetherless and minimally invasive apart added IK-GHK, the resting membrane potential ranged from -30 to 45 mV from delivering the nanoparticles. The application requires solving a series and no stable APs, with pacing, could be recorded. The electronic addition of nanoscience and biophysics challenges. We will discuss our solutions to of IK-GHK (via the Cybercyte) set the resting potential to more hyperpolar- these challenges and present the application in controlling deep brain cir- ized values ( 55 to 70 mV), resulting in the ability to evoke stable APs. In addition to adding an electronic IK-GHK, we also added an electronic cuits in mice. We demonstrate activation of circuits by coupling the heating þ with heat-sensitive cation channels. Our results show robust that motor cor- INa. We blocked endogenous Na channels with TTX, and electronically tex and striatum neurons can be activated and lead to distinct motor behav- addedbackINaþ, along with IK-GHK, which restored our ability to record iors, and that this is repeatable and robust. In addition, we demonstrate evoked APs. This was due largely to changes in the degree of INaþ inactiva- magnetothermal silencing of neurons in the Ventral Tegmental Area, lead- tion. Electronic addition of IK-GHK and INaþ shows the role of non-linear ing to abolishing of a place preference. We evaluated the duration and repe- leak currents in stabilizing neuronal responses to stimulation via tition rate of alternating field dosage required to generate memory retention changes INaþ inactivation and improving the functional responses of hiPSC of the demonstrated aversion. neurons.

BPJ 8745_8748 Wednesday, February 21, 2018 671a

3323-Pos Board B531 Preliminary test included recording of impedance spectroscopy of the cell Application of Electrochromic Thin Films for Electrophysiology culture medium (CCM), 2.5X105 cells/ml concentration, and an increasing Felix Alfonso. concentration of SPIONS (25, 50, 100, 200 and 300 mg/mL). In the experi- Stanford University, Stanford, CA, USA. mental phase, impedance spectroscopy for CCM, mixture of 2.5X105 cells/ Electrodes have been the gold standard for investigating neuronal signaling ml and 25 mg/mL SPIONS, adhesion and spreading of cells were recorded due to their high sensitivity and temporal resolution. However, classical every 2-hour interval within 8 hours for two days. The experimental phase electrodes are invasive and limited by the number of addressable sites. was repeated using other mixtures of cells with an increasing concentration Recently, optical methods have become a sensible approach to measure of SPIONs. the electrical activities of neurons by using electrochromic materials that Result showed that there was no significant difference on the impedance of transduces the electrical signal into an optical signal. In the present CCM overtime. Meanwhile, there was a change in impedance for the work, electrochromic thin films such as Prussian blue, iridium oxide and concentration of cells, different SPIONs concentration and mixtures of PEDOT were investigated to determine which had the best optical properties cells and SPIONS overtime. Also, confirmatory and viability test were to serve as a sensor for the measurement of extracellular action potentials. performed. These materials are known for their biocompatibility, insolubility in The above findings substantiate the importance of thorough investigation of the water, and stability in neutral media. We hypothesized that the electrical interaction of SPIONS to the culture medium and high throughput screening of potential of excitable membranes will modulate the spectral properties the toxicity effect of SPIONs to neuronal cells. The findings may be used to of the materials which, in turn, could be detected by a differential photo- expand basic knowledge on the morphological characteristics of neuronal cells diode detector. The materials were electrodeposited onto indium-tin- and physico-chemical aspect of SPIONs in the academe and research. Finally, oxide-coated glass slides and their optical properties characterized. As a this would also lead to further characterization of neuronal activities for the model for electrophysiology measurements, a clone line of modified HEK development of neuro-biosensors for early detection of neurological diseases 293 cells was used that stably expresses Nav1.3 and KIR2.1 ion channels and other clinical application. and generates spontaneous electrical action potentials. Herein, we demon- strate the ability of the films to detect the extracellular action potentials 3326-Pos Board B534 of HEK 293 cells and evaluate their potential for imaging a network of Characterizing Vesicles using SPR neuronal cells. Ann-Sofie Cans, Hoda Fathali, Thomas Olsson, Fredrik Ho¨o¨k. Chemistry, Chalmers University of Technology, Goteborg, Sweden. 3324-Pos Board B532 Secretory vesicles are storage vehicles of neurotransmitters and hormones in Partial Treatment of In Vivo Single Axons by Mounting a Microfluidic secretory cells. Despite their important biological role, their complex compo- Device Directly sition and release mechanisms are not yet fully understood. To complement Anthony Fan, Alireza Tofangchi, Taher Saif. amperometry measurements and fluorescence-based methods to characterize University of Illinois at Urbana-Champaign, Urbana, IL, USA. neurotransmitter content of secretory vesicles, we here introduce a new Mixing happens slowly under flow conditions with a low Reynolds number method using surface plasmon resonance (SPR) to probe the intravesicular when viscous effect is dominant. This allows multi-channel perfusion content of isolated intact large dense core vesicles from chromaffin cells to flow laminarly even without any physical separations. Furthermore, the and present how using this technique offers real time measurement of vesicle individual flow width, and therefore position, is governed by the respec- content release when subjected the isolated granules to high osmotic pressure. tive volumetric flow rate, enabling flow focusing. This has been used in The observations from these SPR measurements confirm recent ampero- a variety of biological context, including single cells partial treatment metric measurements at live chromaffin cells that neurotransmitters are studies called PARTCELL. PDMS is the most common material for making released from vesicles when subjected to high osmotic pressure. Further, a microfluidics devices. Typically a slab of PDMS with channels patterned comparison of volume exclusion measurements using SPR to the physical di- is plasma-treated such that it can form bonds with a glass slide to seal mensions of the dense core vesicle size as determined by transmission elec- the channels. Suspendable samples (usually dissociated cells) are subse- tron microscopy imaging analysis, the molecular dimension of soluble quently flown into the channels and let adhere to the glass slide before an volume versus the solid volume of membrane and dense protein matrix in experiment commences. This process severely limits the control over sample intact vesicles was determined. placement and sample selection. Here, we propose a novel direct mounting method that enables sample placement and preparation to precede device 3327-Pos Board B535 sealing, thus extending the use of partial treatment to study small living Measurement of Fluid Movement in Scala Vestibuli organisms. Eli Elyas1, William E. Brownell2, Anders Fridberger1. The device was used to treat single axons partially in their proximal 1Clinical and Experimental Medicine, Linko¨ping University, Linko¨ping, segments with Y-27632, a known axonal tension disruptor through Sweden, 2Otolaryngology - Head & Neck Surgery, Baylor College of inhibiting the ROCK pathway. Such treatment allowed us to reduce the Medicine, Houston, TX, USA. tension level in the axons being perfused. Once tension was reduced, The displacement of cochlear structures is now measured with sub- we observed simultaneously a reduction in vesicle density at the distal nanometer precision, but we lack detailed knowledge of the fluid move- presynaptic terminal (PT) of the axons. Switching off the Y-27632 flow ments that drive the structural motion. Here we apply the ‘‘nano-ear’’ can reverse this process and led to the restoration of vesicles at the method in an attempt to make force measurements that match the spatial PTs. Note that the distal PTs were not subjected to any treatment (drug and temporal scales of the displacement measures. The goal is to charac- flow only at proximal segments), thus suggesting that tension along the terize cochlear fluid dynamics under conditions that approach in vivo mea- axons directly correlates with the vesicle density at the PTs. Such funda- sures. We have measured fluid movement in scala vestibuli at while mental experiments cannot be performed without using the direct mounting stimulating the ear with sound. A small orifice was made at the cochlear method. apex of an excised temporal bone mounted onto a holder filled with cell cul- ture medium, while the access to the eardrum was maintained for sound 3325-Pos Board B533 stimulation. 50nm gold nanoparticles were introduced into the perilymph High-throughput Cell Screening for Spions Studies using Impedance Spec- and were trapped with optical tweezers. A digital video camera was used troscopy to detect the vibration of trapped particles, and their 3D movement was sub- Sonia Tan, Ebrahim Ghafar-Zadeh. sequently analysed using an in-house software. The displacement of Reiss- York University, Toronto, ON, Canada. ner’s membrane and outer hair cells was measured by time-resolved Superparamagnetic Iron oxide nanoparticles (SPIONs) being clinically confocal imaging. In the conducted experiments we detected a displacement approved had elevated the optimism of majority in terms of its applicability gradient in scala vestibuli: fluid movement was smaller closer to the Reiss- in both research and clinical domain. Along with this optimism is the use of ner’s membrane, increased as we moved away until the distance of around impedance spectroscopy which provides a non-invasive, fast, and convenient 150mm and then decreased again. Nanoparticle movement peaked at the fre- to use for wide range of biological and chemical applicability. quencies between 160 and 200Hz. The detected peak corresponded to the This study focused on high-throughput test for SPION toxicity using imped- maximal displacement of Reissner’s membrane. As expected, the movement ance spectroscopy of mouse neuroblastoma cells (N2A). The platform con- of the nanoparticle scaled with the amplitude of the acoustic stimuli. Almost sisted of an array of ITO microelectrode incorporated with microfluidics and all models of fluid dynamics in a cochlea assume acoustical transparency connected to impedance spectroscopy system. of the Reissner’s membrane. Our findings challenge this assumption and

BPJ 8745_8748 672a Wednesday, February 21, 2018 require the models be revised. Next, we will use this technique to measure the role of microglial microvescicles (MVs) release in mediating their fluid motion in scala media, the fluid compartment that contains the struc- response to s-GO. tures that directly stimulate the hair cells. 3330-Pos Board B538 3328-Pos Board B536 The MEMS mHammer: Impacting Neuroscience One Cell at a Time Graphene Oxide Nanosheets Target Excitatory Synapses in the Hippocam- Jennifer Walker1, Luke Patterson1, Evelyn Rodriguez-Mesa2, John Foster2, pus: Reversible Down Regulation of Glutamate Neurotransmission In-Vivo Adele Doyle1, Kimberly Foster1. Rossana Rauti1, Manuela Medelin2, Neus Lozano3, Denis Scaini1, 1UCSB, Santa Barbara, CA, USA, 2Owl Biomedical, Inc., Goleta, CA, USA. Kostas Kostarelos3, Laura Ballerini1. Approximately 1.7 million people sustain Traumatic Brain Injuries (TBI) 1Neurobiology Department, SISSA, Trieste, Italy, 2Life Science Department, annually, whether through physical impacts on sports fields or percussive University of Trieste, Trieste, Italy, 3School of Medicine and National blasts on the battlefield. Yet while the response to TBI-level forces and sub- Graphene Institute, University of Manchester, Manchester, United Kingdom. sequent recovery has been extensively studied on the tissue level, less is Neurological disorders significantly impact the quality of life of patients. known on the cellular level. Investigating the damage and recovery mecha- Promising therapeutic paradigms take advantage of nanomaterials to manipu- nisms of NSCs (neural stem cells) will improve the current understanding late the activity of target cells against these diseases. Graphene offers a range of neuroplasticity and may lead to new therapies, as these cells are vital to of unique physicochemical properties that can be tuned for biomedical needs. the regenerative capabilities of the brain. Current systems for applying forces In cultured hippocampal neurons, small graphene oxide nanosheets (s-GO) to individual cells are often limited to low force (sub-mN) and strain-rate (1- were reported to interfere selectively with excitatory synapses, sizing down 10 s1) ranges, and all are low throughput (tens of cells per experiment), glutamatergic activity. Glutamate is the main excitatory neurotransmitter in making them ill-suited for gathering enough data for biologically-relevant the central nervous system, and apart from neurodegenerative diseases, analysis. We have developed a microfluidic MEMS device, the mHammer, growing evidence suggests that glutamate is involved in psychiatric and to subject individual cells to TBI-relevant impacts with high throughput, al- neurological disorders. Localized targeting of the glutamatergic system is lowing us to investigate NSCs’ responses to impact as a function of force and attractive objective, but a mechanistic understanding of s-GO interference duration. The mHammer can impact up to a rate of 80,000 cells per hour at with synapses and the translation in vivo of these evidences are lacking. strain rates ranging from 20,000 to 200,000 s1 and 30 to 60% strain. Using a Here we used cultured neurons and acute hippocampal slices to demonstrate simple Hertz contact model, we estimate the force generation to be in the 1- that s-GO flakes directly impair synaptic vesicle release in excitatory synap- 100 mN range, which falls within characteristic TBI parameters. Initial exper- ses, inducing a vesicle depletion that results, in the long term, in a down regu- iments explored the effects of impact on NSCs at various strain rates and lation of the glutamatergic activity, leaving the inhibitory GABAergic system demonstrated inhibited proliferation commensurate to the rate of strain intact. We further tested in vivo the injection of s-GO in the hippocampus of applied. Further, the influence of strain rates on induced cell necrosis and juvenile rats and patch-clamped single neurons to measure glutamatergic syn- apoptosis is being explored as well as the effect of impact on NSC differen- aptic activity from brain slices isolated after 48 and 72 hours from brain tiation. In conclusion, the mHammer’s capability of impacting thousands of injections. 48h after surgery, acute hippocampal slices isolated from sGO- cells at controlled TBI-relevant force magnitudes will open doors to unprec- treated rats showed a significant reduction in glutamatergic synaptic edented analysis of damage and recovery mechanisms of NSCs and other frequency, while GABAregic one was unchanged, in respect to controls neural cells. (saline injections). These effects were reversed in slices isolated 72h post in- jections. Tissue reactivity, quantified by astrocyte and microglia labelling, was 3331-Pos Board B539 equal or even lower than in controls. s-GO ability to target and silence gluta- Epileptiform Activities in Cultured Human IPSC-Derived Neuronal matergic synapses may have special importance for experimental neurosci- Networks ence research, because in specific brain regions such an activity may Ikuro Suzuki, Aoi Odawara, Naoki Matsuda, Yuto Ishibashi, Remi Yokoi. contribute to neuropathologies. Tohoku Institute of Technology, Sendai, Japan. The functional network of human induced pluripotent stem cell (hiPSC)- 3329-Pos Board B537 derived neurons is a potentially powerful in vitro model for evaluating drug Graphene Oxide Nanosheets and Neural System: From Synaptic Modula- toxicity. Epileptiform activity is one of phenomena in neuronal toxicology. tion to Neuroinflammation To evaluate the dynamics of epileptiform activities and the effect of anti- Mattia Musto1, Rossana Rauti1, Neus Lozano Valdes2, Clara Ballerini3, convulsant drug in cultured hiPSC-derived neurons, we used the high- Loredana Casalis4, Maurizio Prato5, Kostas Kostarelos2, Laura Ballerini1. throughput multielectrode array (MEA) system, where we simultaneously re- 1ISAS, Trieste, Italy, 2University of Manchester, Manchester, United cord extracellular potentials for 16 channels per well across 24-well plates. Kingdom, 3University of Florence, Firenze, Italy, 4ELETTRA Synchrotron We examined chemically evoked epileptiform activity. Epileptiform activities Light Source, Trieste, Italy, 5University of Trieste, Trieste, Italy. were induced by 4-Aminopyridine (4-AP), pilocarpine, chlorpromazine, and Graphene possess extraordinary properties and is extensively explored for pentylenetetrazole (PTZ) . The number of synchronized burst firings were biomedical applications. In neurology, such developments are focusing on increased in a concentration dependent manner at 4-AP, Pilocarpine, and novel neuro-devices, including regenerative interfaces or drug delivery vec- Chlorpromazine administration. On the other hand, the duration and spikes tor systems, based on few-layer graphene sheets. Any further exploitation in in a synchronized burst were increased at PTZ administration. Phenytoin the central nervous system of biomedical devices based on these 2D planar used in anti-convulsant drug suppressed electrophysiological activities. From nanostructures requires deep understanding of their interactions with the these results, we suggest that the electrophysiological assay in cultured human neuronal tissue. We recently reported the ability of graphene oxide nano- iPSC-derived neuron using MEA system has the potential to investigate the sheets (s-GO) to interfere specifically with synapses and to alter glia reac- neuronal toxicity in drug screening. tivity without affecting cell viability, in cultured hippocampal networks. While the specificity of the synaptic alteration might pose the basis for ex- 3332-Pos Board B540 ploiting s-GO for target theranostics applications, the potential tissue reac- 3D Micro Scaffolds for Tailor-Made Three-Dimensional Neural Network tivity raises concerns from a toxicity point of view. To tailor s-GO safe Studies developments we need to model in vitro glial cell responses, and in particular Cornelius Fendler1, Christian Denker2, Gabriele Loers3, Jann I. Harberts1, microglial reactivity in complex tissues challenged by chronic s-GO delivery Robert Zierold1, Markus Munzenberg€ 2, Robert H. Blick1. at high doses. In this work, we design long-term exposure to s-GO in 3D tis- 1Center for Hybrid Nanostructures (CHyN), Universit€at Hamburg, Hamburg, sue cultures. We use mouse organotypic spinal cultures that are ideally suited Germany, 2Institute of Physics, University of Greifswald, Greifswald, for studies involving long-term interference with cues at controlled times and Germany, 3Center for Molecular Neurobiology Hamburg (ZMNH), concentrations. In this preparation, the normal presence, distribution and University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany. maturation of anatomically distinct classes of neurons and glial cells are pre- One of the most important scientific tasks of our time is to understand the com- served to reconstruct the basic cytoarchitecture of spinal cord segments. We plex activities in neural networks (for example in the US BRAIN Initiative and address the impact of treatments with s-GO on premotor synaptic activity the EU Human Brain Projekt). Until recently, it was difficult to simulate neural monitored by single cell patch clamp recordings from ventral interneurons. networks at laboratory scale, since conventional circuits based on 2D-contacts We further investigate by immunofluorescence labeling and confocal micro- differ strongly from biological data processors like the brain, which is a truly scopy the accompanying glial responses. We focus on microglia either in or- three-dimensional (3D) circuit. Huge advances in the additive manufacturing ganotypic spinal cord slices or in isolated primary glial cultures and pure at the nano and micro scale, especially direct laser writing by 2-photon poly- primary microglial cultures, treated with s-GO. In these conditions we test merization, now make the construction of complex resist-based 3D substrates

BPJ 8745_8748 Wednesday, February 21, 2018 673a with a resolution down to 100 nm possible. Therefore, we are able to construct EPs using field intensity twofold lower than the threshold level established. We 3D artificial networks for guided neurite growth: A Nanoscribe Photonic Pro- found that stimulation with 100 EPs at 10 kHz results in steadily triggered APs fessional GT is used to write a pillar structure resembling a medieval castle while 1 kHz failed to produce APs and resulted in sustained TTX insensitive with total dimensions of 600 600 100 mm. Pillars of different height PM depolarization. with cavities on top, which act as attachment sites for the somata of the neurons, are connected to each other through channels to guide the neurites from one pillar to the next in a precise predefined way. The inner diameter of the chan- Posters: Molecular Dynamics III nels, 3.6 mm, is tailored to fit the axon-diameter of 2 - 3 mm. To specifically enhance cell attachment, poly-D-lysin is printed directly into the cavities of 3335-Pos Board B543 the pillars. The substrate is cultured with granule cells from murine cerebella. Efficient Unbiased Sampling of Protein Dynamics using Reinforcement Cell activity after at least 5 days in vitro is shown through basic patch-clamp Learning measurements. The work presented here is a prove of principle for an approach Zahra Shamsi, Diwakar Shukla. to build complex, tailor-made 3D neural circuits. University of Illinois at Urbana-Champaign, Champaign, IL, USA. Understanding protein dynamics is crucial for a fundamental understanding of 3333-Pos Board B541 the biological process and drug design [1]. Molecular dynamics (MD) simula- Focused Ultrasound Evoked Responses in Dorsal Root Ganglion Neurons tion is a powerful tool to help us learn about conformational changes in pro- (DRG) and HEK293 Cells teins. However, even with the use of specialized supercomputers, it is Danny M. Florez-Paz1, Chi-Kun Tong1, Benjamin U. Hoffman1, difficult to simulate proteins with relevant biological timescales [2]. Advances Stephen A. Lee2, Elisa E. Konofagou2, Ellen A. Lumpkin1. in the field of genomics over the last decade, have led to uncovering the sub- 1Physiology & Cellular Biophysics, Columbia University, New York, NY, stantial gene sequencing information, which can be utilized as valuable unused USA, 2Biomedical Engineering, Columbia University, New York, NY, USA. information to enhance the simulation samplings. On the other hand, machine Focused ultrasound (US) is a noninvasive technique to modulate peripheral ac- learning is another fast-growing field, which finds its place in different areas in tivity, and holds promise for the treatment of sensory disorders and inflamma- science. Reinforcement learning (RL) constitutes a significant aspect of the tory diseases. However, the mechanisms underlying US-evoked responses in machine-learning field with numerous applications ranging from finance to sensory neurons are still unclear. Here, we explored the neuromodulatory capa- autonomous vehicles. In this study, we outline a novel computational method bilities of US in sensory neurons and HEK293 cells. First, we developed an to incorporate reinforcement learning concepts along with evolutionary infor- in vitro system integrating an US transducer (3.1 MHz) and a 3D-printed mation to guide the exploration of the long timescale behavior of the proteins recording chamber, combined with calcium imaging and electrophysiological [3]. The algorithm aims to enhance the sampling by identifying reaction coor- techniques to study US-evoked cellular activation. To establish parameters dinates that are relevant for sampling the system. We demonstrate the effective- for activation, we analyzed US in DRG neurons using calcium imaging. We ness of the algorithms by comparing the sampling to long continuous MD found robust, repeatable and reversible calcium responses, that increased simulations and least-counts adaptive sampling on multiple systems such as b with US pressure, over a wide range of sonication pressures. US activated 2-AR GPCR and Src kinase. References: [1] Agafonov, et al. Front. Mol. Bio- 40% of neurons with both small and large diameter cell bodies (N=152). We sci., 2, 1-8, 2015; [2] Shukla, et al. Nat. Commun., 5, 3397, 2014; [3] Shamsi, next recorded US-evoked currents using whole-cell patch-clamp technique. et al. Sci. Rep. InPress; In current-clamp, US evoked action potentials in 33% of sensory neurons 3336-Pos Board B544 (N=21). Using holding potentials from 90 mV to þ50 mV (N=10), current- Rational Development of HBV Capsid Inhibitors Aided by Molecular voltage relation curves did not reverse, suggesting Ca2þ or Naþ conductances, Dynamics however in HEK293 cells, same experiments showed curves reversal close to Anna Pavlova1, Maksym Korablyov2, Chris Chipot3, James Gumbart1. 0 mV (N=8). To implicate specific channels involved in US-evoked currents, 1Physics, Georgia Institue of Technology, Atlanta, GA, USA, 2Massachusetts we tested US in HEK293 cells, followed by perfusion with ruthenium red Institute of Technology, Boston, MA, USA, 3University of Illinois Urbana- (RR; 10mM), a non-specific cation channel blocker (N=5). Three cells showed Champaign, Urbana, IL, USA. reduced currents, whereas two cells showed enhanced currents in the presence Chronic Hepatitis B virus (HBV) infections are a major cause of liver damage of RR, with thresholds of 1.6-1.74 MPa and 1.25-1.37 MPa respectively. These and liver cancer and are presently incurable. A novel approach to combat the results suggest there might be two populations of HEK293 cells and/or chan- virus is by targeting HBV capsid assembly. Two major classes of compounds, nels that have different US activation thresholds, and showing different RR phenylpropenamides and heteroaryldihydropyrimidines (HAP), have been sensitivity. Together, these results suggest multiple ion-channel mechanisms shown to prevent the formation of normal capsids by binding to the intermedi- underlying US-evoke responses in different cells. ates of the capsid assembly. This binding can lead to either assembly into non- capsid structures or accelerated assembly that does not incorporate the viral 3334-Pos Board B542 DNA into the capsid. Unfortunately, both classes of compounds are toxic; Optical Recordings of Action Potentials in E18 Rat Hippocampal Neurons thus, there is significant interest in discovering novel assembly modifiers. Exposed to 10-NS Electric Pulses We have used MD simulations to study a tetramer of HBV capsid proteins, an Iurii Semenov, Shu Xiao, Andrei Pakhomov. important assembly intermediate, with and without bound assembly modifiers. Frank Reidy Research Center for Bioelectrics, Old Dominion University, It was found that the tetramer was highly flexible in solution; snapshots from Norfolk, VA, USA. the simulations were selected for docking using principal component analysis. It is currently unclear if nanosecond pulses can be used to trigger action poten- The highest scoring compounds from docking were tested experimentally. Af- tials (APs) in excitable cells with no accompanied plasma membrane (PM) ter discovering one compound that showed activity against HBV, confirmed electropermeabilization. Present study shows that APs can be triggered in neu- binding to the capsid protein, and low toxicity, we proceeded to dock and rons by 10-ns electric pulses (EPs) with no signs of sustained PM damage test similar compounds. We have discovered several compounds that are struc- related to electropermeabilization. turally distinct from previously known assembly modifiers and that have mod- The experiments were performed on coverslips. Cells were loaded with Fluo- erate activity against HBV. The HBV tetramer was simulated with the new Volt. Dye emission was observed with CCD Camera. Fast imaging at 3000 compounds as well with previously known assembly modifiers bound. It was frames/s was utilized. Pair of 100-mm rod electrodes was used for EPs delivery. found that all active compounds greatly decreased the flexibility of the protein To find threshold of AP generation, neurons were exposed to a single 10 ns complex. Additionally, distinct configurations were sampled for the different pulse in the range from 4 to 93 kV/cm. PM responses were evaluated before þ HAP compounds while the configurations of the tetramer were similar for and after blockage of voltage-gated Na -channels with tetrodotoxin (TTX). our new compounds. Before TTX was added, 4% cells fired APs upon exposure to 4.5 kV/cm and this amount grew to 15%, and 60% with field intensity increased to 5.8 kV/ 3337-Pos Board B545 cm and 8.8 kV/cm, respectively. All APs were 1) reproducible with repeated QwikMD - Gateway for Easy Simulation with VMD and NAMD exposures and; 2) indistinguishable from the APs triggered by conventional Joa˜o Vieira Ribeiro1, Rafael C. Bernardi1, Till Rudack1,2, Klaus Schulten1,3, stimulation in the same neurons. In the presence of TTX 6% neurons responded Emad Tajkhorshid1,4. with moderate PM depolarization to 10.1 kV/cm EPs and 60% of the cells were 1Beckman Institute for Advanced Science and Technology, University of depolarized when field intensity reached 13.4 kV/cm. PM depolarization at Illinois, Urbana, IL, USA, 2Department of Biophysics, Ruhr-Universit€at these conditions was directly proportional to applied field intensity. Repeated Bochum, Bochum, Germany, 3Department of Physics, University of Illinois, ns-EPs applications led to the diminished PM responses. To study phenomenon Urbana, IL, USA, 4Department of Biochemistry, University of Illinois, of APs stimulation by ns-EPs further, we stimulated cells with multiple 10-ns Urbana, IL, USA.

