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Symposium: Biophysical Mechanisms of 68-Symp An Alternative Strategy to Generate Binding Proteins Molecular Evolution Andreas Plueckthun. Biochemistry, University of Zurich, Zurich, Switzerland. 65-Symp The most frequently used binding proteins in research are monoclonal anti- Structural and Functional Constraints on Protein Evolution bodies, made by the >40-year-old hybridoma technology, some with question- Claus O. Wilke. able performance [1]. More recently, recombinant antibodies and non-antibody Department of Integrative Biology, The University of Texas at Austin, scaffolds, selected from synthetic libraries, have started to provide access to Austin, TX, USA. molecularly defined molecules [2]. Nonetheless, all of these approaches require Proteins evolve under constraints determined by their structural and functional one to treat every target as a completely new project. This is unavoidable for properties. These constraints are visible in particular at buried sites in proteins, folded proteins. However, we hypothesized that for unfolded proteins or sites in or near active sites in enzymes, and sites involved in protein-protein in- unfolded stretches (tags, posttranslationally modified tails, denatured proteins terfaces. All these sites experience increased purifying selection and evolve on western blots), termed ‘‘peptides’’ for simplicity, the regularity of the pep- more slowly than do other sites. Here, I will discuss the relative strengths of tide main chain can be exploited. If true, a modular detection system can be these effects, and I will show that even though most active sites of enzymes devised, which would ultimately allow one to generate a sequence-specific are located in the protein core, we can disentangle conservation due to solvent binding protein without experimentation. accessibility and conservation due to enzymatic activity. We also find that cat- The basis of our approach are Armadillo Repeat Proteins [3-9], which bind pep- alytic residues in enzymes exert a long-range effect, causing increased conser- tides in a completely extended way, providing a pocket for each side chain, and vation of residues throughout 80% of a typical enzyme structure. Finally, I will thus access to a modular approach. Combining evolutionary engineering, show that protein-protein interfaces show surprisingly little sequence conserva- NMR, X-ray crystallography and structure-based computation, we have now tion, and that interfaces can diverge substantially yet retain the ability to bind to achieved well crystallizing ArmRPs with bound peptides, picomolar affinities, ancestral binding partners. and a well functioning selection and evolution technology, as well as many biochemical and biophysical analysis technologies for the engineered ArmRPs. 66-Symp Progress in the various aspects will be summarized. Molecular Ensembles Shape Evolutionary Trajectories 1. Bradbury and Pluckthun€ (2015) and 110 co-signatories, Nature 518, 27. Michael J. Harms, Zachary R. Sailer, Lucas C. Wheeler. 2. Pluckthun€ (2015). Annu. Rev. Pharmacol. Toxicol. 55, 489. Institute of Molecular Biology, Department of Chemistry and Biochemistry, 3. Reichen et al., (2016). J. Mol. Biol. 428, 4467 University of Oregon, Eugene, OR, USA. 4. Hansen et al. (2016). J. Am. Chem. Soc. 138, 352 Evolutionary prediction is of deep practical and philosophical importance. One 5. Reichen et al., (2016). Acta Crystallogr. D72, 168. avenue for such a prediction would be to measure the fitness effects of all mu- 6. Reichen et al., (2014). Protein Science 23, 1572. tations to a protein, and then use this information to predict evolution. We at- 7. Reichen et al., (2014). J. Struct. Biol. 185, 147. tempted such a prediction for the simple case of evolving increased 8. Alfarano et al., (2012). Protein Science 21, 1298. thermodynamic stability in a simple protein lattice model. Surprisingly, we 9. Madhurantakam et al., (2012). Protein Science 21, 1015. were unable to predict evolutionary trajectories, even given complete knowl- edge of the effects of all mutations to the ancestral protein. This is a direct consequence of the ensemble of similar structures populated by proteins. The Symposium: DNA Supercoiling effect of a mutation depends on the relative probabilities of conformations in the ensemble, which in turn depend on the exact amino acid sequence of the 69-Symp protein. Accumulating substitutions alter the relative probabilities of conforma- Seeing Supercoiled DNA with