BPJ 8745_8748 674a Wednesday, February 21, 2018

The use of molecular dynamics (MD) simulations has increased significantly curacy of these methods needs to be assessed. Over the years of continuous over the last decade, making it a key methodology in structural biology. Aided development, we have now attained a framework allowing to set up alchemical by MD, researchers were able to resolve atomic structures of multi-protein simulations for amino acid mutations, nucleotide mutations and ligand modifi- complexes from cryo-EM densities; unveil the atomistic details of enzymatic cations in a number of contemporary molecular mechanics force fields. The mechanisms and characterize the binding of small molecules to proteins. To ease with which the setup is automated allows for a large scale scan over hun- achieve all this, the capabilities of MD packages are constantly evolving, dreds of mutations in various biomolecular systems. We explored the accuracy providing a huge variety of complex MD simulation and analysis techniques. of alchemical free energy estimates by mutating amino acids and monitoring Although applicable to a plethora of research problems, a broad usage of MD changes in protein stability, protein-protein and protein-ligand interactions. is often delayed by a steep initial learning curve imposed by nearly every MD Furthermore, we investigated nucleotide mutations in a scan over numerous software. To make MD accessible to a broader audience, we developed an protein-DNA complexes and demonstrated how free energy calculations could intuitive tool named QwikMD(1), which assists the users in the preparation, be utilized to construct DNA binding profiles. Finally, the framework was used execution and analysis of MD simulations. More than just assisting, QwikMD to navigate in a chemical library of arbitrary organic molecules, establish map- ensures reproducibility of results by writing all parameters and steps into two pings between them and carry out free energy calculations assessing the relative log files, one in a script-like format and another in a ‘‘methods section’’ affinities of ligands binding to a protein. The observed calculation accuracy format. The user-friendly graphical interface of QwiKMD allows its user to puts alchemical approaches on top of the list of methods to estimate free energy prepare MD simulations in just a few minutes, in a point-and-click fashion, differences. offering the user a variety of protocols, such as, unbiased MD simulations, Steered MD, MD Flexible Fitting (MDFF), and, most recently, the calculation 3340-Pos Board B548 of the absolute binding free-energy of protein-ligand complexes and hybrid Statistically Reliable Molecular Dynamics Simulations of Transient QM/MM simulations. The later exploits the recently developed VMD and Conformational Changes in the Estrogen Receptor NAMD interface to common quantum mechanics software packages. Barmak Mostofian, Upendra Adhikari, Daniel M. Zuckerman. QwikMD facilitates performing MD simulations for nearly any user, novice OHSU, Portland, OR, USA. or expert. QwikMD also serves as a learning tool, providing the theoretical Typical molecular dynamics (MD) simulations do not reach equilibrium and background of the different MD protocols and options in many ‘‘info hence their trajectory data do not correspond to a well-defined statistical distri- buttons’’. bution. On the other hand, multiple trajectories initiated from the same config- 1. J. V Ribeiro et al., QwikMD — Integrative Molecular Dynamics Toolkit for uration can be averaged to yield well-defined, time-dependent properties. This Novices and Experts. Sci. Rep. 6, 26536 (2016). strategy can be implemented using conventional MD or the weighted ensemble (WE) approach. We apply both methods to study the conformational dynamics 3338-Pos Board B546 of the ligand binding domain of estrogen receptor alpha in several settings - the Computational Epitope Prediction and Screening Precision Antibody wild-type and two mutants important in metastatic breast cancer, as well as apo Therapeutics for Alzheimer’s Disease and ligand-bound conditions. Overall, the simulations enable us to make quan- Xubiao Peng, Ebrima Gibbs, Judith M. Silverman, Neil R. Cashman, titative determinations of when the conformational behavior is statistically Steven S. Plotkin. distinguishable under the various conditions. Department of Physics and Astronomy, University of British Columbia, Vancouver, BC, Canada. 3341-Pos Board B549 Using Molecular Dynamic simulations, we predicted the epitopes on Ab olig- Directing Membrane Poration in MD Simulations with Embedded omers, which would be distinct from those in either Ab monomer or Ab fibril. Mechanical Gizmos Starting from the predicted epitopes, 66 antibodies are raised by passive im- Gregory Bubnis, Helmut Grubmuller. munization in mice, and their binding to Ab monomer, oligomer and plaque, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. along with preferential binding to Ab protein in the cerebrospinal fluid and Membrane topology changes such as poration, stalk formation, hemi-fusion brain homogenate from deceased Alzheimer Disease (AD) patients vs healthy and rupture are essential to cellular function, but their molecular details controls are measured experimentally. These experimental measurements pro- and energy budgets are still not fully understood. Here, we have studied vide up to eight different screening criteria to select lead candidates for clin- the nucleation mechanism of weakly metastable ‘‘pre-pores’’ in pure bilayers ical stages of drug development. The screening criteria do not perfectly using a novel, pseudomolecular ‘‘gizmo’’ that embeds itself into a hydropho- correlate with each other and there is uncertainty in the criteria values as bic membrane during molecular dynamics simulations and provides adjust- well as their importance/weights, making the selection of lead candidates a able controls of pore opening and closure. This bias is subsequently nontrivial maximum-likelihood ranking problem. In other fields, this problem unweighted to obtain the full energetics of the process. We show that the is referred to generally as multiple criteria decision making or MCDM, and pore gizmo can restrain an atomistic DMPC bilayer close to the topological has been applied in various fields including finance and economics, energy transition – which is notoriously hard to sample thoroughly. Furthermore, policy, and environmental science, but not—at least to our knowledge—to from an ensemble of gizmo simulations sampling the full pathway, we recov- the problem of screening candidate drug therapeutics. We employ a stochastic ered the unbiased free energy landscape (via WHAM) and found the pre- MCDM method named ‘‘SMAA-TOPSIS’’ to screen and rank the measured pore’s nucleation barrier to be 18 k_BT high and its metastable basin to large cohort of the antibodies, in order to find lead candidate therapeutics. be 3 k_BT deep. Performing a committor analysis for many barrier crossings We obtain distributions of candidate rankings due to uncertainty in screening (100þ, both directions), we observed that membrane-spanning water-wires measurements, as well as the user-defined weight of importance attributed to form prior to the nucleation barrier, whereas barrier crossing is better each screening criterion. In choosing lead candidates, we propose a quantity captured by the first few lipid headgroups submerging to line the nascent ‘‘topness’’ as the most robust measure of ranking. Our hope is that this toroidal surface. method may enable more systematic screening of candidate therapeutics when it becomes difficult intuitively to process multi-variate screening data 3342-Pos Board B550 that distinguishes candidates, so that additional candidates will be exposed New QMMM Interface to NAMD Probes tRNA Charging Mechanism as potential leads, increasing the likelihood of success in downstream clinical Marcelo C. Melo1, Rafael C. Bernardi2, Klaus Schulten3, trials. Zaida Luthey-Schulten4. 1Biophysics, University of Illinois at Urbana Champaign, Urbana, IL, USA, 3339-Pos Board B547 2University of Illinois at Urbana Champaign, Urbana, IL, USA, 3Physics, A Unified Framework for Alchemical Mutations in Proteins, DNA and University of Illinois at Urbana Champaign, Urbana, IL, USA, 4Chemistry, Ligands University of Illinois at Urbana Champaign, Urbana, IL, USA. Vytautas Gapsys, Bert L. de Groot. Hybrid molecular dynamics simulations combining Quantum Mechanics and Theoretical and Computational Biophysics, Max Planck Institute for Molecular Mechanics (QM/MM) provide a cost-effective way of studying a Biophysical Chemistry, Goettingen, Germany. large range of biological processes. From polarization effects to chemical reac- Rapid advances in the available computing power in recent years have turned tions, a QM/MM approach allows for a precise representation of the dynamics first principles based alchemical free energy calculations into an attractive op- of a biological structure. The most recent version of NAMD interacts natively tion for academic research and industrial applications. Naturally, this develop- with quantum chemistry packages MOPAC and ORCA, allowing for a wide ment imposes the requirement for a reliable and versatile framework to range of quantum chemistry methods, from fast semiempirical to more complex automate the technically involved computations. In addition, the achievable ac- density functional theory and perturbation theory methods. Other quantum

BPJ 8745_8748 Wednesday, February 21, 2018 675a chemistry packages can also be easily employed through a flexible scripted 3345-Pos Board B553 D interface. Taking advantage of the large scalability of NAMD, users can Inference of Calmodulin’s Ca2 -Dependent Free Energy Landscapes via perform simulations with hundreds of millions of atoms in the MM region, hav- Gaussian Mixture Model Validation ing many ‘‘independent’’ QM regions running simultaneously. VMD’s Annie M. Westerlund1, Tyler J. Harpole1, Christian Blau2, QwikMD interface has also been extended to allow fast and easy preparation, Lucie Delemotte1. analysis and visualization of the QM/MM simulations. QwikMD assists the se- 1Physics, KTH Royal Institute of Technology, Stockholm, Sweden, lection of QM regions and automatically handles bonds connecting QM to MM 2Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden. atoms. Users can use default simulation parameters or personalize any aspects A well-estimated FE landscape is critical for inferring information about the of QM/MM calculations. The new interface was used to study the universal re- motion and function of a protein. This estimation, however, is a challenging action of the charging of a tRNA molecule with its cognate amino acid, cata- task given finite molecular dynamics (MD) data. It is especially difficult to es- lyzed by the tRNA synthetase GluRS. The aminoacylation reaction timate FE barriers where sampling is sparse, but these regions are crucial to mechanisms was investigated in order to distinguish between two possible infer FE pathways and kinetics. Furthermore, the accuracy of the FE landscape routes, revealing subatomic details of an essential step in establishing the ge- directly influences the efficiency assessment of different MD sampling netic code. In summary, the ease of access through QwikMD and the modu- methods. larity in NAMD allows this interface to be used in a variety of applications We used a robust FE landscape estimation method based on Gaussian mixture þ and contexts, such as a teaching tool, a day-to-day iterative research interface, model (GMM) and cross-validation to estimate the Ca2 -dependent FE land- a platform for mixing-and-matching QM/MM with free energy and enhanced scapes of calmodulin (CaM). The estimation method was compared to other es- sampling methods, or even as a sandbox for the development of new QM tools timators using a toy model, showing that GMM with cross-validation provides and QM/MM interactions schemes. an estimate not subject to overfitting. Furthermore, the continuous nature of GMM provides better estimates of FE barriers than canonical histogramming. 3343-Pos Board B551 The obtained FE landscapes compared the efficiency of thermally enhanced A Scalable and Efficient Approach to Polarizable Force Fields in Molecu- sampling methods with respect to regular MD. With this methodology, we lar Dynamics Simulations found that diffusion properties determine sampling method effectiveness, 2þ Jonathan P. Coles1, Michel Masella2. such that the diffusion dominated Ca -free CaM is most efficiently sampled 2þ 1Theoretical Biophysics, Technical University of Munich, Garching, by regular MD, while Ca -bound CaM, with its rough FE landscape, is better Germany, 2Laboratoire de Biologie Structurale et Radiobiologie, iBiTec-S, sampled by enhanced sampling methods. CEA Saclay, Saclay, France. 3346-Pos Board B554 Standard biophysical molecular dynamics simulations use complex, fixed- The Combined Force Field-Sampling Problem in Simulation of Intrinsi- charge force field models to capture the atomic interactions of solvated pro- cally Disordered Peptides teins and other organic molecules. These force fields are typically parameter- James Lincoff1, Sukanya Sasmal1, Teresa Head-Gordon2,3. ized to reproduce experimental results and are often very specific to the 1Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA, system being simulated. More flexible models that capture induced polariza- USA, 2Chemical and Biomolecular Engineering, Chemistry, Bioengineering, tion and dynamically account for the changing electrostatic environment of UC Berkeley, Berkeley, CA, USA, 3Chemical Sciences Division, Lawrence proteins, DNA, and ion channels are increasingly recognized as a critical Berkeley National Laboratory, Berkeley, CA, USA. step towards more accurate and predictive simulations. However, such models Molecular dynamics (MD) simulations are a key technique in the study of are generally much more computationally expensive, which has limited their intrinsically disordered peptides (IDPs) because they generate atomic-level adoption. structural ensembles at high time resolution. However, for any simulation there I will present the TCPEp model, a sophisticated, polarizable force field that has are two overlapping questions about the accuracy of the generated ensemble. been implemented in the Polaris(MD) molecular dynamics software package. First, is the force field, which models the interactions of the peptide and water Together with an efficient implementation of the O(N) Fast Multipole Method atoms, accurate to the native physics? Second, did the simulation sufficiently for calculating long-range forces, the expensive cost overhead of using a polar- sample the conformational space of the peptide and generate a converged equi- izable force field has been significantly reduced, allowing simulations of large librium ensemble? To address the force field issue, various groups have pro- molecules to be achieved. Recent work involving a highly scalable and parallel posed and tested modifications to standard fixed-charge force fields that version of FMM will be highlighted along with applications to large optimize them for the more flexible free IDP state. On the sampling side, we biomolecules. had previously developed a new enhanced sampling method, temperature cool walking (TCW), which was shown to converge more rapidly than the stan- 3344-Pos Board B552 dard temperature replica exchange (TREx) at far lower cost. Here, we apply A Refined Free Energy Perturbation Hamiltonian Replica Exchange Mo- both TCW and TREx to investigate the combined effects of sampling method lecular Dynamics Method for Absolute Binding Affinity Predictions and force field selection on simulation results for the amyloid beta peptides Wei Jiang. Ab42 and Ab43, using one standard force field and two IDP-optimized force Argonne National Lab, Lemont, IL, USA. fields. For each force field-sampling method pair we compare the structural 2D Free Energy Perturbation with Hamiltonian Replica Exchange Molecular agreement of each individual peptide and the expected differences between Dynamics (FEP/H-REMD) has been proven efficient to improve the conver- them with experimental results from NMR. Preliminary results illustrate the gence of ligand binding free energy calculations with large surrounding key importance of both force field and sampling method: while the IDP- reorganization [J. Chem. Theory Comput. 2010, 6, 2559]. However, existing optimized force fields outperform standard force fields for each individual sam- protocol is based on 2D replica communications and non-transferable boost pling method, clear differences remain in the ability of each method to effi- potential, and therefore not an economical method for general applications. ciently sample the conformational space regardless of force field. Updating In the present study we propose a refined version FEP/H-REMD, where only one of the two appears likely to be insufficient in the pursuit of more ac- number of replicas is significantly reduced and existing boost potential is re- curate simulations. placed with transferable ones. To avoid complexities of reweighting for the accelerated simulation, boost replicas and lambda windows remain separate 3347-Pos Board B555 in energy postprocessing phase at the expense of minorly increased Toward an Efficient Computational Method to Construct 3-D Atomic Res- computing cost of FEP. High frequency replica exchanges are performed olution Glycosaminoglycan Models to speed up travelling of boost replicas in extended ensemble space. The Elizabeth Whitmore1,2, Hanna Sihler1,3, Olgun Guvench1,2. method is implemented on the basis of generic multiple partition 1University of New England College of Pharmacy, Portland, ME, USA, module of the biomolecular simulation program NAMD, with which an 2University of Maine Graduate School of Biomedical Science and end user can flexibly design customized replica exchange pattern through Engineering, Orono, ME, USA, 3University of New England College of Arts Tcl scripting without touching source code. As three illustrative examples and Sciences, Biddeford, ME, USA. with reliable experiment data, the absolute binding free energies of p-xylene Proteoglycans (PG) are protein-carbohydrate conjugates found in extracellular and n-butylbenzene to the nonpolar cavity of the L99A mutant of T4 lyso- matrix and associated with cells. One of the most abundant PG carbohydrate zyme were calculated. The tests demonstrate that the new protocol chains in the body is the linear glycosaminoglycan (GAG) chondroitin efficiently accelerates the sampling of large configurational reorganization sulfate (CS). CS consists of repeating disaccharide units of b-D-glucuronic around the binding pocket and the convergence of the free energy acid and b-N-acetylgalactosamine. A single CS GAG may contain hundreds computations. of disaccharide units and varying degrees of sulfation. We applied all-atom

BPJ 8745_8748 676a Wednesday, February 21, 2018 explicit-solvent molecular dynamics (MD) simulations to examine the thermo- Replica exchange with solute tempering (REST) significantly reduces compu- dynamics and biophysics of a series of short oligomers (up to 12 disaccharide tational cost in comparison with conventional temperature replica exchange units). Larger systems require more computational resources, so we aimed to molecular dynamics method. We extended the solute selection scheme of find a more efficient method of modeling long, biologically relevant CS poly- this REST, to avoid parameter space trapping problem and associated loss in mers. We therefore developed an algorithm that applies phi/psi angles and conformational search efficiencies happen in REST. The new scheme, which Gibbs free energy from MD analysis of CS disaccharides to construct CS poly- we call generalized REST (gREST), accepts energy term based partitioning mer conformations very rapidly and of arbitrary length. Comparing end-to-end in addition to the particle based one; the solute region can involve only dihedral distances between MD-generated and constructed ensembles produced by our energy terms, for example. We applied this gREST to the folding and free en- program, we noticed consistent differences: the conformational ensemble ergy landscape calculations of Trp-cage small protein system, where only dihe- from MD simulation showed higher end-to-end distances than the ensemble dral energy terms were considered as the solute (dihedral-gREST), and produced by our program. This suggests that there exist long-range interactions compared its efficiency with the most frequently used REST variant, REST2. beyond the disaccharide subunit that impact the backbone flexibility for CS Using this dihedral-gREST, we successfully found the native state of Trp- GAGs. cage in comparable timescale as REST2 with almost a half of computational cost. Moreover, the free energy landscape, calculated with multistate Bennett 3348-Pos Board B556 acceptance ratio (MBAR), of REST2 underestimated the stability of unfolded Finite-Size Effect on the Charging Free Energy in the Alchemical Pertur- state and the convergence rate of the free energy map was slower than that by bation and ‘‘Warp Drive’’ Method dihedral-gREST. In REST2, stable structures including the native one favor Toru Ekimoto, Tsutomu Yamane, Mitsunori Ikeguchi. states with smaller energy scaling coefficients (lower REST temperature, in Yokohama City University, Yokohama, Japan. other words), which is caused by the disturbance in solute and solvent energy With increasing computational power, the binding free-energy calculations compensations due to the energy scaling coefficients. This trapping in the with exact methods, such as the alchemical free energy perturbation (alchem- parameter space eventually resulted in the less accurate free energy map. In ical-FEP), routinely become possible using all-atom molecular dynamics (MD) contrast, dihedral-gREST did not suffer from such a trapping in the parameter simulations. The alchemical-FEP consists of the annihilation process of the space. This generalized variant of REST, gREST, can further extend the capa- bound ligand and the emerging process of the ligand in bulk. The bility of REST scheme and is expected to improve many types of simulations alchemical-FEP can treat buried ligands in deep pocket, and however, accuracy including free energy calculations. for highly charged ligands is poor. One of the reasons is due to the finite-size effect on the charging free energy (CFE) in periodic systems, and the finite- 3351-Pos Board B559 size effect refers to the cell-size dependences of the CFE. In this study, the Towards Realistic Models of Lung Surfactant - MD Simulations with finite-size effect on the CFE in the alchemical FEP is examined using system- Improved Water and Ion Force Fields atically varied cell sizes of the MD unit cell. We show for the phosphotyrosine Pauline Delcroix, Agnieszka Olzynska, Lukasz Cwiklik. peptide (5e) bound to the Src homology 2 domain (þ1e) that the CFEs of both J. Heyrovsky Institute of Physical Chemistry, Czech Academy of Sciences, processes strongly depend on the cell size, and assess the performance of the Prague, Czech Republic. correction scheme formulated by Hummer et al. In addition, we introduce an Lung surfactant lines the gas-exchange interface in the lungs and reduces the alternative perturbation method, termed warp drive method, in which both pro- surface tension at the air-liquid interface to minimize the work of breathing. cesses in the alchemical FEP are done using a coexistence system, and demon- The lung surfactant consists mainly of lipids with a small amount of proteins strate that the calculated value without any correction is in good agreement with and forms a monolayer at the air-water interface connected to bilayer reser- the corrected value calculated by alchemical FEP. voirs. The composition of the lung surfactant and the border conditions of normal human breathing are relevant to characterize the interfacial behavior 3349-Pos Board B557 of pulmonary layers. Here, we focus on the composition of the lung surfactant. Biomolecular Simulations in a Continuum Ionic Solvent with Polarizable We employ classical molecular dynamics simulations at the atomistic resolu- Force Fields, Using Python and GPUs tion of a new more realistic model of the monolayer including DPPC, POPC, Christopher D. Cooper. POPG, and cholesterol. Simulations employ the Slipids force field and TIP3p Department of Mechanical Engineering, Universidad Tecnica Federico Santa parameters for water at the microsecond timescale. The role and behavior of Maria, Valparaiso, Chile. ions present in the water subphase are also addressed. Notably, we employ In molecular simulations, implicit-solvent models use continuum electrostatic scaled-charge parameters for the ions together with the new OPC water force theory to average the solvent degrees of freedom, drastically decreasing the field to improve the description of both ion-lipids interaction and surface ten- amount of computations compared with explicit molecular dynamics. Mathe- sion of the film. The role of cholesterol and the so-called ‘‘Cholesterol Mys- matically, it can be formulated as a coupled system of the Poisson and tery’’ is also investigated. Poisson-Boltzmann equations, interfaced by the biomolecule’s solvent- In parallel to simulations, Langmuir trough experiments are conducted with excluded surface. Usually, these models use point-charge-based (classical) corresponding compositions of lipid films to assess macroscopic properties force fields to parameterize the biomolecule of interest, yet, more elaborate of the monolayers. In combination with microscopic properties obtained force fields that consider higher order multipoles and polarizability have from simulations, it allows us to analyze the effect caused by the presence or emerged lately. Polarizable force fields generate more realistic charge distri- absence of different lipids on lung surfactant properties. butions, but involve larger computations than a classical force field. One pop- ular polarizable force field is AMOEBA, which uses point multipoles up to 3352-Pos Board B560 the quadrupole order to describe the charge distribution, and allows for the Elastic Moduli of Fibrous Proteins from Equilibrium Molecular Dynamics dipole component to polarize. In this work, we present an extension of the Simulation Poisson-Boltzmann solver PyGBe, which couples with the AMOEBA force Russell Hawkins, Daniel Cox. field by solving iteratively for a self-consistent reaction potential. PyGBe is Physics, University of California, Davis, Davis, CA, USA. a boundary element method code, accelerated with a treecode algorithm A technique for determining effective elastic moduli of proteins from equilib- and GPUs, that works from a Python interface. The boundary integral formu- rium molecular dynamics is presented. The technique is in principle appli- lation treats the point multipoles analytically, which is an advantage over cable to any stiff, elongated, beam-like macromolecule. Results for a class volumetric-based methods where very fine meshes are required to effectively of proteins satisfying this condition, known as beta solenoid proteins, is place the point multipoles onto the numerical mesh. We will show results presented. verifying our implementation in comparison with analytical solutions, avail- able for spherical molecules, and computing the solvation energy of larger 3353-Pos Board B561 test proteins. New Automated and High-throughput Tools for the Martini Forcefield Peter C. Kroon, Tsjerk A. Wassenaar, Jonathan Barnoud, 3350-Pos Board B558 Siewert-Jan Marrink. Flexible Choice of Solute in Replica Exchange with Solute Tempering Can Molecular Dynamics, Rijksuniversiteit Groningen, Groningen, Netherlands. Improve Performance of Conformation Search for Small Proteins Recently, the amount of chemical space that can be represented by the Martini Motoshi Kamiya1, Yuji Sugita1,2. coarse-grained model has grown explosively. In particular, non-biological 1Riken Advanced Institute for Computational Science, Kobe, Japan, 2Riken polymers, and protein-ligand complexes are popular research topics. It is strik- Theoretical Molecular Science Laboratory, Wako, Japan. ing that despite this, there are currently no tools available that can efficiently

BPJ 8745_8748 Wednesday, February 21, 2018 677a and conveniently generate topologies for these kind of systems. Currently, Mar- for the efficient calculations and characterization of the reaction coordinates. tinize is a Python script designed and written to make coarse-grained (Martini) Here we try to combine the WE method with another path sampling method, representations of proteins. Later, ad-hoc, additions have extended this to the Lyapunov weighted dynamics, which is based on the idea of instability of a tra- different flavors of the Martini forcefield. However, due to the ad-hoc nature jectory. The latter method has been successfully applied to a Lennard-Jones of these additions, and because the program has not been designed from the cluster and a coarse-grained protein, but as far as we know, there has been ground up to support the flexible addition of new parameters and forcefields, no application to realistic protein systems because of the burden of the it has become very difficult to extend further. We present the advancements instability calculation using a hessian matrix. We try to use an approximate in the development of new tools designed to address these issues, focusing method to calculate the hessian matrix, and apply the WE method in Lyapunov on a new version of Martinize as well as an automatic topology builder for exponent space to several protein systems including chignolin and adenylate the Martini forcefield. kinase.

3354-Pos Board B562 3357-Pos Board B565 An Accurate Computation of a Physical Quantity of a Protein with a Mar- Calculation of Absolute Solvation Shell Entropies from MD Trajectories kov State Model Constructed using a Manifold-Learning Technique via Permutation Reduction Reika Ito, Takashi Yoshidome. Leonard P. Heinz, Helmut Grubmuller.€ Department of Applied Physics, Tohoku University, Sendai, Japan. Department of Theoretical and Computational Biophysics, Max Planck Markov state models (MSMs) are a powerful approach for analyzing the long- Institute for Biophysical Chemistry, Go¨ttingen, Germany. time behaviors of protein motion using molecular dynamics simulation data. For many biomolecular processes, such as cold denaturation or other processes However, their quantitative performance with respect to the physical quantities associated with the hydrophobic effect, a quantitative understanding of the ther- is poor. We believe that this poor performance is caused by the failure to appro- modynamics of solvation is crucial. Typically, it involves a well-balanced priately classify protein conformations into states when constructing MSMs. interplay between enthalpy and entropy contributions. Unfortunately, accurate Herein, we show that the quantitative performance of a physical quantity calcu- absolute entropy values are notoriously difficult to obtain from computer sim- lated using an MSM is improved when a manifold-learning technique is em- ulations. This problem is particularly severe in the case of solvent entropy con- ployed for the classification. The MSM construction using the K-center tributions, since the diffusive nature of the solvent particles leads to a large method, which has been previously used for classification, has a poor quantita- configuration space that needs to be sampled. To solve the sampling problem, tive performance. we exploit the permutation symmetry of the identical solvent particles to compress the phase space volume by a factor of N!. For each frame of an atom- 3355-Pos Board B563 istic simulation, all identical solvent molecules are permuted so that they WESTPA 2.0 Advances in Sampling, Storage, and Analysis of Weighted remain as close to chosen reference positions as possible. Although not altering Ensemble Simulations the physics of the system, the approach ensures that each particle samples only Adam Pratt1, Daniel M. Zuckerman2, Lillian T. Chong3. a small fraction of the full configuration space and thereby reduces the sam- 1University of Pittsburgh, Pittsburgh, PA, USA, 2Biomedical Engineering, pling problem by the Gibbs factor. We employed a mutual information expan- Oregon Health & Science University, Portland, OR, USA, 3Department of sion to obtain absolute solvent entropy values from the permutationally reduced Chemistry, University of Pittsburgh, Pittsburgh, PA, USA. trajectory. The method enables us to assign entropy contributions to the The weighted ensemble path sampling strategy has enabled the generation of different solvation shells and even to individual solvent particles, which renders continuous pathways and calculation of rate constants for rare events such as it a promising tool to enhance our understanding of processes like the hydro- protein folding and protein-ligand binding/unbinding with orders of magnitude phobic effect. greater efficiency than standard simulations. Notably, this strategy is rigorous for any type of stochastic dynamics engine, pruning and replicating trajectories 3358-Pos Board B566 in a way that encourages sampling of under-explored regions without biasing Upside: A New Dynamics Methods Capable of Cooperative De Novo Pro- the dynamics. I will present new features that will be available in the next tein Folding in CPU-Hours John M. Jumper1, Karl F. Freed2, Tobin R. Sosnick3. release of the WESTPA software, including (i) enhanced efficiency of gener- 1 ating pathways and calculating rate constants, (ii) improved schemes for repli- Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA, 2Chemistry, James Franck Inst., University of Chicago, Chicago, IL, cating and combining trajectories to yield a greater diversity of generated 3 pathways, and (iii) a highly scalable framework for storage and analysis of USA, Biochemistry & Mol Biol/Inst. Biophysical Dynamics, University of weighted ensemble simulations. In addition, new tools will be available for Chicago, Chicago, IL, USA. streamlining the setup and analysis of weighted ensemble simulations. Finally, The traditional trade-off in simulations between accuracy and speed is predi- I will discuss challenges that remain for the simulation of long-timescale pro- cated on the assumption that force fields are typically well-parameterized. cesses beyond the ms timescale. We re-examine this trade-off and show that a properly formulated coarse- grain model trained using machine learning methods can rival all-atom models 3356-Pos Board B564 for de novo protein folding and dynamics simulations. We use the contrastive Combining Weighted Ensemble Method and Lyapunov Weighted divergence technique to train the force field directly from short simulation tra- Dynamics: Application to Proteins jectories of 450 proteins beginning near their native structures. This method is Hiroshi Fujisaki1, Kei Moritsugu2, Yasuhiro Matsunaga3, applied to our recently developed Upside model, where the free energy for side Hiromichi Suetani4. chains are rapidly calculated at every time-step, allowing for a smooth energy 1Physics, Nippon Medical School, Musashino, Tokyo, Japan, 2Yokohama landscape without steric rattling of the side chains1. After this contrastive diver- City University, Tsurumi, Yokohama, Japan, 3RIKEN, Kobe, Japan, gence training, the model is able to fold proteins up to 100 residues de novo 4Physics, Oita University, Oita, Japan. on a single core in CPU core-days2. The Upside model’s success argues that Weighted ensemble (WE) method is a convenient and powerful method for simpler models that can be globally parameterized can rival more detailed path sampling and has been used for analyzing dynamic properties of confor- but slower models whose parameterization is more challenging - more mational change of proteins, ligand binding, and chemical reaction networks. complexity does not necessarily equate to higher accuracy! Upside’s ready gen- The basic idea of the WE method is to make bins in order parameter space, eration of Boltzmann ensembles allows for a wide range of studies of protein and run several trajectories in parallel in each bin. Each trajectory has a weight, folding, dynamics and binding. Additionally, in studies that incorporate exper- which can change according to the resampling principle whenever another tra- imental or bioinformatics data, including contact predictions, Upside provides jectory comes into or get away from the same bin. The total weight in each bin an inexpensive Bayesian prior distribution that may be updated using experi- represents a coarse-grained population (or free energy), and its dynamics is mental information. described, for example, by the Fokker-Planck equation if the trajectory is sup- 1Rapid calculation of side chain packing and free energy with applications to posed to be generated by overdamped Langevin dynamics. The WE method is protein molecular dynamics. https://arxiv.org/abs/1610.07277 especially useful when there is an activation process such as conformational 2Trajectory-Based Parameterization of a Coarse-Grained Forcefield for High- change or ligand binding, accelerating the calculation of rare event pathways. Throughput Protein Simulation. http://www.biorxiv.org/content/early/2017/ However, the choice of order parameters is still empirical though it is essential 07/27/169326

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Posters: Computational Methods and ing assembly. To address this, we use a recently developed biophysical model of FN fibrillogenesis (Weinberg et al, Biophys J, 2017), which allows us to Bioinformatics II simulate competing hypotheses for the location and properties of the cryptic FN-FN binding sites and quantify the effect of these molecular alterations on 3359-Pos Board B567 the morphology and biomechanical properties of the assembled fibril. Model An Atomic Four-Body Statistical Potential to Distinguish Native RNA predictions indicate that a single FN-FN binding site located in a domain N-ter- Structures from Nonnative Folds minal of the 10th Type III domain (which contains the integrin-binding motif Majid Masso. RGD) facilitates negligible fibrillogenesis, while a single FN-FN binding site School of Systems Biology, George Mason University, Manassas, VA, USA. in a domain located C-terminal of the 10th Type III domain produces FN fibrils Eighty-five diverse RNA structures were selected for training our multibody that are neither robust nor physiologically similar to actual FN fibrils. In potential (RAMP), and a four-letter alphabet (C,N,O,P) designated atom types. contrast, the inclusion of multiple FN-FN binding sites predicts robust FN fibril Structure coordinate data were subjected to Delaunay tessellation, whereby assembly, which, importantly, minimally depends on the individual mechanical atomic points serve as vertices for a 3D tiling to generate a convex hull of and chemical binding properties in FN with binding sites in all 15 Type III space-filling, non-overlapping, irregular tetrahedra. The four vertices of each domains. tetrahedron identify an interacting quadruplet of atoms, of which there are 35 possibilities. For each atomic quadruplet type (i,j,k,l), its relative frequency 3362-Pos Board B570 of occurrence fijkl in the training set was calculated from the observed tetra- Molecular Dynamics Simulation of the Conformational Changes in the hedra, and a rate pijkl expected by chance was obtained from a multinomial Procaspase 9 Activation reference distribution. Energy of interaction scores for the 35 quadruplet types, Humberto Gasperin1, Claudia G Benı´tez-Cardoza2, 3 4 given by sijkl = - log (fijkl / pijkl), collectively define the four-body potential. Sub- Jorge L Rosas-Trigueros , Absalom Zamorano-Carrillo . sequently for any RNA structure, total potential is computed by adding up 1Bioinformatics, Instituto Politecnico Nacional, Mexico city, Mexico, scores of all tetrahedral-associated atomic quadruplets derived from its tessel- 2Laboratorio de Bioquı´mica y Biofı´sica Computacional, ENMH Instituto lation. RAMP was evaluated by (normalized) scoring of 85 decoy sets using a Politecnico Nacional, Ciudad de Mexico, Mexico, 3Lab Transdiciplinario de leave-one-out procedure, identical to four RASP pairwise potentials in Cap- Sistemas Evolutivos, ESCOM Instituto Politecnico Nacional, Ciudad de riotti, et al. Bioinformatics 27 (2011) 1086-1093. The fraction of native Mexico, Mexico, 4Lab de Bioquı´mica y Biofı´sica Computacional, ENMH RNA structures top-ranked (i.e., lowest normalized total potential) by RAMP Instituto Politecnico Nacional, Ciudad de Mexico, Mexico. (0.82) relative to 500 decoys each was competitive with those obtained using Rational drug design of novel cures for the most common forms of cancer and RASP (0.08,0.35,0.89,0.93), NAST (0.22), ROSETTA (0.62), ROSETTAmin other chronic-degenerative pathologies is still challenging. One strategy could (0.85), and AMBER99 (0.73). The best RASP potential and RAMP were be improving our comprehension of apoptotic mechanisms. Cysteine proteases also used to rank 407 near-native models, built with ROSETTA, for 32 RNA perform a wide variety of roles in lethal intracellular signaling. Particularly, the motifs containing non-canonical base pairs. With ‘‘win’’ defined as lowest family of caspases combines the cysteine protease activity and the cell death normalized score for model having lowest rmsd with native RNA motif, results regulation. Caspases have been classified, according to their mechanism of ac- reflected 12 RAMP wins, 7 RASP wins, and 1 ROSETTAmin win. To the best tion in initiator and effector caspases. The main initiator of the intrinsic of our knowledge, this is the first reported study on an RNA structure-based pathway is Caspase-9, which is produced as a scarcely active zymogen (procas- multibody statistical potential. pase-9). To become a fully active protein, Procaspase-9 must overcome several steps, including cleavage process and conformational changes. In this work, we 3360-Pos Board B568 have performed molecular dynamics simulations, to gain detailed information RNA Secondary Structure Prediction Guided by Chemical Shifts on the activation mechanism of Caspase-9. By exploring the conformational Kexin Zhang1, Aaron Frank1,2. 1 2 space and analyzing the time-course evolution of geometrical and structural Chemistry, University of Michigan, Ann Arbor, MI, USA, Biophysics, characteristics of Procaspase-9, we were able to identify the participation of University of Michigan, Ann Arbor, MI, USA. structural domains of caspase on its self-activation. Furthermore, we describe Characterizing the structures of functional non-coding RNAs is crucial in ratio- relevant interactions that might trigger the gaining of proteolytic activity. nalizing structure-activity relationships. The first step in modeling RNA struc- Last but not least, we demonstrated how an intrinsically disordered region tures is building reliable secondary structure models. Though current secondary (located in the prodomain) might be responsible for promoting the recruitment structure prediction algorithms can be used to predict the secondary structure of by the apoptosome, guiding huge conformational changes associated with acti- an RNA from its sequence, the accuracy of these methods decreases as the vation, and stabilizing the active structure of Caspase-9. complexity of the RNA increases. Moreover, when building models of RNA transient states, which do not correspond to the lowest free-energy structures, 3363-Pos Board B571 additional information is needed to be able to identify the correct secondary Quantification of Sarcomeric Discontinuities in Mouse Ear Muscle using structures. We hypothesize that NMR chemical shifts contain structural infor- Deep Learning mation that can be used to guide RNA secondary structure prediction (like Brad Busse, John Heuser, Glen Humphrey, Joshua Zimmerberg. SHAPE-derived reactivities). In our study, we use machine learning algorithms National Inst. of Health, Bethesda, MD, USA. (such as random forest) to predict nucleotide-level base pairing probabilities When muscle undergoes intense physical activity, it can result in longitudinal directly from chemical shifts and then use the chemical shifts-derived probabil- damage (perpendicular to the sarcomere) to the t-tubule network. Properly ities of each residue to guide RNA secondary structure prediction. Our inves- stained, this becomes visible at the light microscopy level as small longitudinal tigations have revealed that chemical shift-derived base-pairing constraints shifts between myofibrils in otherwise continuous sarcomere structure. We are can improve RNA secondary structure prediction accuracy. Our method should interested in quantifying the presence and severity of these discontinuities in find utility in modeling transient state secondary structures of RNA using the mouse ear muscle under a variety of genetic and environmental conditions. chemical shifts signatures. In the process of acquiring enough imaging data to be experimentally robust, hand segmentation of muscle fibers and the discontinuities therein becomes 3361-Pos Board B569 impractical. We are supplementing our analysis using deep learning, a class Multiple Cryptic Binding Sites are Necessary for Robust Fibronectin of supervised machine learning algorithms popular for computer vision appli- Assembly cations. To generate the necessary training data, we rephrase the segmentation Christopher A. Lemmon, Seth H. Weinberg. task as a large number of spatially-local classification tasks, such that each of Biomedical Engineering, Virginia Commonwealth University, Richmond, the hand-segmented images used for training becomes a set of a few hundred VA, USA. thousand potential training examples. Here we present our latest work in this Assembly of elastic, insoluble fibronectin (FN) fibrils from soluble FN mono- direction. mers is a crucial step in embryonic development and wound healing. However, the mechanism of FN fibrillogenesis is still poorly understood. FN fibril assem- 3364-Pos Board B572 bly requires cell-generated forces, applied to growing fibrils and exposing Multi-Cellular Modelling of Cellular Mechanisms Gives Insights on the cryptic FN-FN binding sites buried in elastic Type III domains. The number Maintenance of Epidermal Tissue Structure and location of cryptic binding sites in Type III domains has been much Claire Miller, James Osborne, Edmund Crampin. debated, and there is increasing experimental evidence that multiple Type III Mathematics and Statistics, The University of Melbourne, Parkville, domains may contain stretch-dependent FN-FN binding sites. The requirement Australia. of cell-dependent forces to generate fibrils limits the ability to recreate fibrils in The inter-follicular epidermis (IFE) forms the outer-most layer of the cell-free experiments, restricting the investigation of the mechanisms underly- skin. Many individual components fundamental to healthy IFE structure are

BPJ 8749_8752 Wednesday, February 21, 2018 679a known: proliferation occurs only in a basal layer; above this layer cells differ- equilibrium conditions. A software package has been created to automate the entiate into keratinocytes forming further distinct layers before they are shed design process. The accelerated proteomicelle assembly simulation was from the surface. However, a definitive understanding of how the balance be- enabled by the Grid Steered Molecular Dynamics (GSMD) technique imple- tween proliferation, differentiation, and cell shedding is maintained in IFE tis- mented in NAMD. By design, no steric clashes or ring-piercing can occur. sue during homeostasis does not yet exist. This method can build proteomicelles of a wide range of detergent aggregation We have developed an agent-based multi-cellular computational model to numbers. It can also automatically adapt to the complex contours of various simulate tissue homeostasis in the skin. Epidermal cells are represented as over- membrane proteins. We demonstrate the effectiveness and robustness of this lapping spheres, and cell divisions are represented as stochastic time-driven new method using various metrics to benchmark against an unbiased proteomi- events. Cell movement is determined by adhesive attractive forces and repul- celle self-assembly simulation. SimShape will facilitate and accelerate the sive forces between other cells and the basal membrane. The magnitude of research on membrane protein-detergent interactions which is crucial for inter- these forces depends upon the types of the interacting bodies, and can vary de- preting experimental membrane protein studies. pending on factors such as cell age and location. Using this model we have analysed the impact of different cell mechanisms and 3367-Pos Board B575 behaviours on the tissue in order to investigate alternate hypotheses around Predicting Drug Delivery Efficiency into Tumor Tissues through Molecu- maintenance of tissue structure. Mechanisms investigated include cell division, lar Simulation of Transport in Complex Vascular Networks 1,2 3 4,2 cell adhesion and stiffness, and cell death. Results of this study provide insights Evan P. Troendle , Ayesha Khan , Peter C. Searson , Martin B. Ulmschneider1,3. on important considerations in modelling this system. In particular, both choice 1 2 of biological assumptions and the mathematical representation of cell division Chemistry, King’s College London, London, United Kingdom, Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA, impact the spatial distribution of proliferative cell populations. We find that the 3 4 cell mechanisms and behaviours critical to maintenance of the tissue width are Medical School, University of Exeter, Exeter, United Kingdom, Institute proliferation rate, spatial adhesion variation, and cell loss. for NanoBioTechnology, Johns Hopkins University, Baltimore, MD, USA. Efficient delivery of anticancer drugs into tumor tissues at maximally effective 3365-Pos Board B573 and minimally toxic concentrations is vital for therapeutic success. At present, Determining Receptor Interaction Kinetics through Single Molecule no method exists that can predict the spatial and temporal distribution of drugs Imaging and Computational Modeling into a target tissue after administration of a specific dose. This prevents accu- Luciana R. de Oliveira, Robel Yirdaw, Khuloud Jaqaman. rate estimation of optimal dosage regimens for cancer therapy. Here we present Biophysics, UT Southwestern Medical Center, Dallas, TX, USA. a new method that predicts quantitatively the time-dependent spatial distribu- Receptor interactions in the plasma membrane are critical for transmembrane tion of drugs in tumor tissues at sub-micrometer resolution. This is achieved signal transduction. Using live-cell single-molecule imaging it is possible to by modeling the diffusive flow of individual drug molecules through the monitor inter-receptor interactions with high spatiotemporal resolution in their three-dimensional network of blood-vessels that vascularize the tumor, and native environment, although only for the labeled subset of receptors, which into surrounding tissues, using molecular mechanics techniques. By evaluating tends to be a small fraction (<10% of the full population). At the same time, delivery into tumors supplied by a series of blood-vessel networks with varying single-molecule localization-based super-resolution microscopy can reveal degrees of complexity, we show that the optimal dose depends critically on the the spatial organization and clustering of almost the full receptor population, precise vascular structure. Finally, we apply our method to calculate the but only as a static snapshot without temporal information. As a result, it has optimal dosage of the cancer drug Doxil into a section of a mouse ovarian tu- remained a challenge to calculate receptor interaction kinetics, especially asso- mor, and demonstrate the enhanced delivery of liposomally administered doxo- ciation rates, from single-molecule data. To make use of the complementary in- rubicin when compared to free doxorubicin. Comparison with experimental formation that these two approaches reveal, we are developing a stochastic data and a multiple-compartment model show that the model accurately reca- mathematical modeling approach that computationally multiplexes dynamic pitulates known pharmacokinetics and drug-load predictions. In addition, it live-cell single-molecule data and static super-resolution data to derive receptor provides, for the first time, a detailed picture of the spatial dependence of association and dissociation rates in their native environment. The approach drug uptake into tissues surrounding tumor vasculatures. This approach is consists of (1) building a mathematical model of receptor dynamics and inter- fundamentally different to current continuum models, and reveals that the actions, mimicking the biological system and data acquisition setup and (2) target tumor vascular topology is as important for therapeutic success as the estimating the unknown model parameters, including receptor association transport properties of the drug itself. This sets the stage for revisiting drug and dissociation rates, by fitting the model to experimental single-molecule dosage calculations. and super-resolution data using the method of indirect inference for stochastic 3368-Pos Board B576 model calibration. To test this approach, we have been simulating trajectories Spatiotemporal Model for Pattern Formation in Phage-Bacteria System of diffusing receptors that interact with each other, considering a wide range of Xiaochu Li1, Floricel Gonzalez2, Birgit Scharf2, Jing Chen2. model parameters, and investigating whether our approach is able to accurately 1Biotrans, Virginia Tech, Blacksburg, VA, USA, 2Biological Sciences, recover the unknown model parameters. Our tests demonstrate that the Virginia Tech, Blacksburg, VA, USA. approach has the sensitivity to determine association and dissociation rates Bacteriophages are viruses that specifically target bacteria. Phages and their as well receptor density and labeled fraction, especially when combining host bacteria form ecosystems, which typically involve intriguing spatiotem- single-molecule and super-resolution data. We are in the process of testing poral dynamics. Previous studies on spatiotemporal dynamics of phage- the framework’s performance in the presence of experimental data error and bacteria systems mostly focused on growth of circular phage plaques on the noise. bacterial lawn. Recent experiments demonstrated intriguing asymmetrical phage plaques when phages were inoculated at the edge of an expanding bac- 3366-Pos Board B574 terial colony. We developed a mathematical model to study the pattern forma- The Simshape Method for Protein-Detergent Interaction Research tion in the phage-bacteria system. We found that negative impacts of the Yuhang Wang, Emad Tajkhorshid. bacterial density on (1) bacterial motility, and (2) phage reproduction could University of Illinois, Urbana-Champaign, Urbana, IL, USA. ‘‘freeze’’ the boundaries of the phage plaque as the bacterial colony expands Detergents have been widely used in membrane protein biophysical and radially outward. Our model sheds light on how spatial niche partitioning biochemical studies, using X-ray crystallography, cryo-electron microscopy, emerges in a host-pathogen system in which the pathogens rely on hosts, not and other methods. However, the effects of detergents on the target proteins only to reproduce, but also to spread spatially. are usually difficult to study at the molecular level using current experimental techniques. Molecular simulations offer an alternative strategy for detailed 3369-Pos Board B577 characterization of protein-detergent interactions. However, modeling a Protonation State Determines Binding Sites of Sodium Channel Inhibitors protein-detergent micelle system (proteomicelle) is very difficult due to its Amanda Buyan1, Delin Sun2, Ben Corry3. amorphous nature and the inherent complexity and diversity of protein struc- 1Australian National University, Acton, Australia, 2School of Pharmacy, tures. Here we propose a new approach termed SimShape which stands for University of Maryland, College Park, MD, USA, 3Research School fo ‘‘Simulated Shape’’. Unlike most current approaches which are based on the Biology, Australian National University, Acton, Australia. direct placement of detergents around the protein, SimShape uses three grid- Voltage-gated sodium channels are transmembrane proteins involved in gener- based external potentials to accelerate the process of detergent aggregation, al- ating action potentials for nerve signalling and muscle contractions. They are of lowing the generation a proteomicelle of a predefined shape within 100 ps of a great deal of interest, as mutations in voltage-gated sodium channels are molecular dynamics simulation. The design of the grid-based potentials was responsible for a variety of disorders, including epilepsy and chronic pain. inspired by the detergent occupancy map of a proteomicelle simulated under Designing better inhibitors and treatments, including ones which are