Atomistic Simulation: A New Twist on a tions, thereby changing the effects of future mutations. This manifests itself as Familiar Structure subtle, but pervasive, multi-way interactions between mutations (high-order Sarah A. Harris1, Agnes Noy2, Thana Sutthibutpong3. epistasis). As mutations accumulate, uncertainty in the predicted effect of 1School of Physics and Astronomy, University of Leeds, Leeds, United each mutation accumulates and undermines prediction. We then developed Kingdom, 2School of Physics, University of York, York, United Kingdom, computational and statistical tools to look for evidence of this high-order epis- 3Theoretical and Computational Science Center, King Mongkut’s University tasis in experimentally determined genotype-fitness maps. We find that high- of Technology Thonburi, Bangkok, Thailand. order epistasis is ubiquitous. This work reveals that evolutionary unpredict- The discovery of the structure of duplex DNA revealed how cells store genetic ability arises—even for fixed environments and under strong natural selec- information. However, we are far from understanding the more complex biolog- tion—as a direct consequence of the thermodynamic ensemble populated by ical question of how this information is regulated and processed by the cell. DNA proteins. supercoiling is generated whenever a gene is transcribed, and complex cellular machinery, such as toposisomerases, are required to modulate the effect of this 67-Symp induced torsional stress. Supercoiling has been implicated in the packaging and Cellular Consequences of Systematic Perturbations of a Highly Conserved 3D arrangement of both prokaryotic and eukaryotic DNA, which in turn has Biological Switch fundamental consequences for transcription regulation and genome stability. Tanja Kortemme. In spite of the ubiquity of supercoiled DNA in cells, no experimental tool has University of California, San Francisco, San Francisco, CA, USA. been able to capture atomically detailed structural information. Small DNA cir- Cellular protein-protein interactions can be highly interconnected. Because of cles containing between 100 and 400 base pairs, however, offer a controllable this complexity, it is often difficult to extract quantitative information on how model system for the systematic exploration of the dependence of DNA structure each interaction contributes to distinct or overlapping cellular functions, and, on supercoiling through cryo-electron microscopy, atomic force microscopy, moreover, how changes to individual interactions result in altered function or and computer modelling. We use atomistic molecular dynamics simulations disease. We are developing an experimental platform for studying perturba- to explore the supercoiling-dependent conformation of small DNA circles. tions to multi-functional network ‘‘hub’’ proteins by combining high- We show that kinks and denaturation bubbles are generated in the DNA by throughput in vivo genetic interaction screening technology (Epistatic MiniAr- high torsional stress, that the compaction of the DNA is highly dependent on ray Profile (E-MAP)) with mass-spectrometry and biophysical assays. Our case salt, and that the DNA adopts writhed structures that are highly dynamic and study protein is the highly conserved multi-functional Gsp1/Ran GTPase which offer additional opportunities for DNA/protein interactions in 3D space. switch that controls key eukaryotic processes. The approach first engineers We then offer an atomistic interpretation for the growing experimental data that defined perturbations to Gsp1/Ran protein-protein interactions by amino acid shows the regulatory role proposed for supercoiling in the genome. point mutations (‘‘edge perturbations’’). The second step determines the func- tional effects of these perturbations at the cellular and organism level in the 70-Symp model S. cerevisiae. We find that E-MAPs have a resolution that enables us Protein-Mediated Loops in Supercoiled DNA Generate Large, Dynamic to identify quantitative functional differences in vivo between individual point Topological Domains mutations, even those between different amino acid substitutions of the same Laura Finzi. residue. Our analysis reveals several classes of observed phenotypes that could Physics, Emory University, Atlanta, GA, USA. be explained by the underlying biophysical perturbations of the on/off balance Proteins that act at a distance along DNA by binding at one site and contacting of the fundamental GTPase switch and considerable allosteric effects in the another create loops that form topological domains and influence regulation. system. Such loops are affected by the torsional state of DNA, which dramatically