BPJ 8749_8752 680a Wednesday, February 21, 2018 subtype-selective, will pave a path for more targeted therapies with minimal amenable for use as fuel molecule with thermodynamic efficiency approaching side effects. A significant number of existing sodium channel inhibitors have 100%. a protonatable amine group with a pKa close to physiological pH, allowing the inhibitor to exist in both a protonated and neutral form in the bloodstream. 3372-Pos Board B580 To determine how a range of pore blocking compounds behave when inside the Computational Toolset for Glycoconjugate Modeling and Simulation pore, we have used molecular simulations with enhanced sampling. This study Sang-Jun Park, Hui Sun Lee, Jumin Lee, Wonpil Im. proposes two distinct binding sites: one for neutral and one for charged com- Biological Sciences, Lehigh University, Bethlehem, PA, USA. pounds, as well as a mechanism of action. This explains why many sodium Characterizing glycoconjugates and protein-glycan interactions in the context channel inhibitors are protonatable: one state aids access to the pore, whilst of three-dimensional structures is important in understanding glycan’s biolog- the other binds and inhibits the channel. This work better informs subtype ical roles and develop efficient therapeutic agents. However, it is challenging to selectivity and will open the doors to new and novel targeted therapies. achieve this goal due to limitations in available experimental approaches and computational tools. To tackle this problem, we have developed GlycanStruc- 3370-Pos Board B578 ture.ORG, a web portal to provide various computational tools. (1) Glycan Computational Study of Butyrylcholinesterase Inhibition by Aryl Alkyl Reader has been developed for automatic detection and annotation of carbohy- Cholinyl Phosphorus Derivatives drates, their chemical derivatives, and glycosidic linkages from PDB files (both Nicholas Humphrey, Mariel Sanchez, Christine Chung, Eric Sorin. in the PDB and mmCIF formats). Molecular system and input generations for California State University of Long Beach, Long Beach, CA, USA. the glycoconjugate simulation are available through CHARMM-GUI, http:// Acetylcholine (ACh) is a neurotransmitter that is released from nerve cells to www.charmm-gui.org. (2) The glycan fragment database (GFDB) provides send signals to other cells, and acetylcholinesterase (AChE) is a highly specific an intuitive glycan sequence search tool that allows users to search for complex enzyme that hydrolyzes ACh to regulate intercellular communication. Howev- glycan structures in the PDB. After a glycan search, each selected glycosidic er, butyrylcholinesterase (BChE), a non-specific AChE imposter, also breaks torsion angle distribution can be displayed and clustering analysis is used to down choline based esters such as ACh. The activity of BChE has been found provide most populated glycan structures, which can be useful for glycan struc- to gradually increase over time in patients with Alzheimer’s disease (AD), ture modeling. (3) GS-align is an algorithm to identify the best structural align- which has been linked to a significant decrease in synaptic ACh levels, thus dis- ment between glycan structure pairs and to provide a length-independent score rupting intercellular communication. In the treatment of AD, discovering inhib- of the structural similarity. This tool is useful to study the relationship between itors that prevent the activity of BChE and that do not interfere with the activity multiple glycoforms and biological functions, and glycan structure prediction. of AChE is vital. Aryl alkyl cholinyl phosphorous (AACP) derivatives are or- (4) Glycan Modeler is a template-based method to predict glycan structures ganophosphates that are expected to reversibly bind within the BChE binding from a given sequence using the PDB glycan structures as fragment template. pocket in the same manner as natural physiological substrates. This study uti- After templates are identified by the random forest algorithm, Glycan Modeler lizes massive flexible inhibitor docking calculations to predict the relative bind- provides two conformational sampling approaches that are assembly of rigid ing affinities between the enzyme and a number of AACP derivatives and to bodies and satisfaction of spatial restraints using by conformational space an- predict the optimal binding orientation(s) of each AACP derivative within nealing (CSA). the BChE active site. When compared to their previously studied dialkyl phenyl 3373-Pos Board B581 phosphate (DAPP) analogs, AACP derivatives are predicted to interact more Cellpacking: Examining Influenza Virulence through Software- strongly with the binding site of BChE, thus serving as more potent inhibitors. Engineered Protein Packing Our results reproduce experimental trends in binding affinity, indicating that Christian Seitz1, Ludovic Autin2, Rommie Amaro1, J. Andrew McCammon1, derivatives with longer alkyl chains show increased inhibitory strength (as Arthur J. Olson2. observed for DAPP derivatives), and the neutralization of the charge of the 1Chemistry & Biochemistry, UC-San Diego, La Jolla, CA, USA, 2Integrative choline group and protonation of the neutral choline group had little effect Structural and Computational Biology, Scripps Research Institute, La Jolla, on inhibitor strength, offering insight into the structural preferences of BChE CA, USA. for specific AACP derivatives. This study thus provides a framework for Computational studies on viruses can reveal a whole host of new information, both future inhibitor design and more rigorous molecular dynamics studies of including protein cooperativity, conformational changes and numerical binding cholinesterase-inhibitor interaction. data. Unfortunately, computational studies on viruses have been lacking in the literature due to their size and realistic time constraints. Thus, there are several 3371-Pos Board B579 holes in the current state-of-the-art to be filled. Currently, it is thought that ATP as a Fuel Molecule: Evolutionary Selection of Magnesium-ATP Inter- influenza A has multiple conformations, including one where hemagglutinin action Mode Facilitates Lossless Chemomechanical Coupling for ATPases (HA) potentially interacts with neuraminidase (NA) to facilitate binding of 1 2 2 € 1 Floris P. Buelens , Hadas Leonov , Bert de Groot , Helmut Grubmuller . sialic acid (SA). However, this cooperativity is not clear as cytoskeletal pro- 1Theoretical and Computational Biophysics, Max Planck Institute for 2 teins may fix the structure and influence the dynamics of HA and NA. One addi- Biophysical Chemistry, Goettingen, Germany, Computational Biomolecular tional inconsistency is that SA preferentially binds to NA, even though binding Dynamics, Max Planck Institute for Biophysical Chemistry, Goettingen, to HA is needed for endocytosis. Decoding the reasons for this preference will Germany. require long molecular dynamics (MD) simulations; one way to realize this is In its physiological setting as the energy currency of life, the adenosine triphos- through software engineering. Investigating viral characteristics as the defining phate (ATP) molecule exists predominantly as a complex with a single magne- science goal drives the development of cellPACK, a software program able to sium ion. Two conformational substates of ATP.Mg2þ can be distinguished: a 2þ incorporate experimental and theoretical data to probabilistically pack proteins ‘closed’ form, with Mg coordinated by all three phosphate groups, and an into a defined space. By creating cellPACK functionalities and linking them to ‘open’ form, where only the two terminal phosphate groups coordinate 2þ simulation programs, accurate protein-protein interactions can be modeled. The Mg . Both states are energetically accessible in solution, albeit separated by first step in this process is building a model; here we built the world’s first full a high energetic barrier. While these conformational substates have been stud- viral model, of the influenza A, H1N1 Cali09 strain, which is needed to better ied before, the physicochemical relevance of this distinction for ATP as an understand intrinsic characteristics of viruses and how cross-species mutations enzyme substrate has not previously been explored. can influence virulence. This model can then be used in MD and Brownian dy- We have examined the distribution of these substates of ATP in the Protein namics (BD) simulations to tease out how protein cooperativity affects influ- Data Bank. Strikingly, we find an apparently exclusive preference for the enza virulence. ‘open’, doubly-coordinated variant among enzymes subject to evolutionary pressure to perform lossless chemomechanical coupling. This contrasts with 3374-Pos Board B582 other enzyme families that are not subject to such evolutionary pressure, which Reconciling Simulated Ensembles of Apomyoglobin with Experimental appear to display no systematic preference. HDX Data We build on this finding with a quantitative examination of the energetics of Hongbin Wan1, Yunhui Ge1, Asghar Razavi2, Vincent Voelz1. ATP hydrolysis, and find that, for the cycle to satisfy the constraints of lossless 1Chemistry, Temple University, Philadelphia, PA, USA, 2Weill Cornell chemomechanical coupling, the use of the the open, double-coordinated Medicine, New York, NY, USA. substate as a fuel molecule avoids a substantial redistribution of interaction en- Hydrogen/deuterium exchange (HDX) is a powerful tool to investigate protein ergy that would be necessary were the closed substate to be used, thus sidestep- conformational dynamics at equilibrium. In the classic model of HDX, each of ping a non-trivial engineering challenge in evolutionary terms. We conclude the amide hydrogens of a protein can either exist in an ‘‘open’’ conformation, that, of the two ATP substates that could have been chosen, the evolutionary where HDX can occur with the exchange rate kint, or in a ‘‘closed’’ conforma- process for chemomechanical converting enzymes has favoured the one most tion, where HDX rate, kex, gets much slower. The extent of protection for a

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residue i, is characterized by a protection factor,PFi = kex,i/kint,i, which is ware is available on our webpage [4]. [1] Dimura, M., et al., Quantitative FRET related to the effective free energy difference between open and closed states, studies and integrative modeling unravel the structure and dynamics of biomol- DG, through ln(PFi )=bDGi. However, it has been challenging to make direct ecular systems. Current Opinion in Structural Biology, 2016. 40: p. 163-185. connections between molecular simulations of native-state protein dynamics, [2] Sindbert, S., et al., Accurate distance determination of nucleic acids via For- both in predicting HDX PF from trajectory data, and in using experimental pro- ster resonance energy transfer: implications of dye linker length and rigidity. J. tection factors as restraints in simulated ensembles. In this study, we propose an Am. Chem. Soc., 2011. 133(8): p. 2463-2480. [3] Kalinin, S., et al., A toolkit expression, ln(PF)= bc þ bh þ b0, to predict the protection factors and benchmark study for FRET-restrained high-precision structural modeling. based on the average number of heavy-atom contacts and hydrogen Nature Methods, 2012. 9(12): p. 1218-1227. [4] http://www.mpc.hhu.de/ bonds observed in simulations. The model parameters are trained on software.html ultra-long, all-atom trajectories of proteins with experimentally measured pro- tection factors (BPTI and ubiquitin). Using Bayesian inference to propagate the 3377-Pos Board B585 uncertainties in model parameters, we then use this model to refine conforma- Towards Real-Time Holographic Three-Dimensional Imaging with Ma- tional ensembles of apomyoglobin at neutral pH. To minimize bias, we simu- chine Learning 1 2 1 lated apomyoglobin using a range of weak to strong amide exposure restraints. Lindsey Peng , Anaya Srivastava , Christopher M. Yip . 1Chemical Engineering, University of Toronto, Toronto, ON, Canada, We applied a transition-based reweighting method, TRAM, to construct a 2 Multi-ensemble Markov model from the combined simulation data, and then Biochemistry, University of Toronto, Toronto, ON, Canada. used our Bayesian Inference of Conformational Populations (BICePs) algo- Real-time visualization of cellular and molecular dynamics is critical in rithm to refine a conformational ensemble that best matched the experimental achieving a flexible, adaptive imaging platform capable of automatically iden- structural data. The resulting ensemble shows a partially-disordered F/H helix, tifying regions of interest and proactively modifying acquisition parameters and atomically detailed information that was previously missing from 2D NMR In holographic microscopy, the time-intensive steps of sample and / or stage studies. manipulation are replaced by the computational time required to accomplish the volume reconstruction. A key challenge in real time visualisation of 3375-Pos Board B583 time-lapse holographic microscopy arises when long-term studies are under- Improved Specificity Prediction of Small Molecule Myosin Inhibitors way, such as the study of biofilm development, which may take place over hours or days. In this case, computing 3-D positions and reconstructions is a through Ensemble-Based Molecular Docking CPU-intensive process. To address this, efforts are underway to apply GP- Dmitrij Malcev1,2, Adrian Kishonti3, Georgios Tsiavaliaris2, GPU based approaches that allow for parallelization of the process. In the Hans-Joachim Kno¨lker3, Matthias Preller2,4. 1Organic Chemistry, Leibniz Universit€at Hannover, Hanover, Germany, absence of GPU platform, an attractive alternative is to consider the use of arti- 2Institute for Biophysical Chemistry, Hannover Medical School, Hanover, ficial neural networks(ANN) to bypass the repeated matrix multiplication asso- Germany, 3Department Chemie, Technische Universit€at Dresden, Dresden, ciated with each reconstructing distance. Here, we demonstrate how supervised 4 ANN can be used by determining the algorithms necessary to estimate the z-po- Germany, Centre for Structural Systems Biology, Deutsches Elektronen- sition of particles from a single 2-dimensional in-line hologram. With only a Synchrotron (DESY), Hamburg, Germany. Computational calculations of small molecule-protein interaction using molec- small cost of one-time training, the newly-found algorithm would bypass the ular docking algorithms are powerful tools for the study of ligand recognition iterative calculations required to search through the dataset to identify the final and drug discovery. However, accounting for protein flexibility remains still a position of objects in each newly acquired hologram. This in turn will help real- challenge. Here we show the combination of physics-based molecular dy- ise real-time visualisation of holographic imaging and achieving a adaptive im- namics simulations with time-efficient docking that yield a highly improved aging system where executive decisions on the speed and volume of imaging interest can be made during acquisition. correlation of the in silico predictions with experimentally determined inhibi- tory potencies of halogenated carbazoles towards different isoforms of the mo- Posters: Single-Molecule Spectroscopy II tor protein myosin. Analysis of protein dynamics revealed marked structural changes of the allosteric target pockets in different isoforms. Ranking of the 3378-Pos Board B586 predicted binding affinities of a series of halogenated carbazoles towards Efficient and Fast Purification Method for Fluorescent Dye-Labeled Oligo- different myosin isoforms using molecular docking against clustered ensembles nucleotides of protein conformations correlated well with IC50 values, measured using a So Young Bak1, Jihee Hwang1, Sohyeon Bae1, Soonkyu Lim2, spectroscopy-based ATPase assay, and gave high potencies of carbazole deriv- Younggyu Kim2, Seong Keun Kim1. atives in the low micromolar range. Our results demonstrate the superior, but 1Chemistry, Seoul National University, Seoul, Republic of Korea, still resource-efficient performance of our combined molecular dynamics - mo- 2LumiMac. Inc., Seoul, Republic of Korea. lecular docking method in computing the specificity behavior of small mole- Fluorescent dye labelling is an essential and general procedure in Biophysics. cules between different myosin isoforms, and highlight the importance of Especially in single-molecule spectroscopy, labeling the oligonucleotides with including protein flexibility in the in silico predictions. fluorescent dyes and purifying the excess to enhance the signal-to-noise ratio are unavoidable. Common purification methods such as gel filtration, ethanol 3376-Pos Board B584 precipitation and HPLC consume large amounts of time and cause a huge sam- Integrative Molecular Modelling of Biomolecules Guided by FRET ple loss. In order to improve the efficiency and to simplify the procedure of Experiments removing the unlabeled free dyes, here we introduce a new purification method. Christian A. Hanke, Mykola Dimura, Thomas-Otavio Peulen, Through this new method the labeled oligonucleotides and the unlabeled free Holger Gohlke, Claus A.M. Seidel. dyes can be separated and purified by pH-control. This innovative method is € € Heinrich-Heine-University Dusseldorf, Dusseldorf, Germany. easy, fast and efficient which is applicable to various classes of dyes regardless Fo¨rster Resonance Energy Transfer (FRET) experiments provide valuable in- of charge, size and structure with a high recovery yield and purification result. formation on the dynamics and the tertiary and super-tertiary structure of dy- namic biomolecules. We demonstrated that FRET experiments together with 3379-Pos Board B587 computational methods, combined in hybrid approaches, can be employed No More ‘‘Wiggles’’: Decoherent Acousto Optic-Based High-Resolution for computational structural modeling of biomolecules with high accuracy Tweezers Combined with Multi-Color Fluorescence and precision [1]. Here we present a methodology for FRET-based structural Cho-Ying Chuang, Andrew Baker, Miles Whitmore, Matthew Comstock. modeling of proteins and nucleic acids. The methodology can help in designing Michigan State University, East Lansing, MI, USA. FRET experiments by providing suggestions for the most informative labeling Recent advances in hybrid single molecule methods that couple angstrom- positions and FRET pairs for structural studies, allowing to reduce the number resolution optical trapping with single molecule fluorescence (high-resolution of necessary measurements [1]. Furthermore, the methodology includes the fleezers) have demonstrated the utility of measuring protein activity and screening and scoring of an ensemble of conformations obtained by e.g. molec- conformation simultaneously e.g., revealing detailed helicase conformation ular dynamics or coarse-grained simulations, or homology modeling against coupled to DNA unwinding. Here we present advancements in multi-color experimental (FRET) data. This allows to identify conformers from the high-resolution fleezers methods. First, we have developed a high-resolution ensemble which best agree with experimental data. As FRET experiments fleezers instrument with three-color confocal fluorescence microscopy. To are label-based, the methodology takes into account the positional uncertainty reduce photobleaching caused by the optical traps, timeshared dual optical traps of the attached labels via accessible volume calculations [2, 3]. Furthermore, were interlaced and synchronized with three fiber coupled fluorescence excita- we present the potential of FRET-restrained simulations to refine structural tion lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting models in order to reach optimal agreement with experimental data. The soft- fluorescence detectors. We report performance with a set of fluorophores

BPJ 8749_8752 682a Wednesday, February 21, 2018 varying both optical trap and fluorescence excitation intensity. In the fleezers, ing a simple model assuming identical equilibrium constants for all oligomers the Cy5 fluorophore dramatically outperforms other commonly used fluoro- except the 24-mer, revealing a stability difference on the order of 5 kJ/mol. phores. Second, we have devised a method that completely removes acousto- Using the same approach, we are also able to reveal the function and ener- optic device trap positioning errors. Acousto-optic devices have been applied getics behind the powerful HIV inhibitor banana lectin. We demonstrate high- extensively in optical tweezers because of their flexibility and speed, however ly cooperative binding of banlec to HIV envelope protein and quantify the these devices have well-known trap positioning inaccuracies (‘wiggles’) that associated equilibrium constants. Taken together, these results demonstrate limit their usefulness, particularly for high-resolution applications. We show the unique capability of iSCAMS to directly and natively study oligomeric that these inaccuracies are due to interference patterns within the device sound distributions and dynamics of biomolecules in solution. fields. We have devised a method that removes these inaccuracies by reducing the coherence of the sound fields by directly controlling and randomizing the 3382-Pos Board B590 phase of the radio frequency control. We demonstrate that the trapping inaccur- Interferometric Scattering Mass Spectrometry (ISCAMS): Single Mole- acies are completely eliminated, and that no additional measurement noise is cule Mass Imaging in Solution added, for both constant trap position and force-ramp measurements. We Daniel Cole, Gavin Young, Nikolas Hundt, Philipp Kukura. show that this random phase method is applicable both to acousto-optic modu- Physical and Theoretical Chemistry, The University of Oxford, Oxford, lator and deflector type devices and can be easily integrated via a software up- United Kingdom. grade into existing instruments. The advent of single molecule detection and imaging with optical microscopes has transformed our ability to interrogate structure and dynamics on nanoscopic 3380-Pos Board B588 length scales. Most single molecule imaging techniques rely on detection of Probing p53 Activation by Live-Cell Single-Molecule Chromatin Binding fluorescence as a means of distinguishing the molecule of interest from the Measurements background. To broaden the scope of single molecule techniques, recent efforts Alessia Loffreda1,2, Emanuela Jacchetti2, Sofia Antunes2, Paolo Rainone2,3, have been aimed at label free imaging modalities, including approaches based Tiziana Daniele2, Tatsuya Morisaki4, Marco E. Bianchi5, Carlo Tacchetti2,6, on plasmonically-enhanced whispering gallery mode detection and light scat- Davide Mazza1,2. tering, such as interferometric scattering microscopy (iSCAT). 1Imaging Department, European Centre for Nanomedicine, Milan, Italy, Here, we demonstrate that numerical aperture based differentiation of back- 2Experimental Imaging Centre, San Raffaele Scientific Institute, Milan, Italy, ground light from scattered light with a partially transmissive mask placed in 3Institute of Molecular Bioimaging and Physiology, National Researches the back focal plane of a high numerical aperture objective enhances the Council, Segrate, Italy, 4Department of Biochemistry and Molecular Biology, contrast of scatterers near an interface, thereby enabling routine label-free Colorado State University, Fort Collins, CO, USA, 5Chromatin Dinamics imaging of single proteins in an iSCAT microscope. Combination of this Unit, Universita` Vita – Salute San Raffaele, Milan, Italy, 6Experimental concept with a compact microscope construction strongly suppresses Imaging Centre, Universita` Vita – Salute San Raffaele, Milan, Italy. external perturbations resulting in a detection noise floor of 6 kDa at 10 Live-cell microscopy revealed the transient nature of transcription factors (TFs) Hz frame rate. Using this instrument, we show that the iSCAT signal of a binding to chromatin. However, it is still not clear whether the duration of these single protein in solution is directly proportional to its molecular weight. binding interactions can be tuned in response to an activating stimulus, and We achieve a mass accuracy comparable to native mass spectrometry of whether such modulation can control the transactivation of target genes. To 1% and a mass precision of 1.8% of the molecular weight, enabling not cover this issue we focused on the tumor-suppressor p53 by combining live-cell only mass quantification, but also direct observation of small molecule bind- single molecule tracking and single cell in-situ measurements of transcription. ing. Our approach is equally applicable to biomolecules composed of amino We show that the fraction of chromatin bound p53 and its residence time are acids, lipids, carbohydrates and combinations thereof, paving the way for increased following genotoxic stress. Moreover, we found that the modulation quantitative mass spectrometry in solution. of p53 residence time on chromatin requires post translational modifications (PTMs) and correlates with the induction of transcription of target genes. These 3383-Pos Board B591 results allowed us to depict a model in which the modification state of the TF, Single Molecule Fluorescence Fluctuation Analysis of Fluorescent Probes that accompanies its increase, determines the TF transcriptional activity by tun- in Crowded Environments: Molecular Size and Shape Dependence ing its residence time on target sites. Our findings could have an important rele- Hong Bok Lee, Megan Currie, Hannah Leopold, Erin D. Sheets, vance for those cancers that express an inactive wild type form of p53, like Ahmed A. Heikal. neuroblastoma (NS), in which has been recently reported a defect in PTMs Chemistry and Biochemistry, University of Minnesota - Duluth, Duluth, MN, of p53. Thus, we are currently performing SMT measurements in NS cell USA. line to verify whether p53 binding kinetics are affected and we aim to restore Living cells are crowded with heterogeneous distributions of macromolecules the proper PTMs in order to rescue p53 binding kinetics and its transactivation which influence protein folding, molecular transport, and biochemical reac- potential in these cancers. tions. In addition to crowding, the viscosity in living cells varies based on the cellular location. We hypothesize that the translational diffusion coeffi- 3381-Pos Board B589 cient of fluorescent probes deviates from the Stokes-Einstein model of random Direct Observation of Oligomeric Protein (Dis)Assembly and Drug- walks in a manner that depends on both the molecular size and shape of those Induced Cross-Linking at the Single Molecule Level with Interferometric probes. Scattering Mass Spectrometry (ISCAMS) To test this hypothesis, we used fluorescence fluctuation analysis to measure the Gavin Young, Nikolas Hundt, Anna Olerinyova, Ayla Ansari, Daniel Cole, translation diffusion coefficients of rhodamine green 110, enhanced green fluo- Philipp Kukura. rescent protein (EGFP), water-soluble quantum dots, and a family of Department of Chemistry, University of Oxford, Oxford, United Kingdom. mCerulean-linker-mCitrine constructs with variable linker lengths and flexibil- The self-assembly of biomolecules plays a central role in the physiological ities. The mCerulean-linker-mCitrine systems represent fluorescence resonance and pathological function of proteins. Similarly, the role of drugs is often energy transfer (FRET) probes that were rationally designed to be genetically- to perturb protein-protein interactions, thereby altering native function and encoded sensors of compartmentalized macromolecular crowding. Nanomolar regulation. The frequently associated polydispersity of the involved species, concentrations of these probes were prepared in PBS buffer enriched with var- however, poses a distinct challenge for analytical techniques. The state-of- ied concentrations of glycerol (0-900 g/L) and Ficoll-70 (0-300 g/L). The bulk the-art in terms of mass resolution requires expert operation and functions viscosity of these environments was independently measured using a viscom- only in the gas phase in the form of native mass spectrometry, while eter. For fluorescence fluctuation analysis of the size and shape dependence solution-based techniques are limited in mass resolution, accuracy and preci- of these fluorescent probes, we employed different modalities of fluorescence sion. Single molecule methods, while in principle applicable, suffer from the correlation spectroscopy (FCS) to quantify the diffusion coefficient in both ho- need for the introduction of external labels and a lack of precision as a result mogeneous (glycerol-enriched) and heterogeneous (Ficoll-70-enriched) envi- of imperfect labeling efficiencies and photochemical and photophysical ef- ronments. Our results suggest that the deviation from the Stokes-Einstein fects. Here, we show that interferometric scattering mass spectrometry (iS- model is dependent upon the size of the fluorescent probe, as well as the con- CAMS) is capable of characterising protein polydispersity and oligomeric centration of crowding agents. Currently, we are using global fits to quantify (dis)assembly in solution in a label-free manner, one molecule at a time. the observed trends in the measured translational diffusion coefficients. Taken To illustrate this, we investigated the disassembly of the 24-mer small heat together, these single-molecule studies of size-dependent fluorescent probes in shock protein, HSP16.5. We observed an increase in the proportion of small biomimetic crowding help elucidate the effects of homogeneous and heteroge- oligomers compared to the predominant 24mer with decreasing monomer con- neous environments on translational diffusion as a necessary step towards centration. Critically, we were able to model the oligomeric distributions us- in vivo studies.

BPJ 8749_8752 Wednesday, February 21, 2018 683a

3384-Pos Board B592 structure, proximities closer than 2 nm are not possible. Thus, CET between Versatile Tools towards Real Time Single Molecule Biology FPs at physiological temperatures is thought to be impossible. Nonetheless, us- Jordi Cabanas-Danes, Rosalie P.C. Driessen, Avin Ramaiya, Philipp Rauch, ing two-photon excitation and time-correlated single photon counting we have Andrea Candelli. observed both anomalous ultra-fast energy transfer and strong antibunching be- LUMICKS, Amsterdam, Netherlands. haviors in FP assemblies composed of mVenus, mClover, or mNeonGreen. Our Biological processes performed by proteins interacting with and processing experiments suggest stronger than expected coupling between FPs. Thus, we DNA and RNA are key to cell metabolism and life. Detailed insights into these speculate that CET between FPs at physiological temperature may be possible. processes provide essential information for understanding the molecular basis of life and the pathological conditions that develop when such processes go 3387-Pos Board B595 awry. The next scientific breakthrough consists in the actual, direct, real-time Single Molecule Study of ATAD5-Induced Unloading of PCNA observations and measurements of the individual mechanisms involved, in or- SeungWon Lee, Eunji Ryu, Sukhyun Kang, HaJin Kim. der to validate and complete the current biological models. Single-molecule UNIST, Ulsan, Republic of Korea. technologies offer an exciting opportunity to meet these challenges and to study The loading and unloading steps of the DNA clamp, proliferating cell nuclear protein function and activity in real-time and at the single-molecule level. Here, antigen (PCNA), which acts as a processivity factor for DNA polymerase, are we present our efforts for further enabling discoveries in the field of biology crucial for the precise control of DNA replication. The underlying molecular and biophysics using both the combination of optical tweezers with single- mechanisms for these processes are yet to be well understood. Recently it molecule fluorescence microscopy (C-TrapTM). We show the latest applica- was discovered that ATAD5 protein is involved in the unloading of PCNA tions of these technologies that can enhance our understanding not only in from the chromatin. We studied the molecular mechanism of this process the field of DNA/RNA-protein interactions but also in the fields of molecular from single molecule approaches. By site-specific fluorescent labeling of motors, protein folding/unfolding, cell membranes and genome structure and PCNA proteins, which form trimeric rings around DNAs by the clamp loader, organization. These experiments show that the technological advances in replication factor C (RFC) complex, we could directly observe their loading hybrid single-molecule methods can be turned into an easy-to-use and stable and unloading dynamics at the ssDNA-dsDNA junction, together with their instrument that has the ability to open up new venues in many research areas. diffusion along the DNA. Upon loading on the DNA junction, PCNA was sta- bly bound at the preferred 3’-end ssDNA-dsDNA junction and also made tran- 3385-Pos Board B593 sient diffusion dynamics away from this site, as observed from our single Fluorescence Correlation Spectroscopy with Nanowire Waveguide Illumi- molecule fluorescence resonance energy transfer (smFRET) measurements. nation for High Concentration Conditions By substituting the first subunit of five-subunit RFC complex with ATAD5, Olaoluwalotobi Thomas, Arstanbek Tulekeyev, Justin Isaac, Huizhong Xu. its function can be switched to the unloading of PCNA. Interestingly, PCNA Dept of Physics and Astronomy, San Francisco State University, San trimer was unloaded by ATAD5 complex not all at the same time but one- Francisco, CA, USA. by-one in concentration-dependent manner. This suggests an intriguing mech- Fluorescence correlation spectroscopy (FCS), a technique based on correlating anism of PCNA unloading, in which the incomplete PCNA complex can be the fluorescence signal from a small sample volume, has been commonly used held on the DNA while being serially detached. By additionally labeling to study biological processes such as photophysical phenomena, binding ki- ATAD5 protein, we could reveal more precise mechanism from three-color netics, and intracellular dynamics. In a conventional FCS setup, the sample vol- smFRET measurements. We present our preliminary results on these attempts. ume is defined by a focused laser beam for excitation and a confocal pin hole 3388-Pos Board B596 for collection resulting in a size of about one femtoliter. Evanescent waves Single-Molecule DNA Unzipping Reveals Asymmetric Modulation of the from total internal reflection have been used to obtain a smaller sample volume, Transcription Factor EGR-1 by its Binding Site Sequence and Context as a result of the improved confinement along the longitudinal axis for the illu- Hadeel Khamis1, Sergei Rudnizky1,2, Omri Malik1, Allison Squires3, mination. However, applying FCS under physiological conditions where the Amit Meller4, Melamed Philippa2, Ariel Kaplan2. molecule concentration is typically in the mM range requires an even smaller 1Physics, Technion, Haifa, Israel, 2Biology, Technion, Haifa, Israel, sample volume. Here we propose to apply FCS with nanowire waveguide illu- 3Chemistry, Stanford University, Stanford, CA, USA, 4Biomedical mination with nanometer-sized lateral and longitudinal confinement. Nanowire Engineering, Technion, Haifa, Israel. waveguides, made of a dielectric nanowire with a diameter of 50 nm Binding of transcription factors to regulatory elements is a central step in the embedded inside a metal film, allow for the efficient transmission of visible complex regulation of gene expression, and its perturbation is linked to light by exploiting surface waves traveling along the dielectric-metal boundary. numerous disease states. Egr-1 is an inducible transcription factor that binds Such waveguides can generate a lateral illumination area of about 50 nanome- to 9-bp response elements via three zinc finger domains in response to a variety ters in diameter. In addition, a longitudinal confinement of around 20 nanome- of stimuli, such as hormonal signals and stress. In our work, we use single- ters is achieved with the rapidly-attenuating near fields exiting from the molecule DNA unzipping with optical tweezers to study the binding properties waveguide. The strong confinement in both lateral and longitudinal directions of Egr-1 to its binding sites, using as a model the promoter of the Lhb gene. We thus lead to a sample volume on the order of one zeptoliter, allowing for FCS find that both the core 9 base pairs bound to Egr-1 in each of the binding sites on measurements in the mM concentration range. By combining Brownian dy- Lhb, and the base pairs flanking these sites, modulate the affinity and structure namics simulations with the illumination profile obtained from finite element of the protein-DNA complex. The effect of the flanking sequences is asym- method simulations of a zinc oxide nanowire waveguide in a silver metal metric, with a stronger effect for the sequence flanking the triplet in contact film, we numerically calculate the correlation function to demonstrate the with zinc finger 3. Next, using a novel method to characterize the dissociation use of this method in the study of molecular dynamics under high concentration time of Egr-1 at the single molecule level, we show that a local, mechanical conditions. perturbation of the interactions of zinc finger 3 is able to destabilize the com- plex more effectively than a similar perturbation acting on the interactions of 3386-Pos Board B594 ZF1. Taken together, our findings suggest a novel functional role for ZF3 in Anomalous Ultra-Fast Energy Transfer Suggests Coherent Energy Trans- the interaction of Egr-1 with other proteins. fer between Fluorescence Proteins Youngchan Kim1, Grace H. Taumoefolau1, Henry L. Puhl1, 3389-Pos Board B597 Tuan A. Nguyen1, Paul S. Blank2, Steven S. Vogel1. Cholesterol Promotes Cytolysin a Activity by Stabilizing the Intermediates 1NIH/NIAAA, Rockville, MD, USA, 2NIH/NICHD, Bethesda, MD, USA. during Pore Formation Two assumptions for FRET experiments using fluorescent proteins (FPs) are Pradeep Sathyanarayana1, Satyaghosh Maurya2, Monisha Ravichandran2, that FPs act independently (i.e. very weak coupling) and that they behave Ganapathy K. Ayappa3, Sandhya S. Visweswariah4, Rahul Roy3. like classical organic fluorophores. When conventional fluorophores are in 1Center for Biosystems Science and Engineering, Indian Institute of Science, close-proximity (< 1 nm) and/or are cooled to temperatures approaching abso- Bangalore, India, 2Indian Institute of Science, Bangalore, India, 3Chemical lute zero, stronger coupling is possible. Under these conditions coherent energy Engineering, Indian Institute of Science, Bangalore, India, 4Molecular transfer (CET) may enable multiple fluorophores to behave as a single quantum Reproduction, Development and Genetics, Indian Institute of Science, entity. CET is thought to play a key role in photosynthesis, and vis-a`-vis tech- Bangalore, India. nology, may enable quantum computing. CET manifests as ultra-fast long-dis- Pore-forming proteins (PFP) are cell membrane rupturing proteins and form the tance energy transfer within fluorophore assemblies. Antibunching, a uniquely largest class of proteins that mediate bacterial virulence. PFPs are secreted as quantum mechanical behavior, is consistent with CET. Physiological tempera- water-soluble monomers that bind strongly to the lipid membrane of eukaryotic tures extinguish CET by promoting rapid collisional dephasing of fluorophore cells. They do not contain a signature transmembrane motif, but nevertheless vibrational modes. Moreover, because FP fluorophores are encased in a ß-barrel adopt structures that traverse the membrane, thereby allowing the passage of

BPJ 8749_8752 684a Wednesday, February 21, 2018 molecules from within the cell to the exterior, resulting in host cell lysis. The on the nanometer scale. Our goal is to develop the use of quantum dots as do- conformational transitions of a PFP from a water-soluble structure to a distinct nors for quantitative single-molecule FRET experiments. membrane-associated form are not understood in mechanistic detail. In this study, we use single molecule tracking and spectroscopy to understand the dy- 3392-Pos Board B600 namics of Cytolysin A (ClyA), a prototypical a-PFP from E. coli, on bilayer Conformations and Single-Molecule Dynamics of Nitric Oxide Synthase 1 2 3 lipid membranes. Binding of ClyA to PEG-cushioned supported bilayer was Carey K. Johnson , David C. Arnett , Brian C. Smith . 1Chemistry, University of Kansas, Lawrence, KS, USA, 2Chemistry, rapid and reached saturation within a few seconds. Diffusional analysis of par- 3 ticle trajectories showed existence of mobility states that could be assigned to Northwestern College, Orange City, IA, USA, Biochemistry, Medical two distinct structural states that match structures of the protein in the monomer College of Wisconsin, Milwaukee, WI, USA. form and the protomer state. In the presence of membrane cholesterol, the pop- Nitric oxide synthase (NOS) functions by transfer of electrons sequentially from ulation of the slower moving species increased, with the concomitant decrease FAD to FMN in the reductase domain of the enzyme and then from FMN to the in the fast mobility population. Analysis of transition probabilities by Hidden heme in the oxygenase domain of the opposing member of a homodimeric com- Markov Model revealed that the conversion from fast to slower mobility state plex. Efficient electron transfer is activated by the calcium signaling protein was due to the conformational transition from a peripherally-associated protein calmodulin (CaM) and requires close proximity of electron-transfer donors and conformation to a membrane-inserted conformation respectively. Furthermore, acceptors. The sequence of electron transfers therefore necessitates multiple a hitherto unknown cholesterol interaction domain was discovered in the trans- conformational states of the enzyme, suggesting that the activity of the enzyme membrane helix of ClyA and validated by mutational studies. Therefore, we is conformationally gated. We have detected the presence of multiple conforma- propose a molecular mechanism for selective pore formation in eukaryotic tional states of NOS by time-resolved detection of fluorescence from a fluoro- membranes which is driven by conformational selectivity in the presence of phore attached to CaM. Fluorescence is quenched by FRET to the heme groups cholesterol. of the enzyme, and the extent of quenching depends on the conformational state of the enzyme. Single-molecule intensity trajectories reveal multiple fluorescence 3390-Pos Board B598 states with dynamics on the millisecond to second time scales. Analysis suggests DNA Stability after Oxidative Damage sequential conformational interchange, with the longest-lived state being highly Micah J. McCauley1, Leah Furman2, Catherine A. Dietrich3, Caitlin J. Cain2, quenched, consistent with a conformation in which CaM is in close proximity Diana Seminario3, Ioulia Rouzina4, Megan E. Nunez2, Mark C. Williams1. with the heme groups. Analysis of conformational dynamics is underway. 1Department of Physics, Northeastern University, Boston, MA, USA, 2 3393-Pos Board B601 Department of Chemistry, Wellesley College, Wellesley, MA, USA, The Other Histone: Probing the Role of Linker Histone in a Chromato- 3Department of Chemistry, Mount Holyoke College, South Hadley, MA, 4 some USA, Department of Chemistry and Biochemistry, The Ohio State Madhura De, Kathrin Lehmann, Katalin To´th. University, Columbus, OH, USA. Biophysics of Macromolecules, Deutsches Krebsforschungszentrum, One of the most common DNA lesions is created when reactive oxygen spe- Heidelberg, Germany. cies modify guanine bases to create 8-oxoguanine. 8-oxoguanine may bind The chromatosome, or the smallest unit of chromatin, comprises of the nucleo- to the opposing cytosine in the anti conformation or to an opposing adenine some (core histone octamer þ 146 bp DNA surrounding it in 1.65 turns) with in the syn conformation. To elucidate the free energy of 8-oxoguanine-con- additional stretches of linker DNA associated with the linker histone protein taining base pairs, DNA hairpin stability was quantified using optical twee- (LH). Although recent cryo-EM and X-Ray crystallographic studies have shed zers and compared to a predictive model of base-pair energies. In contrast to some light on the localization of the LH, the linker DNA specificity of LH still either a canonical guanine-cytosine or adenine-thymine pair, an 8-oxogua- remains unclear. In this study, we have employed single molecule FRET spec- nine-cytosine base pair shows modest destabilization of several kBT. The troscopy to observe the disassembly of the LH induced by increasing salt concen- amount of destabilization is comparable to the destabilization induced by tration. Core histone octamers were reconstituted on 210 bp Widom 601 DNA a thymine-guanine mispair but less than a thymine-cytosine mispair. Further- labelled at the linker arms with the fluorophores Alexa488 and Alexa594. We more, while the 8-oxoguanine-cytosine base pair is not predicted by the measured FRET between the fluorophores on the two linker DNA arms in the model, the energies of the guanine-thymine ‘wobble’ and thymine-cytosine presence and absence of LH and observed that the LH compacts the chromato- mismatch base pairs match their expected values. Notably, the measured en- some by bringing the two linker arms closer together in a salt dependent manner. ergy of 8-oxoguanine-adenine matches the model for the guanine-adenine Further analysis by salt-dependent electrophoretic mobility shift assays of the mispair, indicating that oxidative damage does not further destabilize this chromatosome in the presence and absence of LH showed faster migration of mismatch. These single molecule results support earlier findings that oxida- the chromatosome in the presence of LH, supporting the hypothesis that the tive damage changes the local backbone conformation, altering the direction LH is compacting the chromatosome. This compaction is profound in the salt of the guanine dipole and disrupting stacking interactions, while leaving range 5 to 150mM, as seen by both FRET and EMSA. Moreover, both single base pairing intact. molecule FRET experiments and EMSA revealed that presence of LH increases heterogeneity of the chromatosome population. By labelling the chromatosome 3391-Pos Board B599 at different positions we are currently addressing certain key issues: the linker Quantification of Single-Molecule FRET between Quantum Dots and DNA specificity of the LH, salt induced dissociation of the LH, and possible in- Organic Dyes fluence of the LH on chromatosomes reconstituted using mutated core histone Nooshin Shatery Nejad, Candice M. Etson. octamers. We hope that this system will help pin-point the localization and Physics, Wesleyan University, Middletown, CT, USA. role of the LH in freely diffusing mononucleosomes. Single-molecule Fo¨rster resonance energy transfer (FRET) is a useful tech- nique for studying inter- and intra-molecular dynamics in biophysics. FRET ef- 3394-Pos Board B602 ficiency is highly sensitive to distance, with half-maximal energy transfer Energy Landscape Analysis of the Full-Length SAM-I Riboswitch using occurring at an inter-dye distance on the order of 5 nm, which makes it possible Single-Molecule FRET Spectroscopy to quantify distances changes on the molecular scale. FRET efficiency also de- Christoph Manz1, Andrei Yu Kobitski1, Ayan Samanta2, Bettina G. Keller3, pends on the spectral overlap and quantum yield of the donor and acceptor mol- Andres J€aschke2, G. Ulrich Nienhaus1. ecules, and therefore the choice of dye pair is critical to the success of any 1Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, 2Heidelberg experiment. Most organic dyes that are used for single-molecule imaging University, Heidelberg, Germany, 3Freie Universit€at Berlin, Berlin, have bleaching lifetimes in the tens of seconds at most, which limits their Germany. use to relatively fast processes. Quantum dot nanocrystals can emit for thou- Binding of the ligand S-adenosyl-L-methionine (SAM) produces major struc- sands of seconds without entering a long-lived dark state, which should tural changes in the SAM-I riboswitch (RS) and thereby regulates gene expres- make them good candidates for use as a FRET donor for longer single- sion via transcription termination. As yet, the conformations and motions molecule experiments, but there has been little quantitative study of the energy governing the function of the full-length Bacillus subtilis yitJ SAM-I RS transfer efficiency at the single-molecule level. In this work, we use dsDNA to have not been deeply investigated. We have studied its conformational energy couple individual quantum dot donors to fluorescent dye acceptors. We then landscape as a function of Mg2þ and SAM ligand concentrations using single- immobilize these FRET pairs on a functionalized glass coverslip and image molecule Fo¨rster resonance energy transfer (smFRET) microscopy. smFRET them using total internal fluorescence (TIRF) microscopy with additional optics histograms of differently FRET-labeled constructs were so complicated that that allow us to image the donor and acceptor simultaneously. The length of the they could only be resolved with the help of kinetic experiments on immobi- DNA between the pair can be varied from 11 to 32 base pairs, allowing us to lized riboswitches and hidden Markov modeling (HMM) analysis. At least observe and quantify the energy transfer efficiency over a range of distances four conformational states were identified, both in the presence and the absence