BPJ 8532_8543 Sunday, February 18, 2018 13a modulates topology, driving the DNA from extended and accessible, to more localization microscopy as a function of the measured photophysical parame- compact and genetically secured forms. Furthermore, the response of the ters of the probe such as photobleaching quantum yield, count rate per mole- DNA filament to supercoiling is biased by accessory factors. For example, small cule, and intensity of saturation. The model was used to predict the molecules like polyamines, which neutralize the negative charge repulsions dependence of resolution on acquisition parameters such as illumination inten- along the phosphate backbone, enhance flexibility and promote writhe over twist sity and time per frame, demonstrating an optimal set of acquisition parameters in response to torsion. On the other hand, stiffer DNA antagonizes looping and for a given probe for a variety of measures of resolution. The best-possible res- bending. Recent experiments quantitatively reveal the extent to which nega- olution was then compared for Dendra2 and other widely used probes, tively supercoiling DNA lowers the free energy of looping, and could therefore including Alexa dyes and quantum dots. This work establishes a framework bias the operation of genetic switches. A new role for DNA supercoiling has for determination of the best possible resolution using a localization micro- emerged; it synergizes with protein-mediated looping to create large, dynamic scope to image a particular fluorophore, and suggests that development of topological domains that extend beyond the length of the loop. Such domains probes for use in super-resolution localization microscopy must consider the may coordinate gene regulation and other DNA transactions across spans in count rate per molecule, the saturation intensity, the photobleaching yield, the genome that exceed the separation between the protein binding sites. and, crucially, management of bright/dark state transitions, in order to optimize image resolution. 71-Symp Organisation and Function of DNA Supercoiling in the Human Genome 74-Plat Nick Gilbert. Pushing the Boundary of Storm Resolution: Seeing the Actin Lattice in University of Edinburgh, United Kingdom, Edinburgh, United Kingdom. Muscle 1 2 3 72-Symp Sheema Rahmanseresht , Kyounghwan Lee , Jeffrey Robbins , David M. Warshaw1, Roger Craig2, Michael J. Previs1. The Role of DNA Topology and Conformation in Gene Regulation, In Vivo 1 1 1 1,2 Department of Molecular Physiology and Biophysics, University of David Levens , Fedor Kouzine , Laura F. Baranello . 2 1Laboratory of Pathology, Center for Cancer Research, NCI, NIH, Bethesda, Vermont, Burlington, VT, USA, Division of Cell Biology and Imaging, 2 Department of Radiology, University of Massachusetts Medical School, MD, USA, Cell and Molecular Biology, Karolinska Institute, Stockholm, 3 Sweden. Worcester, MA, USA, Department of Pediatrics and the Heart Institute, The transient DNA unwinding exposing the bases templating RNA polymeri- Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA. zation, and the translocation of the transcription machinery (and chromatin fac- Muscle cells are composed of interdigitating arrays of myosin and actin fila- tors) around the double helix generate high levels of torsional stress ments that slide past one another to shorten cellular length. In striated muscle (supercoiling). Unless this stress is disposed of or accommodated within the cross-sections, myosin and actin form hexagonal lattices that have been well DNA and chromatin, it can accrue to levels high enough to impede or event ar- defined by electron microscopy and x-ray diffraction. The spacing between rest transcription and other genetic processes. Yet low levels of supercoiling neighboring actin filaments within the lattice is 25 nm which is at the boundary assist DNA melting, facilitate looping between remote elements and stabilize of STochastic Optical Reconstruction Microscopy (STORM) resolution. We nucleosomes. Hence, the generation and removal of supercoils must be regu- combined STORM with a single particle averaging technique to resolve actins’ lated. Dynamic supercoils represent transient torsional stress due to the ongoing hexagonal lattice in rat skeletal and mouse cardiac muscles. Muscles were cranking of DNA fibers by a molecular motor, such as RNA polymerase. dissected and fixed with paraformaldehyde and glutaraldehyde. Muscles were Dynamically supercoiled DNA rapidly relaxes to the unstressed state when then frozen in liquid nitrogen with sucrose as a cryoprotectant and thinly sliced, the motor is off. Fixing the ends of a DNA or chromatin segment, whether producing 200-400 nm transverse cryosections. Sections were thawed, then by looping or tethering to an immobile structure, mathematically defines a placed on glass coverslips and incubated with phalloidin-Alexa647 or static level of supercoiling within the embraced topological domain; this level phalloidin-Alexa488 to label actin. 