BPJ 8749_8752 Wednesday, February 21, 2018 685a of SAM. We determined their Mg2þ-dependent fractional populations and Posters: Biosensors II conformational dynamics, including state lifetimes, interconversion rate coef- ficients and equilibration timescales. Riboswitches with terminator and antiter- 3397-Pos Board B605 minator folds were found to coexist under all conditions; SAM binding induced Evanescent Raman Spectroscopy of Bio-assemblies with Gallium Nitride only a gradual increase in the population of terminator states. Conformational Waveguide Structures transitions were much faster with bound SAM, which may be crucial for off- Alfons Schulte1, Samuel Borges1, Lee Chow1,2, W.S. Chen3, switching during the brief decision window before expression of the down- Shiang-Fu Huang3,4, Ming-Jer Jeng3, Liann-Be Chang3. stream gene. 1University of Central Florida, Orlando, FL, USA, 2National Taiwan University, Taipei, Taiwan, 3Chang Gung University, Taoyuan, Taiwan, 3395-Pos Board B603 4Chang Gung Memorial Hospital, Taoyuan, Taiwan. Revealing the Mechanism of Amyloid Fibril Formation by Combined Sin- Integrated evanescent Raman spectroscopy is an enabling technology for appli- gle Molecule FRET and Kinetic Modeling cations in biomedical diagnostics and bio-sensing. We investigate composite Jie Yang1, Alexander J. Dear2, Thomas C.T. Michaels2, structures where gallium nitride (GaN) provides the substrate and the guiding Christopher M. Dobson2, Tuomas P.J. Knowles2, Sarah Perrett1, Si Wu1. layer for a biomolecular film whose optical response and molecular conforma- 1Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, tion is probed. GaN waveguides are created by standard lithography and induc- 2Department of Chemistry, University of Cambridge, Cambridge, United tively coupled plasma etching processes. The waveguide structures are Kingdom. characterized with scanning electron and optical microscopy. The conversion of native soluble proteins into insoluble amyloid fibrils that A visible laser beam is coupled into the gallium nitride waveguide and the evanes- arerichincrossb-sheet structure is associated with a variety of neurodegen- cent field is employed to excite Raman scattering in the biomolecular film. erative diseases. It has recently become apparent that smaller oligomers Emerging applications of evanescent wave excitation for characterization and formed during the early stages of amyloid fibril formation may be primarily spectroscopy of bio-assemblies on patterned semiconductors will be discussed. responsible for the toxicity in amyloid diseases. Traditional ensemble tech- niques have limitations in characterization of oligomers due to the metastable 3398-Pos Board B606 and heterogeneous nature of these prefibrillar species. Single molecule fluo- Integration of Nanopore and Nanoelectrode for Single Entity Detection rescence spectroscopy allows the conformational dynamics of individual bio- and Manipulation molecules to be investigated, revealing properties that may be averaged in the Popular Pandey, Jin He. ensemble experiment. It is therefore extremely powerful for identification and Florida International U, Miami, FL, USA. characterization of the low-populated, heterogeneous and transient species Nanopore and ultrasmall electrode are emerging electrochemical methods for formed during fibril assembly. Combining single molecule fluorescence single entity studies. However, sensitivity and the selectivity of these methods with kinetic modeling can provide quantitative information regarding the are still limited to single molecular level. In recent years, multimode sensing microscopic steps in amyloid formation. Here we have applied single mole- methods have emerged as a new trend for detection and analysis of single en- cule fluorescence resonance energy transfer to investigate in detail the inter- tities. In this study, we have demonstrated that the two methods (nanopore molecular assembly and aggregation process of the yeast prion protein Ure2. sensing and nanoelectrode sensing) can be integrated together to study single Oligomerization during the initial lag phase was observed, and two types of nanoparticle (NP) in solution by using a multifunctional nanopipette with Ure2 oligomers with different assembly modes were identified. Furthermore, both nanopore and nanoelectrode at the nanopipette apex. Initially, as a model using theoretical analysis combined with single molecule and ensemble ki- system, we’ve used negatively charged, non-polarizable different sized poly- netic data, we describe the formation and depletion pathway of oligomers, styrene (PS) NPs. Electrophoretic force generated by externally applied bias and propose a multi-step mechanism for Ure2 fibril formation, in which initial is used to steer the NPs and to selectively interact with the nanopore and nano- oligomerization is followed by conformational conversion to b-sheet contain- electrode. This interaction of NPs produces various types of single NP events, ing oligomers that are then able to grow to form mature amyloid fibrils. We including translocation through nanopore, collision at the nanopore orifice, and also show directly that secondary nuclei do not form, and that fragmentation collision at the nanoelectrode. Based on the complementary and correlated is therefore responsible for the autocatalytic behavior of Ure2, in contrast to ionic current from the nanopore and potential signals from the nanoelectrode, the mechanism of the amyloid formation of Ab peptide. These results shed we can unambiguously differentiate these single NP events. NPs assembly light on the potential origin of differences in cytotoxicity between functional near the nanopipette tip can be achieved by applying DC and AC dielectropho- and disease-related amyloids. retic forces. This NPs assembly can be manipulated and investigated in detail by both nanopore and nanoelectrode. We further demonstrated that this method 3396-Pos Board B604 can be applied to study biological entities such as virus NPs and proteins. Topology-Dependent Dynamics of Single DNA Molecules Jaein Jang1, Rafal Fudala2, Ryan Rich3, Zygmunt Gryczynski2,4, 3399-Pos Board B607 Stephen D. Levene1,5. Impedimetric Fingerprinting and Structural Analysis of Isogenic E.coli 1Department of Biomedical Engineering, University of Texas at Dallas, Biofilms using Multielectrode Arrays Richardson, TX, USA, 2Department of Molecular Cell Biology and Genetics, Erkuden Goikoetxea1,2, Denis Routkevitch3, Ami de Weerdt1, University of North Texas Health Science Center, Fort Worth, TX, USA, Jordan J. Green3, Hans Steenackers1, Dries Braeken2. 3Department of Mathematics, Computer Science, and Physics, Texas 1Centre of Microbial and Plant Genetics, KU Leuven, Heverlee, Belgium, Wesleyan University, Fort Worth, TX, USA, 4Department of Physics and 2Life Sciences & Imaging Department, imec, Leuven, Belgium, Astronomy, Texas Christian University, Fort Worth, TX, USA, 5Department 3Ophthalmology, Oncology, Neurosurgery, and Materials Science & of Biological Sciences and Physics, University of Texas at Dallas, Engineering, John Hopkins University, Baltimore, MD, USA. Richardson, TX, USA. Microbial biofilm contamination is a persistent problem in clinics and industry. We present the data on the conformational dynamics of circular DNA mole- However, the biofilm structure, its extracellular matrix and its formation pro- cules with defined levels of topological complexity, characterized by the cess are hardly understood to date. Characterization of the biophysical structure knot type K and linking number Lk. Covalently closed DNAs covalently con- of biofilms would help understand these microorganisms as well as pave the jugated to specific fluorophores were investigated using fluorescence correla- way to the design of efficient tailored antimicrobial strategies. At present, there tion spectroscopy (FCS) and fluorescence cross-correlation spectroscopy are limited options to investigate biofilms outside the lab, as most in situ tech- (FCCS) measurements. We investigate in detail the respective effects of super- niques lack sensitivity and resolution. In contrast, impedance sensors provide a coiling and DNA knotting using Lk-topoisomer pools and specific DNA-knot fast, label-free and highly sensitive manner to characterize biofilms, although types that were respectively generated by topoisomerase-I relaxation and Cre mostly large and thus less precise electrodes have been used. Here, we em- site-specific recombination. Previous Brownian-dynamics simulations by Lan- ployed 60 mm-sized electrode arrays (MEAs) to map the structure of biofilms gowski et al. (1994) suggest that increasing superhelicity leads to progressively formed by wild type (WT(curliþ/celluloseþ)) Escherichia coli TG1 and the faster dynamics on long time scales, corresponding to translational diffusion, isogenic DcsgD (curli-/cellulose-), DcsgB (curli-/celluloseþ) and DbcsA but also on short time scales, corresponding to segmental motions within super- (curliþ/cellulose-) mutants. 24 hours of growth, the resistance at 2Hz increased coiled DNA. We investigate here whether DNA knotting similarly affects by 3.4% and 0.3% for the curli producing strains (WT and DbcsA), yet it translational and internal motions. The acceleration of DNA dynamics with decreased by 5.7% and 4% for the curli non-producing strains (DcsgD and increased supercoiling may have implications for processes in cells, such as DcsgB). The imaginary impedance at 2Hz decreased for all the strains by transcriptional regulation and DNA recombination, both of which are depen- 7.2%, 6.9%, 5.1% and 2.5% (WT, DbcsA, DcsgB and DcsgD, respectively). dent on the dynamics of juxtaposition involving multiple DNA sites. Interestingly, the variation of impedance within each biofilm, resulting from

BPJ 8749_8752 686a Wednesday, February 21, 2018 physiological heterogeneity, was significantly different for each biofilm and Reference: 1. U.S. Patent Application/WO2010/039941 titled ‘‘Bionanosensor most pronounced in the WT. Depending on the strain, the biofilm attachment Detection Device’’ European Patent #09818504.4-1403. phase lasted between 6 and 10h, and was characterized by an increase in the 2. U.S. Patent Application WO/2013/020106 titled ‘‘ Dynamic Thermal Inter- interfacial resistance, as opposed to the decrease in medium resistance observed face Material filed 2012 during the maturation phase. Overall, impedance-based MEA assays proved effective to differentiate between biofilms with varying structure, detect spatial 3402-Pos Board B610 diversity and explain biofilm life-cycle in terms of attachment and maturation. Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization 3400-Pos Board B608 Benjamin Cressiot1, Sandra Greive2, Wei Si3,4, Tomas Pascoa2, An Encapsulated Droplet Interface Bilayer Array for the High-throughput Mehrnaz Mojtabavi5, Maria Chechik2, Huw Jenkins2, Xueguang Lu6, Optical Measurement of Lipid Membranes with Single Bilayer Resolution Ke Zhang6, Aleksei Aksimentiev3, Fred Antson2, Meni Wanunu1. Divesh K. Baxani, William D. Jamieson, David A. Barrow, Oliver K. Castell. 1Physics, Northeastern University, Boston, MA, USA, 2Chemistry, Cardiff University, Cardiff University, Cardiff, United Kingdom. University of York, York, United Kingdom, 3Physics, University of Illinois at Droplet interface bilayers (DIBs) represent a recently developed method of Urbana-Champaign, Urbana, IL, USA, 4Jiangsu Key Laboratory for Design generating stable artificial lipid membranes suitable for electrophysiology and Manufacture of Micro-Nano Biomedical Instruments and School of, and optical interrogation. Prepared from the contacting of two aqueous droplets Southeast University, Nanjing, China, 5Engineering, Northeastern in oil, in the presence of dissolved lipid, DIBs are usually confined to a hydro- University, Boston, MA, USA, 6Chemistry and chemical biology, phobic medium. Recent advances have created freestanding, hydrogel- Northeastern University, Boston, MA, USA. encapsulated droplet interface bilayers (eDIBs) that are aqueous compatible, Nanopore-based sensors for nucleic acid sequencing and single-molecule sensing self-supporting and can withstand mechanical handling. Microfluidic methods typically employ lipid-embedded protein channels that contain a beta-barrel struc- allow for their rapid generation, paving the way for high throughput measure- ture or a hydrophobic domain. In this work, we describe a strategy for inserting ments on individually addressable lipid bilayers. hydrophilic proteins with tunnel-like structures into lipid membranes. We demon- Here, we report on the mass preparation of eDIBs using a 3D-printed microflui- strate here that conjugating a porphyrin moiety to a single-point cysteine mutation dic device and the proof-of-principle demonstration of high-throughput optical in a circular 12-subunit assembly of the thermophilic bacteriophage G20c portal membrane screening with single bilayer resolution. Individual eDIBs are pro- protein enables its insertion into lipid bilayers. X-ray diffraction, transmission duced and output into wells of a 96-well plate. Optical measurements are made electron microscopy, and thermal/chaotrope denaturation studies delineate this reporting on membrane leakage to monitor membrane integrity by fluorescent protein as a stable and suitable candidate for sensing studies. We characterize measurement. Addition of bilayer disrupting agents, such as detergents, to in- here the electrical properties of lipid-embedded channels of a single-mutant dividual wells enables parallel measurement of membrane activity using a stan- porphyrin-anchored protein assembly, as well as a triple mutant in which we dard fluorescent plate reader. This platform provides sufficient sensitivity to have engineered an increased tunnel diameter. Finally, we use a series of cyclo- measure the leakage of dye through membrane spanning pores in otherwise dextrins to size the inner constriction of the pore, as well as to provide insight intact bilayers, as well as direct detection of bilayer failure. into the electroosmotic flux direction, revealing asymmetric transport that This high-throughput, scalable and automatable approach to arrayed bilayer possibly originates from the portal’s DNA-ratchet function. Our results, backed measurements offers exiting opportunities for application with further by molecular dynamics simulations, pave the way for use of other non- fluorescent-based membrane, or protein, assays. The segregated nature of hydrophobic protein candidates as nanopore-based sensors. each droplet allows for the separation of contents either side of the bilayer. In- dividual bilayers can easily be independently addressed, affording the opportu- 3403-Pos Board B611 Controlling Molecular Conformation before Passage through a Nanopore nity to screen a wide parameter space with single bilayer resolution. The ability 1 1 2 3 to reconstitute membrane proteins into DIBs and eDIBs creates many exciting Vincent Tabard-Cossa , Kyle Briggs , Gregory Madejski , Martin Magill , Konstantinos Kastritis3, Hendrick de Haan3, James McGrath2. opportunities for the optical screening of membrane proteins and high 1 2 throughput biophysical measurements. University of Ottawa, Ottawa, ON, Canada, University of Rochester, Rochester, NY, USA, 3University of Ontario Institute of Technology, Oshawa, ON, Canada. 3401-Pos Board B609 We present a device to pre-stretch DNA before translocation through a solid- Substrate Optimization of Carbon Nanomaterial Based DNA Hybridiza- state nanopore, taking advantage of a recently developed ultra-thin nanopo- tion Detection System rous membrane positioned within nanometer distances from a sensing nano- Sethan K. Jasti1, Shawn M. McGinley2, Franzel Pena1, Samuel Opper1, pore fabricated by controlled breakdown. This nanofiltered nanopore Ewa S. Kirkor3, Saion K. Sinha4. 1 essentially creates a nanopore device with two pores in series, which reveals Electrical & Computer Engineering, University of New Haven, West Haven, fundamental physics of polymer translocation previously hidden in the noise. CT, USA, 2Biology & Environmental Science, University of New Haven, 3 4 We show experimental results that demonstrate that the fluctuations in the West Haven, CT, USA, Anchor Science LLC, Branford, CT, USA, Physics passage time of translocating DNA molecules can be reduced by controlling & Electrical Engineering, University of New Haven, West Haven, CT, USA. the molecular conformation during capture by a nanopore. Furthermore, this Rapid advancements in patient diagnostics are taking place in the medicine, reduction in spread is found to be independent of the pore dimensions and particularly in point of care testing (POCT). Herein, we describe the improved stability, enabling consistently narrower passage time distributions and substrate design of a novel bionanosensor1 developed with wireless capabilities more reliable polymer size separation regardless of the characteristics of for optimal POCT and field applications. The selective affinity to various biolog- the nanopore. Remarkably the presence of the nanofilter upstream of the ical molecules, make carbon nanomaterials (CNs) usable for a vast array of ap- sensing nanopore can also help to suppress folding of translocating DNA, plications throughout various fields. Bionanosensors utilize biomolecules for ensuring single-file molecular passage. These unique attributes will offer sig- determining the presence of an analyte through the process of signal transduc- nificant practical advantages to many solid-state nanopore-based technolo- tion. DNA hybridization is a biological technique that can be utilized to deter- gies, including sequencing, genomic mapping, and barcode detection for mine the presence of a specific sequence or gene. DNA and CNs interactions diagnostics. dependent upon DNA and CNs structures. The bionanosensor is composed of 2 our patented carbon nanomaterial composite applied to two substrates, papers 3404-Pos Board B612 of different density, porosity and thickness, first one was thin and very porous Modulation of Ionic Conductivity of Lipid Bilayer-Based Nanoscopic paper, the second was the Whatman paper with 2.5 micron porosity and manu- Channels by Pre-adsorbed Charged Macromolecules as a Tool for their facturer- specified filtration rate. Arrays of such bionanosensors were fabricated Detection and Quantification and used for detection of hybridization reaction at appropriate for sequence tem- Ksenia Chekashkina1,2, Timur Galimzyanov2,3, Peter Kuzmin2, perature. Few minutes after a single-stranded DNA (ssDNA) was applied to the Galina Pozmogova1, Dmitriy Klinov1, Pavel Bashkirov4. sensor a complementary or non-complementary DNA sequence was added. A 5 1Scientific Research Institute of Physical-Chemical Medicine, Moscow, minute long, direct current (DC) measurement was made under constant voltage Russian Federation, 2A.N. Frumkin Institute of Physical Chemistry and source to record the signal. The signal to noise ratio and percent difference be- Electrochemistry, Russian Academy of Sciences, Moscow, Russian tween hybridizing and non-hybridizing experiments are calculated. Our results Federation, 3National University of Science and Technology MISiS, show that Whatman filter paper substrate showed best result : greater difference Moscow, Russian Federation, 4Scientific Research Institute of Physical- between hybridizing and non-hybridizing experiments , 3 dB improvement in Chemical Medicine, Zhukovsky, Russian Federation. signal to noise ratio. This bionanosensor is expected to perform affordable and Investigation of transport phenomena in the nanoscopic channels (pores) hav- time efficient analysis of nucleic acid samples. ing the characteristic cross dimension of less than 100 nm is of utmost

BPJ 8749_8752 Wednesday, February 21, 2018 687a importance for diverse areas of applied biology, medicine and technology. One gated to polystyrene beads have been reported for the detection of miR-204 of the mainstream lines of development of nanofluidic systems and applications and miR-210 related to the clear cell Renal Cell Carcinoma (ccRCC). Electro- today is creation of biosensors capable of sensing single molecules and manip- osmotic flow (EOF) was induced as the driving force to transport PNA-beads ulating them in a controllable manner. Molecules can be detected based on the harboring target miRs to the tip of the pore (sensing zone) which resulted in measurements of ionic currents through appropriately sized channels: entry into pore blockades with unique and easily distinguishable serrated shape electrical a channel of a molecule with the effective cross-dimension comparable to that signals. However, in the case of the control experiments ionic current blockades of the channel lumen is accompanied by decrease of the ionic current recorded with right-angled shape were detected. The results showed 1 to 10 fM concen- at a given transmembrane potential. The transport properties of such channels tration detection limit and 97.6% detection accuracy in 87 experiments. This can be modulated by coating their walls with lipid bilayers, two-dimensional simple, PCR-free, and robust platform has a technological appeal to be evolved fluids capable of sustaining transport processes within them. In our present into a quantitative measurement tool for analysis of miR biomarkers in basic work, we made use of this property of the membranes to develop a method and clinical research by correlating the dwelling time of the particles transloca- for detecting and controllably transporting single-stranded DNA molecules tion through the pore with the concentration of the RNA oligomers bound to through channels formed by lipid membrane cylinders with the luminal radius their surface. of 5-7 nm. Entry of a DNA molecule into such a channel in the conditions of low (10 mM) ion strength proved to be accompanied by detectable increase 3407-Pos Board B615 of its ionic conductivity in a manner dependent on the direction of the electric Probing MspA Porin with PEGs: Size-Dependent Partitioning vs. Specific field gradient. The amplitude of the conductivity increment can be credibly Binding 1 2 3 3 used to quantify the number the DNA molecules within the channel. Besides Philip A. Gurnev , David Hoogergheide , Jens Gundlach , Andrew Laszlo , 1 that, adsorption of DNA molecules on the lipid bilayer surface was shown to Sergey Bezrukov . 1Section on Molecular Transport, National Institute of Child Health and render the membrane cylinder the properties of a voltage-dependent channel 2 with ion selectivity. The work was financially supported by the Russian Science Human Development, NIH, Bethesda, MD, USA, Center for Neutron Fund No 17-75-30064. Research, National Institute of Standards and Technology, Gaithersburg, MD, USA, 3Department of Physics, University of Washington, Seattle, WA, 3405-Pos Board B613 USA. Asymmetric Dynamics and Current Signals of DNA Entering and Exiting We evaluate the effect of differently sized poly(ethylene)glycols, PEGs, on a Strongly Confining Nanopore conductance of Mycobacterium smegmatis porin A (D90N/D91N/D93N/ Kaikai Chen, Nicholas A.W. Bell, Ulrich F. Keyser. D118R/E139K/D134R mutant, M2 MspA), designed as a nanopore sensor Cavendish Laboratory, University of Cambridge, Cambridge, United for DNA sequencing. Unlike other cases of beta-barrel pores, such as Kingdom. alpha-hemolysin and mitochondrial VDAC, M2 MspA pore is characterized In nanopore sensing, changes in ionic current are used to analyse single by a smooth dependence of PEG partitioning into its lumen on the polymer molecules in solution. The translocation dynamics of polyelectrolytes is molecular weight. Asymmetry of M2 MspA channel manifests itself in asym- of particular interest given potential applications such as DNA sequencing. metric polymer partitioning from the opposite channel entrances, with the In this study, we determine how the dynamics and current signatures of characteristic cutoff sizes of PEGs with molecular mass of 8000 and 2000. voltage driven DNA translocation can be affected by the nanopore geom- This is in good agreement with the crystallography data indicating 4.5 nm etry and hence the available configurational space for the DNA. Using the and 2.5 nm diameters for the corresponding channel openings. We note inherent geometrical asymmetry of a conically shaped nanopore, we that PEG interaction with the M2 MspA pore is more pronounced when the examine how DNA dynamics and current signals depend on the direction- polymer is applied from the side of the narrow opening. This is observed as ality of transport. The total translocation time of DNA when exiting the a gradual increase of current noise with the PEG molecular weight. Applying extended conical confinement is significantly larger compared to the buffer conditions that induce high polymer-pore attractions, we resolve indi- configuration where the DNA enters the pore from the open reservoir. vidual events of PEG molecule retention in the M2 MspA pore. Overall, our By using specially designed DNA molecules with positional markers, we data provide for better understanding of the M2 MspA pore geometry and demonstrate that the translocation velocity progressively increases as the describe another model system for probing polymer dynamics in confined DNA exits from confinement. We show that a hydrodynamic model can ac- nano-volumes. count for these observations. The current signatures also depend on the DNA translocation direction and we used a finite simulation method to 3408-Pos Board B616 explain the observed current signatures. Our analysis shows that over a Model-Free Observation of Polypeptide Translocation Success Rate wide range of geometries, voltages, and salt concentrations, we are able through a Nanopore 1 2 2 to understand the ionic current signals and dynamics of DNA in asym- David P. Hoogerheide , Philip A. Gurnev , Daniel Jacobs , 2 2 metric nanopores, enabling signal optimization in molecular sensing Tatiana K. Rostovtseva , Sergey M. Bezrukov . 1Center for Neutron Research, National Institute of Standards and applications. 2 References [1] N. A. W. Bell, K. Chen, S. Ghosal, M. Ricci, and U. F. Keyser. Technology, Gaithersburg, MD, USA, Eunice Kennedy Shriver National Nature Communications, 8(380), 2017. [2] K. Chen, N. A. W. Bell, J. Kong, Y. Institute of Child Health and Human Development, National Institutes of Tian, and U. F. Keyser. Biophysical Journal, 112(4):674-682, 2017. [3] K. Health, Bethesda, MD, USA. Chen, M. Juhasz, F. Gularek, E. Weinhold, T. Yu, U. F. Keyser, and N. A. Elucidating the motion of uniformly charged polymers in voltage-biased W. Bell. Nano Letters, 2017, 17, 5199-5205. nanoscale channels has been a scientifically and technologically fruitful en- terprise. Non-uniformly charged polymers such as polypeptides present a 3406-Pos Board B614 new set of challenges and opportunities, as they do not in general move Amplification-Free Detection of Micrornas Related to Clear Cell Renal through a nanopore unidirectionally or translocate the channel-containing Cell Carcinoma Utilizing a Novel Nanopore-Based Sensor membrane with unity probability. Here we demonstrate a single-molecule, Yuqian Zhang1, Ankit Rana1, Maria F. Czyzyk-Krzesk2,3, model-free experimental technique to track the motion of a polypeptide in Leyla Esfandiari1,4. a channel and determine whether each polypeptide ultimately translocates 1Department of Electrical Engineering and Computer Science, University of through, or retracts from, the channel. The technique relies on the heteroge- Cincinnati, Cincinnati, OH, USA, 2Department of Cancer Biology, neity of the charge density along the polypeptide and its effect on the selec- University of Cincinnati, Cincinnati, OH, USA, 3Department of Veterans tivity of the channel. In a nanopore under an electrolyte concentration Affairs, VA Research Service, Cincinnati, OH, USA, 4Department of gradient, the modulation of the channel selectivity is observable in real Biomedical Engineering, University of University of Cincinnati, Cincinnati, time as a change in the ionic conductance. For a polypeptide with different OH, USA. charge densities at the N- and C-termini, the ionic current at the end of a MicroRNAs (miRs) are small noncoding RNAs that play an important role in single molecule capture ‘‘event’’ reports on which end of the molecule exits gene regulation. Recent studies have shown the correlation of the miRs expres- the nanopore, and hence the direction of escape. As a demonstration, we sion level to carcinogenesis. Currently, qRT-PCR technology is considered as report experimental observations of the interaction of a ‘‘diblock copol- the ‘gold standard’ for miR detection due to its high sensitivity and specificity. ymer’’-like neuronal intrinsically disordered protein, a-synuclein, with However, this technique requires time-consuming and expensive amplification mitochondrial voltage-dependent anion channel (VDAC). The voltage- steps along with labeling and enzymatic reactions. In this work, a new dependent translocation probability derived from the experiments shows nanopore-based detection scheme utilizing a borosilicate micropipette and an that a-synuclein is bound to membrane surfaces with a distribution of bind- assay of complementary gamma-peptide nucleic acid (g-PNA) probes conju- ing energies that strongly depends on lipid species. These results have broad