100 nm multicolor beads were added as fidu- cannot be changed excepted by breaking one strand and swiveling a newly cial markers. Stochastic blinking of the fluorophores was captured using total in- created end around the unbroken strand (as performed by topoisomerase 1) ternal reflection microscopy. The fluorescence of each individual molecule was or creating a double-stranded break, or by breaking both strands and passing fitted and their positions were corrected for drift using the fiducial markers, al- through an unbroken double-stranded segment before resealing the strand(s). lowing for a localization precision of 8 nm. The resultant optical reconstructions Because the generation and removal of supercoils must be actively managed of actin were visually interrogated for the presence of hexagonal structures. A to fine-tune genetic transactions, the activity of topoisomerase 1 is coordinated mask was applied with 50 nm spacing between hexagonal centers and its local- with the progression of RNA polymerase II through the transcription cycle, and ization was refined by cross-correlation analysis. Individual actin hexagons were is adjusted by the direct action of transcription and chromatin factors. Further- cropped from the lattice and averaged to provide a 2-fold improvement in the more, DNA and chromatin structure is susceptible to context-and sequence resolution. By combining STORM with single particle averaging methods we dependent, torsion-driven changes in conformation. Moreover, there are factors were able to define the actin hexagonal lattice and its spacing with 4 nm resolu- that read these sequences and structures and so provide a tool to monitor and tion. We are currently extending this technique to 2-color imaging in order to control local gene activity in real time. resolve the spatial relationships between other contractile proteins that are known to interact with actin within these hexagonal arrays.

Platform: Optical Microscopy and 75-Plat Superresolution Imaging: Methods I Fast Super Resolved Imaging of Live Cells Using Superresolution Optical Fluctuation Imaging 2.0 (SOFI-2.0) 73-Plat Xiyu Yi1, Sungho Son2, Shimon Weiss1,3. The Role of Probe Photophysics in Localization-Based Superresolution 1Chemistry and Biochemistry, University of California, Los Angeles, Los Microscopy Angeles, CA, USA, 2Department of Ecology and Evolutionary Biology, Francesca Pennacchietti1, Travis J. Gould2, Samuel T. Hess1. University of California, Los Angeles, Los Angeles, CA, USA, 3California 1Dept. of Physics and Astronomy, University of Maine, Orono, ME, USA, Nano Systems Institute, University of California, Los Angeles, Los Angeles, 2Dept. of Physics and Astronomy, Bates College, Lewiston, ME, USA. CA, USA. Fluorescent proteins are used extensively for biological imaging applications; Superresolution Optical Fluctuation Imaging (SOFI) [1] has been widely photoactivatable and photoconvertible fluorescent proteins (PAFPs) are used acknowledged and advanced over the past years. Comparing to other exten- widely in super-resolution localization microscopy methods such as FPALM sively adopted super resolution techniques such as PALM [2], STORM [3], and PALM. However, their optimal use depends on knowledge of not only their STED [4] and SIM [5], advantages of SOFI include compatibility with different bulk fluorescence properties, but also their photophysical properties at the sin- imaging platforms, suitability for a wide variety of probes, flexibility in imag- gle molecule level. We have used fluorescence correlation spectroscopy (FCS) ing conditions, and a user-determined trade-off between spatial- and temporal- and cross-correlation spectroscopy (FXCS) to quantify the diffusion, photo- resolutions. SOFI therefore holds great promise for utilization in a broad area of bleaching, fluorescence intermittency, and photoconversion dynamics of Den- applications and for ‘democratizing’ and lowering the ‘barrier-of-entry’ of su- dra2, a well-known PAFP used in localization microscopy. Numerous dark per resolution imaging to non-expert practitioners. The theoretical resolution states of Dendra2 are observed both in inactive (green fluorescent) and active enhancement of SOFI scales as the square root of the cumulant order n, and (orange fluorescent) forms; the interconversion rates are both light- and pH- once combined with a post-processing deconvolution algorithm, the resolution dependent, as observed for other PAFPs. The dark states limit the detected enhancement factor increases up to n. Several practical approaches for address- count rate per molecule, which is a crucial parameter for localization micro- ing fundamental challenges and limitations of high order SOFI will be dis- scopy. We then developed a new mathematical estimate for the resolution in cussed, including pixel intensity dynamic range expansion, associated

BPJ 8532_8543 14a Sunday, February 18, 2018 artifacts, point-spread function (PSF) estimation, and deconvolution. Together, Highly Inclined and Laminated Optical sheet (HILO) microscopy is an optical this set of practical approaches constitutes what we dub as ‘SOFI-2.0’. The po- technique that employs a highly inclined laser beam to illuminate the sample wer of SOFI-2.0 will be demonstrated for focal-adhesion and actin stress fiber with a thin sheet of light that can be scanned through the sample volume. HILO dynamics (at super resolution) in live cells. is an efficient illumination technique when applied to fluorescence imaging of [1]Dertinger, T.; Colyer, R.; Iyer, G.; Weiss, S.; Enderlein, J.; Proceedings of thick samples owing to the confined illumination volume that allows high contrast the National Academy of Sciences of the United States of America 2009, 106 imaging while retaining deep scanning capability in a wide-field configuration. (52), 22287-22292. The restricted illumination volume is crucial to limit background fluorescence [2]Betzig, E.; Patterson, G. H., Sougrat, R.; Lindwasser, O. W.; Olenych, S.; originating from portions of the sample far from the focal plane, especially in Bonifacino, J. S.; Davidson, M. W.; Science 2006, 313 (5793), 1642-1645. applications such as single molecule localization and super-resolution imaging. [3]Hell, S. W.; Wichmann, J.; Optics Letters 1994, 19 (11), 780-782. Despite its widespread use, current literature lacks comprehensive reports of [4]Gustafsson, M. G. L.; Journal of Microscopy-Oxford 2000, 198, 82-87. the actual advantages of HILO in these kinds of microscopies. Here, we thoroughly characterize the propagation of a highly inclined beam 76-Plat through fluorescently labeled samples and implement appropriate beam 3D Single-Molecule Super-Resolution Microscopy in Mammalian Cells shaping for optimal application to single molecule and super-resolution imag- Using a Tilted Light Sheet ing. We demonstrate that, by reducing the beam size along the refracted axis Anna-Karin Gustavsson, Petar N. Petrov, Maurice Y. Lee, only, the excitation volume is consequently reduced while maintaining a field Yoav Shechtman W.E. Moerner. of view suitable for single cell imaging. We quantify the enhancement in Department of Chemistry, Stanford University, Stanford, CA, USA. signal-to-background ratio with respect to the standard HILO technique and We present tilted light sheet microscopy with 3D point spread functions apply our illumination method to dSTORM super-resolution imaging of the (TILT3D), an imaging platform that combines a novel, tilted light sheet illumi- actin cytoskeleton. We define the conditions to achieve localization precisions nation strategy with long axial range point spread functions (PSFs) for comparable to state-of-the-art reports, obtain a significant improvement in the low-background, 3D super-localization of single molecules as well as 3D image contrast, and enhanced plane selectivity within the sample volume due to super-resolution imaging in thick cells. Numerous light sheet designs have the further confinement of the inclined beam. been implemented for super-resolution imaging, but these designs can have drawbacks in certain situations such as being incompatible with imaging of flu- 79-Plat orophores close to the coverslip using high numerical aperture imaging objec- Graphene Biointerfaces for Optical Stimulation of Genetically Intact Cells tives. Some previous setups require complicated optical and electronic Alex Savtchenko1, Volodymyr Cherkas2, Alexander