BPJ 8749_8752 688a Wednesday, February 21, 2018 general implications for the interactions of peripheral proteins with lipid methodology provides a new tool to investigate the single-molecule catal- membranes. ysis of redox enzymes, which will lead to a better understanding of the enzymatic mechanisms and biocatalytic processes. 3409-Pos Board B617 Revealing Multiple Transition Paths during the Unfolding/Folding of Indi- 3412-Pos Board B620 vidual Peptides in a Confined Nanopore Nanopore-Based Detection of Microcystin-LR Yilun Ying, Shaochuang Liu, Yitao Long. Shuo Zhou, Wanyi Xie, Shixuan He, Peng Tang, Daming Zhou, East China University of Science and Technology, Shanghai, China. Deqiang Wang. A fundamental question in peptide folding is how the peptide fleets through a Chongqing Institute of Green and Intelligent Technology, Chinese Academy set of transition states which dominate the dynamics of biomolecular folding of Science, Chongqing, China. path. Owing to their rapid duration and sub-nm structure difference, however, Microcystin-LR (MC-LR), a possible human carcinogen, is considered as they have always been oversimplified because of limited instrumental resolu- one of the most poisonous toxins released in cyanobacterial water blooms. tion. Moreover, the most experiments indicate a single fold pathway while The maximum recommended level of MC-LR in drinking water is 1 mg/L the simulations suggest peptides owns the preference in multiple pathways. Us- according to the World Health Organization (WHO). Current detection ing the electrochemical confined effect of a solid-state nanopore, we measured methods for MC-LR are chromatography and Enzyme-Linked Immuno- the multiple transit paths of peptide inside nanopores. Combining with Markov Sorbent Assay (ELISA). However, the former is expensive and requires modelling, this new single-molecule technique is applied to clarify the 5 tran- professional training while the latter demands the time-consuming labeling. sition paths of the b-hairpin peptide which shows 4 nonequilibrium fluctuating Hence, a nanopore biosensor is designed for the determination of MC-LR, stages. These results enable experimental access to previously obscured peptide which should be a highly sensitive and selective detection method based on dynamics which is essential to understand the misfolding in peptides. The sta- the specific advantages of the nanopore-based single-molecule techniques, tistical analyzing of each peptide from high throughput show that 78.5% of the such as rapid, label-free, etc. Besides, water sample tests and interference peptide adopt the pathways I during their folding/unfolding in a nanopore while studies are conducted to explore the potential applications of the nanopore 21.5% of the peptide undergoes the hidden folding/unfolding of transit path- techniques on environmental monitoring and protection. ways II-V. These measurements provide a first look at the critical experiment picture of the mechanical folding/unfolding of peptide, opening exciting ave- 3413-Pos Board B621 nues for investigating transition paths. Direct Quantification of Metabolites from Bodily Fluids using Nanopores Nicole S. Galenkamp, Misha Soskine, Carsten Wloka, Giovanni Maglia. 3410-Pos Board B618 Chemical Biology 1, University of Groningen, Groningen, Netherlands. Electro-Osmotic Capture and Ionic Discrimination of Small Peptides and Crucial steps in the miniaturization of biosensors are the conversion of a bio- Proteins with FRAC Nanopores for Single-Molecule Protein Sequencing logical signal into an electrical current as well as the direct sampling of biolog- Gang Huang. ical fluids. Here we show that protein sensors in combination with a biological Groningen Biomolecular Sciences & Biotechnology Institute, Groningen, nanopore Cytolysin A (ClyA), acting as an electrical transducer, can accurately Netherlands. quantify metabolites from tiny amounts of bodily fluids with internalized sub- Biological nanopores are nano-scale sensors employed for high-throughput, strate binding proteins (SBDs). The single-molecule nature of the sensor allows low-cost and long read-length DNA sequencing applications. The analysis continuous sampling of multiple analytes simultaneously without the need of and sequencing of proteins, however, is complicated by the folded structure calibration for signal drift. Crucially, no adverse effects of other analytes in and non-uniform charge of proteins. Here we show that an electro-osmotic the bodily fluids tested could be discerned as the nanopore act as a nanoscale flow through Fragaceatoxin C (FraC) nanopores1 can be engineered to allow molecular filter, allowing direct sampling of small volumes of biological sam- the entry of polypeptides at a fixed potential regardless of the charge compo- ples with no sample preparation. Incorporation of the nanopore into portable sition of the polypeptide. We further use the nanopore currents to discrim- electronic devices will allow developing sensitive, continuous and non- inate peptide and protein biomarkers from 25 kDa down to 1.3 kDa invasive sensors for hundreds of metabolites for point-of-care, home diagnos- including polypeptides differing by one amino acid2. Even smaller differ- tics and wearable devices. ences among peptides shorter than 10 amino acid could also be detected with FraC nanopores. On the road to nanopore proteomics, our findings 3414-Pos Board B622 represent a rationale for amino acid analysis of folded and unfolded poly- Tea’s Antioxidant Potency by the Detection of Optical Absorption Spec- peptides with nanopores and pave the way for single-molecule protein troscopy with DNA-Encased HIPCO Carbon Nanotube Hybrids sequencing. Lijun Wang, Kazuo Umemura. [1]. Wloka C, Mutter NL, Soskine M, Maglia G. Alpha-Helical Fragaceatoxin Tokyo University of Science, Tokyo, Japan. C Nanopore Engineered for Double-Stranded and Single-Stranded Nucleic There is an interest in developing a simple method to quantitatively detect gene Acid Analysis. Angew Chem Int Ed Engl 55, 12494-12498 (2016). and inspect food, which is also expected to be applied in nano biosensors. Here, [2]. Huang G, Willems K, Soskine M, Wloka C, and Maglia G. Electro- a sensitive nanoprobe, double-stranded DNA-encased HiPco carbon nanotube Osmotic Capture and Ionic Discrimination of Peptide and Protein Biomarkers hybrid has been developed to detect antioxidant potency of selected samples with FraC Nanopores. Nature Commun. Accepted (i.e., different tea) against hydrogen peroxide (H2O2), owing to its different responses under irradiation of different wavelengths. Besides, since the 3411-Pos Board B619 main component of tea is catechin, epigallocatechin gallate (EGCg) is Dynamics of Single-Enzyme Activity in a Nanopore Confinement used as standards. In this work, near-infrared absorption (NIR-ABS) and Yao Lin, Yilun Ying, Rui Gao, Yitao Long. NIR-photoluminescence (PL) were carried out to record the optical absorption East China University of Science and Technology, Shanghai, China. spectroscopy with the hybrid. It is found that the selected tea suggests excellent Enzymatic reactions play a crucial role in controlling and performing a antioxidant potency in the case of the presence of 0.03wt% H2O2. Moreover, number of biological processes. The ability to directly probe catalysis of the catechin plays a major role in antioxidant potency of tea. This work pro- a single enzyme can potentially enable a deeper understanding of the enzy- vides a simple detection method for evaluating the antioxidant potencies of matic mechanisms that are not distinguishable in ensemble measurements. samples against H2O2 by using DNA-SWCNTs hybrids for biological and Herein we report the direct observation of the electrocatalytic process of a food applications. single enzyme molecule in nanopore confinement. Horseradish peroxidase (HRP), a heme-containing enzyme that is widely used in immunoassays Posters: Micro- and Nanotechnology II and bioassay, was chosen as a model enzyme in our studies. We used nanopore-based techniques to electrochemically capture a single HRP 3415-Pos Board B623 molecule in its confined space, which enable long-term observation of New Insights into the Dynamics and Energetics of Phage T4 Injection Ma- only a single enzyme molecule. Then the bipolar electrochemical reactions chineray using a Continuum Model catalyzed by HRP enzyme were occurring at the two poles of the polarized Ameneh Maghsoodi1, Anupam Chatterjee2, Ioan Andricioaei2, gold layer. The enzyme-catalyzed electrochemical reactions resulting in Noel Perkins1. 1 easily distinguished ionic current signals enable us to monitor the catalytic Mechanical Engineering, University of Michigan, Ann Arbor, MI, USA, 2 process of single enzyme molecules. Using this system, we investigated the Department of Chemistry, University of California, Irvine, Irvine, CA, USA. single HRP enzyme catalysis in the confined spaces of nanopore and found Bacteriophage T4, from the family Myoviridae, is one of the most common the heterogeneous catalytic activity between individual enzymes. This tailed viruses that infects E.coli. Phage T4 is composed of three major

BPJ 8749_8752 Wednesday, February 21, 2018 689a protein structures; 1) a large capsid containing the viral genome, 2) a con- will be done by creating an on-chip competition assay based on C-peptide tractile tail structure connected to the capsid which generates the driving conjugated to 5-TAMRA (C-peptide*) and changes in fluorescence anisot- force to pierce the host membrane and conveys DNA from the capsid to ropy. Second, we aim to measure oxygen consumption rates of individual the host, and 3) a baseplate equipped with fibers that recognize and bind islets. Oxygen is consumed by the electron transport chain and can be to the host. The contractile tail consists of a rigid tail tube surrounded by used to identify perturbations in oxidative phosphorylation. We are using an elastic six-helical-stranded sheath. During injection, the sheath un- RuII(bpy)3 as an optical sensor to measure oxygen with excitation and dergoes a large conformational transition from a high-energy extended state emission maxima at 450 and 600 nm, respectively. Lastly, we aim to mea- to a low-energy contracted state, thereby releasing energy needed for sure extracellular acidification rate as a readout of glycolytic rate and/or the tail tube to penetrate the host. While the atomic structure of phage oxidative phosphorylation. We will use HPTS, a pH sensitive dye, in solu- T4 is largely known, the dynamics of the injection process is not, including tion. Each of these sensors is spectrally and/or spatially resolved from one time scale and energetics of injection. To fill that gap, we propose a dy- another. This strategy will allow us to simultaneously measure these re- namic model of the entire phage T4 to simulate the dynamics of the injec- sponses to fully index the function of individual islets and engineered tion process. The simulation follows in two stages; first, we employ micro-tissues. This multiparametric characterization could inform the pro- molecular dynamic (MD) simulations to calculate the elastic stiffness con- duction of engineered islets as well as assist in the screening of tissue prior stants and internal friction of the sheath strands. Second, we employ those to transplantation. material properties in a continuum model of the entire virus. The continuum model treats the tail sheath as a six-interacting elastic helical strands that 3418-Pos Board B626 are coupled to a massive cylinder representing the capsid and to a rigid A 16384 Electrode 1024 Channel Multimodal Cmos MEA for High rod representing the tail tube. The resulting model predicts that the driving throughput Drug Screening energy of injection process is about 5500kT, the time scale of sheath Dries Braeken, Carl Van, Den Bulcke, Carolina Mora Lopez, contraction is on the order of milliseconds, and the internal friction of Veerle Reumers. sheath strands is a main source of energy dissipation during sheath Life Sciences, Imec, Leuven, Belgium. contraction. Drug attrition is a major concern for the pharmaceutical industry. Besides the loss of the large investment needed to develop novel drugs, many lives 3416-Pos Board B624 have been lost due to lethal side effects. Cardiotoxicity accounts for more 2-Photon Lithography for Nanofluidic Lab-on-Chip Devices than one third of the drug withdrawals from the market. Although the Oliver Vanderpoorten1, Pavan K. Challa1, Quentin Peter1, Food and Drug Administration has set guidelines to tackle this issue, there Jerome Charmet2, Nathan Curry1, Tuomas P.J. Knowles1, is still room for improvement on the methodology of safety screening. Clemens F. Kaminski1. Multi-electrode arrays (MEAs) are a candidate technology to screen in vitro 1University of Cambridge, Cambridge, United Kingdom, 2University of cardiac parameters because of its non-invasive recording of cardiac beating Warwick, Warwick, United Kingdom. rate and electrical field potential duration. Here we present a novel active The misfolding of proteins inside neuronal cells is known to be linked to MEA chip featuring 16,384 electrodes, 1024 simultaneous readout channels neurodegenerative diseases such as Alzheimer’s and Parkinson disease. and 4 different electrode sizes to perform extracellular and intracellular Two promising approaches used by researchers in this field are: The usage recording from cardiomyocytes. Biocompatible TiN (height of 300nm) elec- of lab-on-chip devices to characterize proteins ‘‘in vitro’’ from a biophysics trodes were processed on top of the in-house designed circuits. The circuit point of view and fluorescence microscopy to study the protein aggregation noise for the recording mode was 7.550.6 mVrms and 1252.4 mVrms for in living cells in scaffolds ‘‘in vivo’’. The length of Amyloid-b 42 (M1-42) extracellular and intracellular recording, respectively. Electrode impedance aggregates is connected to their toxicity and integrated nanofiltration at 1kHz was determined using the built-in impedance circuitry and was 4.2, methods to separate proteins in a microfluidic process pipeline are not avail- 2.2, 1.1 and 0.5 MU for electrode areas of 8.75, 10.5, 45.5 and 121mm2, able in biological research laboratory without access to expensive electron respectively. Further, we recorded extracellular signals of 1.4 mV and intra- beam lithography systems as in clean-room facilities. Therefore, we present cellular signals of 12.7mV on average (peak to peak) from primary rat car- a novel soft lithography method to produce nanometric structures with two- diomyocytes cultured for 3 days in vitro on the chip. Finally, applying 1mM photon lithography (2PL) for nanofluidic chip fabrication. We demonstrate a nifedipine, a drug to treat hypertension and angina, on the cells changed the system capable of producing master wafers for nanofluidic devices and use shape and duration of intracellular action potential significantly. The novel these to measure the diffusion of fluorescently labelled Amyloid-b 42 in platform thus allows for high throughput electrical activity monitoring and nanochannels. [1] We also show the fabrication of 3D cell scaffolds for drug screening of cardiomyocytes, and can be used for toxicity screening or more realistic 3D cell cultures, to improve studies of e.g. the protein Tau drug development. in Alzheimer’s and its interplay with Amyloid-b 42. [2][3] To accelerate lab-on-chip development we overcome the current microscale fabrication 3419-Pos Board B627 capabilities by combining mask whole-wafer UV-lithography with locally Effects of Hypoxia on Breast Cancer Extravasation in a 3D Microvascular 2P-written nano-sized functional features and make rapid nanofluidic chip Network prototyping possible. Jiho Song1,2. 1 [1] Erik C. Yusko, et al., Nature Nanotechnology 6, 253260 (2011) BioSym, Singapore MIT Alliance Research and Technology, Singapore, 2 [2] Carla Davanzo et al., Nature 515, 274278 (13 November 2014) Singapore, Biological Engineering, Massachusetts Institute of Technology, [3] Clemens F. Kaminski, Gabriele S. Kaminski Schierle, Neurophoton. 3(4), Cambridge, MA, USA. 041807 (2016). Hypoxia within the tumor microenvironment plays an important role in regu- lating breast cancer progression and is associated with metastasis and patient 3417-Pos Board B625 mortality. Lack of oxygen in the tissue triggers changes in gene expression Design of a Multiparameter Islet-on-a-Chip Device to Measure the Func- mediated by hypoxia-regulated proteins, i.e. hypoxia inducible factors (HIFs) tional Variability of Individual Pancreatic Islets that impact cancer in numerous ways: angiogenesis, metabolic reprogramming, Romario Regeenes1, Afifa Saleem2, Huntley Chang1, epithelial-mesenchymal transition (EMT), invasion, and metastasis. Recent ad- Jonathan V. Rocheleau1,3. vances in microfabrication technologies have allowed for the development of in 1IBBME, University of Toronto, Toronto, ON, Canada, 2Division of vitro platforms that recapitulate cellular components and functions relevant to Engineering Science, University of Toronto, Toronto, ON, Canada, cancer metastasis. Previously, we engineered 3D vascular networks with hu- 3Physiology, University of Toronto, Toronto, ON, Canada. man umbilical endothelial cells (HUVECs) seeded in fibrin gels and cultured Type 1 diabetes results from autoimmune destruction of pancreatic islets, to allow signaling with human lung fibroblasts (HLFs). HUVECs spontane- small micro-tissues (150 mm) that are primarily composed of insulin ously formed networks within 24 hours and the engineered perfusable vessels secreting beta-cells. Islet transplantation is a current treatment option, but were mature and perfusable after 4 days. Despite the importance of hypoxia in will never be universally available due to limited supply of donor tissue. tumour progression, as well as advances in modeling angiogenesis and vasculo- Tissue engineered islets could help bridge this gap and provide an opportu- genesis, these models have not previously been used to study hypoxia in met- nity to treat many more people. However, the success will depend heavily astatic tumours. Also, most pathology and gene-expression studies are on the quality and uniformity of the engineered tissue. To inform in the performed on primary tumours, and little is known about hypoxia at the meta- design, we are creating a microfluidic device to assay multiple readouts static site. We, therefore, sought to investigate how hypoxia affects cancer of function on individual islets and engineered micro-tissues. First, we metastasis from in vitro blood vessels. In this study, we show that three breast aim to measure C-peptide release as a proxy for endogenous insulin. This cancer cell lines cultured in normoxia, hypoxia, and with knock-down HIF

BPJ 8749_8752 690a Wednesday, February 21, 2018 hypoxia cells, exhibit changes in extravasation rates 6 hours after perfusion into shown to have a considerable antitumor efficacy in various human cancer the networks. Hypoxia is shown to activate genetic programs that facilitate cell models (i.e., breast, liver, and pancreatic) genografted in mice. Compared to survival, while at the same time, promoting invasion and metastasis. a saline control, Gd@C82(OH)22 treatment restricted both tumor growth and Continuing studies will examine mechanisms mediating the role of hypoxia migration even at a much lower dose from that of conventional drugs. A in metastasis, and these may lead to new and innovative therapeutic strategies detailed study revealed that Gd@C82(OH)22 specifically inhibits cancer metas- to limit metastatic disease. tasis by effectively down-regulating several key angiogenic factors including MMP2 and MMP9 in both mRNA/protein expression and enzyme activity 3420-Pos Board B628 levels with more weight on MMP9. In our study, we more focused on the nano- Characterization of Nanoscale Her2-Containing Clusters at the Cell Mem- particular binding dynamics and specificity, and their relevance to enzyme brane inhibitory mechanism using atomistic molecular dynamics simulations. Briefly, Elena Ambrosetti, Alessandro Bosco, Ana Teixeira. we found that Gd@C82(OH)22 interaction to MMPs are distinctive from the Karolinska Institutet, Stockholm, Sweden. conventional MMPi’s targeting the Zn2þ-catalytic site, in that the nanoparticle The spatial distribution of proteins on the cell surface has emerged as a key interacts with MMP selectively at the ligand specificity S1’ loop, the most regulator of membrane proteins function. Protein assemblies are of biological unique place among member proteins of MMP family in terms of sequence and therapeutic importance: the nanoscale spatial ordering and dynamic distri- length and identity, as strongly implicating a possibility of allosteric inhibitory bution of proteins at the cell membrane tune the complex molecular networks mechanism for Gd@C82(OH)22 on MMPs. Furthermore, we investigated the they regulate in normal physiology and in disease. Therefore, there is a need to molecular origin of Gd@C82(OH)22 preference to MMP9 compared to develop high-throughput methods to understand how the organization of pro- MMP2, and their inhibitory mechanism difference. Our study not only demon- teins on a nanoscale level regulates receptor signaling and cellular phenotypes. strates how nanoparticles can function as a potential ‘‘nanodrug’’, but also pro- In this work, we present the development of a non-microscopy based super- vides a molecular insight for a specific MMP inhibitor design based on resolution method for unbiased analysis of protein nanoclusters at the cell nanoparticles. membrane, studying the frequency in which specific types of protein appear in the proximity of the protein of interest. We setup a DNA nanotechnology- 3423-Pos Board B631 based approach, using DNA nanostructures to decipher the position and the Quantitative Investigations Reveal New Antimicrobial Mechanism of Sil- identity of proteins within the assembly. STORM-TIRF super-resolution mi- ver Nanoparticles and Ions croscopy analysis and advanced biochemical assays were combined to vali- Prabhat Khadka1, Mohammad Haque1, Venkata Rao Krishnamurthi1, date the methodology and to obtain exhaustive characterization of protein Isabelle Niyonshuti2, Jingyi Chen2, Yong Wang1. clusters. We focused here on clustering of the membrane receptor Her2. 1Department of Physics, University of Arkansas, Fayetteville, AR, USA, Although Her2 has well known roles in driving tumor progression, the mo- 2Department of Chemistry, University of Arkansas, Fayetteville, AR, USA. lecular mechanisms by which Her2 can lead to diverse cellular outcomes are Silver in various forms, including silver nanoparticles (AgNPs) and ions still unclear. Since Her2 forms homo- and hetero-dimers and oligomers, we (Agþ), has been found to show promising antimicrobial activities, which aim to provide a precise portrait of the spatial organization of Her2- may open new avenues to fighting against antibiotic resistant microbes. containing clusters and to understand how it correlates with downstream However, although exciting progress has been made, the molecular mecha- signaling and cellular outcomes. Our approach has the potential to nism of the antimicrobial effect of AgNPs and Agþ remains a subject of contribute to developing a new paradigm in targeting receptor signaling at debate. Our lab has been investigating the action mechanisms of AgNPs the nanoscale, focusing not on the membrane receptors per se, but on their and Agþ using experiment-based quantitative modeling and super- spatial organization. resolution fluorescence microscopy. Using kinetic growth assays and þ 3421-Pos Board B629 colony-forming-unit assays, it was found that AgNPs and Ag caused elon- Membrane Curvature Dependent F-Actin Polymerization at Nano-Cell gation of lag time in the bacterial growth, while the max growth rate re- Interface mained unaffected. We developed a quantitative model to account for and þ Hsin-Ya Lou1, Wenting Zhao2,3, Bianxiao Cui1. predict the actions of AgNPs and Ag ; predictions from the model agree 1Department of Chemistry, Stanford University, Stanford, CA, USA, 2School well with the experimental results. In addition, the parameters in our model of Chemical and Biomedical Engineering, Nanyang Technological were determined experimentally, providing alternative ways to MIC/MBC University, Singapore, Singapore, 3Materials Science and Engineering, for characterizing antimicrobial activities of Ag. In addition, using super- þ Stanford, Stanford, CA, USA. resolution fluorescence microscopy, we found that Ag and AgNPs do Polymerization of actin plays an important rule in several biological pro- not directly cause DNA condensation, in contrary to a previous hypothesis cesses including polarization, endocytosis, migration, etc. Developing an suggested by TEM results. Instead, enhancement of expression and spatial efficient method of manipulating F-actin dynamics in life cells is important reorganization of histone-like nucleoid structuring (H-NS) proteins was for studying these actin-related biological processes. Several studies have observed, which is expected to, in turn, cause DNA compaction and conden- shown that nanoscale structures are able to induce F-actin polymerization sation. By varying the treatment time and concentration of Ag, we obtained at the sites of nano-cell interface. However, the detail mechanism is still un- the quantitative dose- and time-dependence of Ag’s antimicrobial effect. clear. Current studies focus extensively on focal adhesion-induced F-actin Our data indicate that the previous hypothesis is incorrect, or at least incom- polymerization and the corresponding mechanotransduction, but not all plete, and a new mechanism for Ag’s antimicrobial activities via nucleoid- F-actin polymerization are correlated with focal adhesion sites. Here we associated proteins. Furthermore, using long-term live-cell imaging, we introduce another mechanism that nanostructures promote F-actin polymer- found that exposure to Ag results in morphological changes in the bacteria, ization by creating nanoscale membrane curvatures. Nanostructures with which eventually caused membrane abruption and cell burst. Our study pro- curvature below 400 nm diameter are able to effectively trigger Arp2/3 vides the first dynamic data on Ag’s action mechanism via membrane nucleated F-actin polymerization via recruiting the curvature sensitive pro- damage. tein FBP17. This study help provide a new angle to understand the interac- tion at the cell-material interface. 3424-Pos Board B632 Site-Selective RNA Splicing Nanozyme: Dnazyme and RTCB Conjugates 3422-Pos Board B630 on a Gold Nanoparticle 1 1 1 2 Molecular Insight of Metallofullerenol GD@C82(OH)22 in Cancer Anti- Jessica R. Petree , Kevin Yehl , Kornelia Galior , Roxanne Glazier , Metastasis: In Silico Modeling of Nanodrug Brendan Deal1. Seung-gu Kang. 1Chemistry, Emory University, Atlanta, GA, USA, 2Biomedical Engineering, Computational Biology Center, IBM Thomas J. Watson Research Center, Georgia Tech, Atlanta, GA, USA. Yorktown Heights, NY, USA. Modifying RNA through either splicing or editing is a fundamental biolog- Nanotechnology have been playing a critical role in tackling incurable diseases ical process for creating protein diversity from the same genetic code. like cancers with some approved and more in clinical inspection. A variety of Developing novel chemical biology tools for RNA editing has potential to physicochemical amenability and functional modularity endowed unprece- transiently edit genes and to provide a better understanding of RNA dented clinical potentials to nanomaterials in mulita-dimensional applications biochemistry. Current techniques used to modify RNA include the use of ri- such as in novel diagnostics and therapeutic tools for cancer therapy. Despite bozymes, adenosine deaminase and tRNA endonucleases. Herein, we report decades of effort on nonconventional nanomedicines, however, relevant phar- a nanozyme that is capable of splicing of virtually any RNA stem-loop. This macological mechanisms at a molecular level remains largely unknown. nanozyme is comprised of a gold nanoparticle functionalized with three en- Recently, Gd-encaged endohedral metallofullerenol Gd@C82(OH)22 was zymes: two catalytic DNA strands with ribonuclease function and an RNA