Kleschevnikov1, components or many custom-made parts which are often expensive and diffi- Gary Braun3, Aliaksandr Zaretski4, Darren L. Lipomi1, Ke Wei5, cult to build and operate, and thus may not be easily accessible to the general Elena Molokanova6. research community. We alleviate many of these issues by tilting the illumina- 1University of California, San Diego, CA, USA, 2Bogomoletz Institute of tion plane and detecting the 3D position of single molecules using engineered Physiology, Kiev, Ukraine, 3StemCell Technologies, Vancouver, BC, long axial range PSFs. Because the axial positions of the single emitters are en- Canada, 4GrollTex, San Diego, CA, USA, 5School of Life Sciences and coded in the shape of each single-molecule image rather than in the position or Technology, Tongji University, Shanghai, China, 6NTBS, San Diego, CA, thickness of the light sheet, the light sheet need not be extremely thin. TILT3D USA. is built upon a standard inverted microscope and has minimal custom parts. The Non-invasive stimulation of cells is crucial for the accurate examination and result is simple and flexible 3D super-resolution imaging with tens of nm local- control of their function both at cellular and system levels. To address the ization precision throughout thick mammalian cells. We validated TILT3D for need for truly non-invasive stimulation, we present a pioneering optical stimu- 3D super-resolution imaging in mammalian cells by imaging mitochondria and lation platform that does not require genetic modification of cells, and instead the full nuclear lamina using the double-helix PSF for single-molecule detec- capitalizes on unique optoelectronic properties of graphene, including its abil- tion and the recently developed Tetrapod PSFs for fiducial bead tracking and ity to efficiently convert light into electricity, zero-band gap, and strong live axial drift correction. We think that TILT3D in the future will become electron-electron interactions. an important tool not only for 3D super-resolution imaging, but also for live We tested our graphene-based optical stimulation platform using stem cell- whole-cell single-particle and single-molecule tracking. derived neurons and cardiomyocytes. Our experiments revealed exceptional biocompatibility of graphene-based biointerfaces (G-biointerfaces) as evi- 77-Plat denced by the formation of intricate neuronal networks with synaptic connec- Imaging Complex Protein Machines by High-Throughput Localization tions, and strong adhesion of cardiomyocytes to the graphene-based surface. Microscopy While examining light effects on functional activity of cells on G-biointerfaces, Joran Deschamps, Markus Mund, Jonas Ries. we discovered that light induces fast and reversible changes in the cell mem- Cell Biology and Biophysics, EMBL, Heidelberg, Germany. brane potential, leading to action potential generation or increase in action po- Single-molecule localization microscopy (SMLM) has become a popular tool in tential frequencies. We determined that our optical stimulation platform can cell biology as it allows imaging cellular structures with a precision of tens of enable fast, reversible, simultaneous stimulation of many cells at once, and nanometers. However, acquiring superresolved images with SMLM is inher- for extended periods of time. The kinetics of this process is controlled by light, ently slow, requiring up to hours for a single image and careful user intervention and light intensities for graphene-mediated and optogenetic stimulation are in in between experiments. This effectively limits the throughput of SMLM to a the same range. handful of images of the structure of interest per study. We overcame this limi- To evaluate the mechanism of graphene-mediated stimulation, we monitored tation by developing a fully automated microscope for high-throughput superre- photocatalytic activity, potential redox processes, and thermal effects in solution (HT-SRM). This microscope is optimized for stability and capable of light-illuminated G- biointerfaces. Our results strongly suggest that optical performing unsupervised SMLM imaging over the course of days. It is controlled stimulation via G-biointerfaces is consistent with capacitive effects of by an open-source plugin for Micro-manager, allowing the design of complex ‘‘clouds’’ of photogenerated ‘‘hot’’ ballistic electrons from graphene. experiments. In addition, it features a flat illumination system, providing homo- In summary, graphene-mediated optical stimulation could become a