BPJ 8749_8752 Wednesday, February 21, 2018 691a ligase. The nanozyme cleaves and then ligates RNA targets, performing a dependent stability and membrane permeability were investigated as well. splicing reaction that is akin to the function of the spliceosome. Our results The drug release kinetics was monitored by UV VIS spectroscopy in different show that the three-enzyme reaction can remove a 19 nt segment from a 67 environments. nt RNA loop with up to 66% efficiency. The complete nanozyme can This research was supported by the UNKP-17-3-III-SE-17 new national excel- perform the same splice reaction at 10% efficiency. These splicing nano- lence program of the ministry of human capacities and by the OTKA Founda- zymes represent a new promising approach for gene manipulation that has tion (Grant No. K115259). potential for applications in living cells. 3427-Pos Board B635 3425-Pos Board B633 Generalized Langevin Dynamics for Stealth Nanoparticle Adhesion to Characterizing Large-Pore Protein Crystals for Advanced Material Membrane Surface Applications Yu-Wen Wu, Hsiu-Yu Yu. Luke F. Hartje1, Brian E. Munsky2, Hieu T. Bui3, David A. Andales2, chemical engineering, National Taiwan University, Taipei City, Taiwan. Christopher D. Snow1,2. Stealth nanoparticles (SNP) are promising materials for nanocarriers in 1Biochemistry and Molecular Biology, Colorado State University, Fort vascular targeted drug delivery due to their capability of avoiding the Collins, CO, USA, 2Chemical and Biological Engineering, Colorado State recognition by the immune system. Although the long-circulation time of University, Fort Collins, CO, USA, 3School of Biomedical Science, Colorado the SNP can be generally achieved, the delivery efficiency of such nanopar- State University, Fort Collins, CO, USA. ticles is also influenced by various physiological factors such as vessel- With rapidly growing interest in therapeutic macromolecules, high-density in- wall-mediated hydrodynamic interactions, biological recognition between formation storage, and advanced biofunctional fabrics comes the need for new its targeting agents and membrane receptors, as well as cell membrane materials capable of guest macromolecular storage and metered release on the morphology. In this work, we model the SNP as a composite nanoparticle nanoscale level. One novel possibility for such materials are engineered large- consisting of a hard core coated with a porous polymeric brush with end- pore protein crystals (LPCs). Composed of numerous chiral constituents, LPCs ligands. In the presence of a harmonic binding potential between the func- are ordered biologically derived nanoporous materials exhibiting hexagonal tionalizing ligands and membrane receptors, the nanoparticle center-of- close-packed pores greater than 8 nm. These substantial pores distinguish mass motion, structural relaxation of the coated polymers, and membrane LPCs from typical nanoporous scaffolds, enabling engineered LPC materials thermal undulations are simultaneously resolved using coupled generalized to readily uptake, immobilize, and controllably release macromolecular guests. Langevin equations (GLE). The mean squared displacement, velocity auto- Furthermore, LPCs can be designed to contain varying functional groups by correlation function, and position autocorrelation function of the SNP for means of genetic mutation or post-translational modification. The chemical di- various distances to the vessel wall, coated brush heights, and binding po- versity and functional versatility of LPCs make them promising targets for use tentials are examined. We demonstrate that the interplay between the ther- as nanostructured scaffolds with potential applications in drug delivery, bio- modynamic effects and hydrodynamic interactions substantially impacts the sensing, enantiomer separations, and multifunctional textiles. transient relaxation of both the velocity and position of the SNP, as evi- This work highlights our efforts to: experimentally and computationally inves- denced by the distinct behaviors observed at different characteristic time tigate macromolecular transport and interaction energies within an LPC envi- scales. ronment using time-lapse confocal microscopy, bulk equilibrium adsorption, 3428-Pos Board B636 and hindered diffusion simulation; assess the cytocompatibility of various Micro Magnetic Arrays for Micromanipulation at the Molecular and cross-linking chemistries for the production of biostable LPC materials for Cellular Scale use in biologically sensitive environments; and create multifunctional textiles Koceila Aizel1, Chiara VIichario1, Elie Balloul1, Cornelia Monzel1, by covalently attaching cross-linked protein crystals to cellulose fibers in Emilie Secret2, Loı¨c Toraille3, Mathieu Coppey1, Maxime Dahan1. woven cotton fabrics. 1Institut Curie, Paris, France, 2UPMC, Paris, France, 3Universite Paris Sud 11 By pursuing this research, we hope to better understand LPC materials and Orsay, Paris, France. leverage that knowledge to design advanced nanostructured devices for appli- The use of magnetic fields for studying a variety of biological mechanisms has cations in biotechnology and nanomedicine. gained increasing attention among the biophysical community since they can be used to non-invasively apply physical cues. With the use of bio- 3426-Pos Board B634 functionnalized Magnetic Nanoparticles (MNPs), it has become possible to Novel Biocompatible Poly(Aspartamide) Based Drug Conjugates specifically target and manipulate individual molecules in living systems in or- 1 1 € 2 3 David Juriga , Peter Laskawy , Zeliha Guler , Krisztina Ludanyi , der to remotely actuate cellular events. To successfully displace MNPs or apply 1 2 3 Angela Jedlovszky-Hajdu , Sezai A. Sarac , Imre Klebovich , mechanical forces, high magnetic field gradients need to be generated. Such Miklos Zrinyi1. 1 gradients necessitate using microsized magnetic elements. Usually, micro mag- Biophysics and Radiation Biology, Semmelweis University, Budapest, netic tips are used for that purpose but they are limited in terms of throughput Hungary, 2Department of Chemistry, Istanbul Technical University, Istanbul, 3 and reproducibility. Thus, we have developed Micro Magnetic Arrays Turkey, Department of Pharmacy, Semmelweis University, Budapest, (MMAs), for in vitro and in vivo experiments, which can be combined with mi- Hungary. cropatterning technniques. The magnetic properties of the soft ferromagnetic Drug-polymer conjugates are novel class of nano-carriers for effective drug microelements made out of permalloy (typical size 10-100 microns) and depos- administration. Drug molecules can be covalently conjugated to macromole- ited on glass coverslips were carefully characterized using different measure- cules in such a way that their release is primarily controlled by the rate of chem- ment methods (including optical magnetometry using NV centers) to further ical or enzymatic cleavage of polymer-drug bonds. Furthermore protection of optimize the performances of the arrays. As a result, we were able to microfab- the drug from deactivation as well as from premature degradation, minimiza- ricate structures capable of generating magnetic field as high as 1T and gradi- tion of the cytotoxicity and enhancing tissue compatibility can be achieved. ents above 104T/m, capable of successfully manipulating within cells MNPs as Several polymers have been recently investigated as candidates for polymeric small as 10nm in diameter. prodrugs. For good polymer candidate it is essential that the polymer used is neither inherently toxic nor immunogenic whereas biodegradability is also 3429-Pos Board B637 favorable. Release of drug or drug analogues from the macromolecular back- A Low Voltage Insultaor-Based Nanopipette Dielectrophoresis Device for bone might be induced either by chemical or by enzymatic reactions. Rapid Nanoparticles Entrapment As several enzymes can be found in the human body which cleaves amide Leilei Shi, Ankit Rana, Leyla Esfandiari. bonds, poly(amino acids) are promising candidates to prepare polymer-drug Electrical Engineering and Computer Science, University of Cincinnati, conjugates. In this work poly(aspartamide) based conjugates were synthesized Cincinnati, OH, USA. with different chemical constitutions with dopamine as conjugated drug. The The ‘‘insulator-based’’ DEP (iDEP) devices have become attractive to manip- conjugates were synthesized from poly(succinimide) by nucleophilic addition. ulate and separate biomolecules. However, they depend on complicated fabri- The drug release from the different conjugates was investigated in the presence cation procedures and high operational voltage 100 V/cm. Utilizing a glass of different enzymes and the kinetics of release was described. To prepare drug nanopipette as an iDEP device provides simple and cost-effective fabrication delivery system oppositely charged polymers were prepared from poly(succini- procedure while the applied voltage can be reduced significantly due to their mide). Oppositely charged poly(a-amino acid)-based polyelectrolytes interact small conical geometry. The majority of the nanopipette-DEP devices utilize electrostatically forming polymer nanocomplexes with a diameter 100-200 alternative currents (AC) for inducing the non-uniform electric field and nm. The size and the shape of the nanocomplexes were characterized by thus, no electroosmosis (EOF) flow participates in conveying molecules to DLS and different microscopic techniques. The pH and salt concentration the pipette tip. Here, we present a novel nanopipette-iDEP device that can

BPJ 8749_8752 692a Wednesday, February 21, 2018 trap particles under significantly lower Direct Current (DC) compared to the of silicon. A change in temperature changes the resonant wavelength condition conventional iDEP schemes. The electrokinetic forces and the trapping mech- of a silicon micro-resonator. Temperature near the surface of nanoparticles anisms were systematically studied by investigating the relationship between attached to these resonators can then be measured based on this optical readout. the nanoparticles trapping and physical aspects of the system including the This approach allows us to measure the heat dissipation rates of nanoparticles ionic strength of the solution, the electric field strength, and the pore geometry present on a surface. Silicon resonators have been shown to have temperature and size. sensitivities of few tens of mK and response times of 6 ms, making them suit- The results indicate that the minimum required voltage to effectively trap par- able candidates for precise measurement of nanoscale heating. ticles can be as low as 1 V/cm. The quantity of trapped particles was deter- mined by the net electrokinetic force comprise of the DEP, EP and EOF 3432-Pos Board B640 forces and the initial velocity of the particles before entering the trapping Gel-Based and Single Molecule Nanoswitch-Linked Immunosorbent As- area, which depends on the pore geometry and the ionic strength. This proof- says (NLISA and smNLISA) for Sensitive and Specific Protein Detection Clinton H. Hansen1,2, Johanna Blass1,2, Darren Yang1,2, Wesley P. Wong1,2. of concept study paves the way to further utilize the nanopipette-iDEP device 1 2 for entrapment of the biomolecular entities from bodyfluids or cell culture me- Boston Children’s Hospital, Boston, MA, USA, Harvard Medical School, dia in biosensing applications. Boston, MA, USA. Protein detection and quantification play critical roles in basic research and 3430-Pos Board B638 clinical practice. Current detection platforms range from widely used ELISAs Modulation of the Drag Force Exerted by Microfluidic Flow on Laser- to more sophisticated and expensive approaches such as digital ELISA. Despite Trapped Particles: A New Method to Assess Surface-Binding Kinetics, An- advances, there remains a need for a method that combines the simplicity and alyte Size, and Solution Viscosity cost-effectiveness of ELISA with the sensitivity and speed of modern ap- Wooten D. Simpson III, Volkmar Heinrich. proaches in a format suitable for both laboratory and rapid, point-of-care appli- Biomedical Engineering, University of California - Davis, Davis, CA, USA. cations. Building on recent developments in DNA structural nanotechnology, We have designed and validated a platform capable of assessing minuscule we introduce two detection platforms based on easily-constructed DNA nano- changes of the viscous drag force that different fluid environments exert on in- devices that change conformation upon binding to a target protein. In the dividual, micrometer-sized particles. Based on a combination of optical twee- nanoswitch-linked immunosorbent assay (NLISA) this conformational change zers and a microfluidic device, this platform allows us to expose a laser-trapped is read out by gel electrophoresis. In the single-molecule NLISA (smNLISA), particle to a series of microenvironments, created through microfluidic laminar nanoswitches are tethered to a surface with a microsphere on the free end, and flow, and to monitor biochemical interactions on the surface of the particle. In the conformational change corresponding to a difference in length is read out addition, we can determine the viscosity of the different environments with by stretching under hydrodynamic flow. NLISA is surface-free and includes excellent resolution. a kinetic-proofreading step for purification, enabling both enhanced sensitivity We have used this setup to examine the interaction between human IgG and and reduced cross-reactivity. We demonstrate fM-level detection of prostate- both protein-A as well as protein-G. At saturation, bound IgG added an specific antigen in biological fluids, as well as reduced cross-reactivity between apparent thickness of 12.65.77(SD)nm and 16.051.4(SD) nm to protein-A- different serotypes of dengue and also between a single-mutation and wild-type and protein-G-coated beads, respectively. This is in agreement with the ex- protein. NLISA is less expensive, uses less sample volume, is more rapid, and pected size of human IgG of 10-16 nm dependent on orientation. Moreover, with no washes, includes fewer hands-on-steps than ELISA, while also 7 1 achieving superior sensitivity. In smNLISA, the states of the DNA nano- we measured equilibrium constants of KA=4.21x10 M for the protein-A:IgG 7 1 switches are read out at the single-molecule level with a tethered bead assay. interaction and KA=7.94x10 M for the protein-G:IgG interaction, in agree- ment with literature values. Flow stretches out the nanoswitch in two directions for an accurate conforma- Finally, we measured the viscosity of different solutions of bovine serum albu- tional measurement, with the measured length used to distinguish between min, human serum, and glycerol with mPa$s resolution. Measurements were bound and unbound constructs. Stretching out also enables mechanical proof- performed in a microfluidic flow chamber and in microwells on a glass slide. reading, in which force disrupts nonspecific binding to enable accurate quanti- The microwells allowed us to carry out comparative viscosity measurements fication and reduced background for aM-level detection. with very small volumes of solution, less than 50 mL. The results obtained in 3433-Pos Board B641 each case agree with published models. Micro-Patterned Coverslips Using Thermal Nanoimprint Lithography for This platform opens up a new playing field for the study of biochemical surface Drift Correction for Super-Resolution Fluorescence Microscopy interactions. Its potential applications include the investigation of biomolecular Yeoan Youn1,2, Yuji Ishitsuka2,3, Chaoyi Jin1,2, Paul R. Selvin1,2. interactions at the surface of live biological particles, such as individual bacte- 1Biophysics and Quantitative Biology, University of Illinois Urbana ria, fungal cells, or large viruses, and the determination of the viscosity of Champaign, Urbana, IL, USA, 2Center for the Physics of Living Cells, costly solutions or small samples. University of Illinois Urbana Champaign, Urbana, IL, USA, 3Department of Physics, University of Illinois Urbana Champaign, Urbana, IL, USA. 3431-Pos Board B639 Super-resolution fluorescence microscopy is a technique which enables sub- Measuring Nanoscale Heating using Silicon Photonic Thermometry diffraction-limited imaging of biological structure and dynamics in vitro and Sruthi Polali1, Fan Ye2, Jacob Robinson3. 1 in vivo. However, it requires correction of stage drift as the imaging requires Electrical Engineering/Applied Physics, Rice University, Houston, TX, extended acquisition time to obtain. Micro-patterning is a possible solution for USA, 2Electrical Engineering, Rice University, Houston, TX, USA, 3 the issue. However, the selection of materials is limited by autofluorescence, Electrical Engineering, Bioengineering, Rice University, Houston, TX, chemical properties and cytotoxicity of the materials, and complexity of the USA. fabrication process. Here, we introduce a simple micro-patterning technique Genetically encoded ion channels that respond to magnetic fields -‘Magnetoge- which is called thermal nanoimprint lithography (T-NIL), for which the material netics’ — enable wireless stimulation of specific neurons deep in the brain and used has low auto-fluorescent background and a high bio-compatibility in terms provide a powerful tool for studying neural correlates of behavior in freely of cell adhesion and viability. We have cultured various types of cells, including moving animals. Recently, several magnetogenetic proteins were constructed mammalian cell lines and primary-culture neurons with cell viability. We find using heat-sensitive channels such as TRPV1/4 attached to biogenic iron stor- the viability and fluorescence quality comparable to that of bare glass coverslips. age protein, ferritin. Magnetic fields have been proposed to activate these chan- After drift correction with the patterned glass coverslips, the drift correction pre- nels via a thermal mediated pathway. Ferritin can be heated in an alternating cisions were 1.6 nm and 1.3 nm for lateral axes (x and y), and 5.9 nm for axial magnetic field through relaxation losses or in a low frequency field via the mag- axis. We apply this method to acquire 4-color fluorescence image of live netocaloric effect. Classical heat transfer laws predict that because nanopar- mammalian cells and super-resolution images of glutamate receptors and post- ticles dissipate heat quickly and efficiently to the surroundings, the small synaptic density proteins in live hippocampal neurons. amount of heat produced in ferritin would not be sufficient to gate channels. However, experimental evidence has emerged recently that suggest that tem- 3434-Pos Board B642 peratures of magnetically heated iron oxide nanoparticles are many degrees Reconstructing Nanoscale Structures from Sequence Topology of Spatial higher than that of the bulk, and that the heat dissipates much slower than ex- Networks of Barcoded DNA pected. The actual dissipation rates have been shown to depend on whether the Ian T. Hoffecker, Giulio Bernardinelli, Larsen Vornholz, Yunshi Yang, nanoparticles are in a suspension or on the surface of a cell membrane. Bjo¨rn Ho¨gberg. Here, we propose a novel method for measuring temperature in the vicinity of Karolinska Institutet, Stockholm, Sweden. magnetic nanoparticles and their heat dissipation rates using silicon photonic Nanoscale spatial information such as the distribution of membrane proteins thermosensors. This method relies on temperature dependent optical properties and other biological structures can be obtained with super resolution and

BPJ 8749_8752 Wednesday, February 21, 2018 693a electron microscopy, however such methods often lack throughput and the sta- 3436-Pos Board B644 tistical power needed to characterize populations of cells or diagnose diseases. Nanocrescent Optical Antennas for Ultrafast Photonic PCR Proximity detection strategies conversely fall short in the structural detail that Doyeon Bang, Jonghwan Lee, SoonGweon Hong, Min Sun Song, they can provide. Our method, Spatial Inference from DNA Topology Luke P. Lee. (SIFFDT), addresses this gap by utilizing the pairwise association of DNA BIOE, UC Berkeley, Berkeley, CA, USA. barcodes to record the spatial adjacency of probes that neighbor each other. Us- Ultrafast photonic polymerase chain reaction (PCR), which thermal cycling uti- ing next gen sequencing to recover the pairwise association, detailed nanoscale lizing rapid plasmonic conversion of the light to heat drastically reduced time- spatial networks of probes can be reconstructed. We implemented an in silico consuming thermal cycling step. However, to enable rapid precision medicine reconstruction algorithm and demonstrate that sequences of barcodes can be in the point-of-care (POC), quantitative and multiplexed realtime PCR is neces- processed to return a visual representation close to the true structure of clusters sary. Here we present a hexagonally packed nanocrescent optical antenna array of probes. In addition, we performed basic proof of concept experiments for the for realtime ultrafast photonic PCR. We theoretically demonstrated that each generation of spatially adjacent barcode pairs. The technique represents a nanocrescent antennae have an internal cavity with 0.1 fL volume and liquid microscopy-free approach to extracting nanoscale spatial information that within the cavity is heated up from 25 Cto95 C in nanosecond scale. We also could have broad utility in cell biology and diagnostic medicine. experimentally demonstrated that hexagonally packed nanocrescent cavity heater array exhibited fast heating in the macroscale (22.4 5 3.1 C). In order 3435-Pos Board B643 to demonstrate real-time PCR on the chip, we integrated hexagonally packed Real Time Actuation of a DNA Based Robotic Arm nanocrescent array to the microfluidic chip. We believe that this hexagonally Enzo Kopperger1, Jonathan List1, Sushi Madhira2, Florian Rothfischer1, packed nanocrescent optical antenna impact both life science and ultrafast pre- Don C. Lamb2, Friedrich C. Simmel1. cision molecular diagnostics. 1Physics, Technical University of Munich, Garching, Germany, 2Chemistry, Ludwig Maximilian University of Munich, Munich, Germany. 3437-Pos Board B645 Since the early days of nanotechnology, scientists have dreamt of nanoscale Addressing the Stability of Polygonal DNA Nanostructures In Vitro and mechanical systems that resemble macroscale industrial assembly lines. Molec- In Vivo ular self-assembly with DNA molecules, used for the bottom-up creation of Christina Kolonelou, Alessandro Bosco, Bjo¨rn Ho¨gberg, Ana Teixeira. complex molecular constructs, has been shown to hold large potential to Karolinska Institute, Stockholm, Sweden. push towards a realization of this vision. Starting out from simple mechanical DNA nanostructures have shown great potential to investigate fundamental mechanisms, increasingly intricate systems have been demonstrated in the past biological questions. We have previously developed DNA nanostructure- decade. A wide collection of mechanical elements, potentially used as compo- based tools to control the display of protein ligand assemblies with nano- nents for even more elaborate systems have been developed over the past years. meter- scale precision. These types of functionalized DNA nanostructures These elements were based on a variety of different concepts to facilitate can provide the means to assess the biophysical properties of membrane re- conformational changes, sliding motion and rotation. We here demonstrate a ceptor activation and to control cellular outcomes. Moreover, DNA nano- DNA-based molecular robotic arm integrated on a square base plate created structures can be utilized in vivo, for example in drug delivery targeting with the DNA origami technique, expanding the repertoire of available building tumors in cancer research. However, their structural integrity in physiolog- components for DNA assembled nanomachines. A flexible connection between ical conditions is a challenge that remains to be addressed. Here, we pro- base plate and arm allows rotation of the arm with respect to the specifically pose a novel method based on proximity ligation assay that enables us to addressable base plate. Based on a one pot folding approach, the structure effi- monitor the stability of polygonal DNA nanostructures in vitro and ciently self-assembles. With the help of single-molecule fluorescence tech- in vivo. We designed polygonal DNA nanostructures decorated with short niques, diffusive arm rotation and temporary fixation at different docking protruding DNA sequences, which serve as a template for the polymeriza- positions on the base plate is observed on a millisecond time scale. Utilizing tion of rolling circle amplification products (RCP). RCPs are detected by the high intrinsic charge of our DNA nanoconstruct we also demonstrate the hybridization of a fluorescently labeled detection oligonucleotide. The dis- use of electrical fields to actively control the arm’s angle relative to the base tance between the short protruding DNA sequences determines whether the plate. Actuation of the robot arm only requires milliseconds, which is many or- rolling circle amplification reaction takes place or not. Thus, a fluorescent ders of magnitude faster than established methods for the operation of synthetic signal indicates that the DNA nanostructure integrity is unaffected. This DNA nanomachines. For future work, the system promises to act a as versatile method can have a range of applications in assessing the stability of rapid prototyping platform for the development of components for molecular DNA nanostructures and evaluate their suitability for applications in vivo assembly lines and highly parallelizable nanoscale force spectroscopy. and in vitro.

BPJ 8749_8752