next- geneous statistics across large fields of view. We used this microscope to auto- generation technology enabling many innovative applications, including (a) matically acquire 100000 sites of clathrin-mediated endocytosis in yeast. This non-invasive studies of activity- and voltage-dependent processes in the brain high statistical power allowed us to resolve fine details of the endocytic machin- and heart; (b) activity-depended production of mature stem cell-derived cells; ery. This application demonstrates that HT-SRM enables system-wide imaging (c) improving functional integration of stem cell-derived cells into damaged tis- of the structural organization of complex protein machines. sues; (d) restoring vision; (e) enabling optical pacemakers. 78-Plat 80-Plat Optimization of Highly Inclined Optical Sheet Illumination for Super- A New Method (Sigma-SHREC) for Two-Color Fluorescent Distance Mea- Resolution Microscopy surements with Nanometer Accuracy Tiziano Vignolini1, Lucia Gardini2,3, Valentina Curcio1, Stefan Niekamp, Jongmin Sung, Walter Huynh, Ronald D. Vale, Marco Capitanio1,2, Francesco Saverio Pavone1,2. Nico Stuurman. 1LENS - European Laboratory for Non-linear Spectroscopy, Sesto Fiorentino Cellular and Molecular Pharmacology, University of California, San (FI), Italy, 2Department of Physics and Astronomy, University of Florence, Francisco, San Francisco, CA, USA. Sesto Fiorentino (FI), Italy, 3National Institute of Optics - National Research Understanding the spatial arrangement of macromolecules is crucial for Council, Firenze, Italy. discerning their molecular mechanisms. By labeling single molecules or

BPJ 8532_8543 Sunday, February 18, 2018 15a complexes at defined sites with a pair of chromatically distinct fluorescent sions. Furthermore, such structures have also been observed in a range of living probes, it is possible to obtain static or dynamic distance information using organisms. Although, reverse cubic or hexagonal lipid aqueous phase can be fluorescent microscopy. Churchman et al. (2006) have previously shown that used to entrap smaller biomolecules, it is still challenging to encapsulate bioac- most distance measurements in the lower nanometer range no longer follow tive macromolecules, such as proteins. Here, we will present a novel lipid sys- a Gaussian distribution and they developed a maximum-likelihood (MLE) tem able to form highly swollen sponge phases (L3), with aqueous pores up to based method (named SHREC) that fits distance measurements with a probabil- 13 nm of diameter. We will show that this structure is preserved even in excess ity distribution function to estimate the true distance. However, using both aqueous solution, where they form sponge-like nanoparticles (L3 NPs) in which experimental data and Monte-Carlo simulations, we show that the MLE esti- two enzymes of different sizes, Aspartic protease and beta-galactosidase (34 mates distances inaccurately when the true distance and localization errors KDa and 460 KDa, respectively), could be included. To reveal the nature of are of similar order. We overcame this problem by incorporating additional in- the interaction between the enzymes and the lipid matrix, we studied the formation, including localization and registration errors, and are able to deter- adsorption of both proteins on the lipid layers formed by the L3 NPs. The results mine small distances with nanometer accuracy. Thus, we have named this new will be discussed in terms of the ability of these nanoparticles to encapsulate method sigmaSHREC. Moreover, we developed a piecewise affine based im- and release of the proteins in the lipid matrix. age registration procedure that routinely yields sub-nanometer registration er- rors over the entire field of view. To ensure high-throughput and consistent 83-Plat measurements we implemented all analysis tools as a mManager-plugin. The Artificial Tunneling Nanotubes between Cells accuracy of our method was confirmed by a two-color measurement of the Kaori Sugihara. head-to-head distance of rigor-bound homodimeric kinesin-1. We found 7.9 University of Geneva, Geneva, Switzerland. 5 1.2 nm close to the EM measured distance of 8.0 nm. In addition, we applied Previously, we have discovered a new type of lipid nanotube self-assembly this method to understand the conformational heterogeneity of the coiled-coil made of the main lipid component of bacterial cell membranes, 1,2-dioleoyl- of Bicaudal-D, a dynein adaptor protein. Together, our new methods not sn-glycero-3-phosphoethanolamine (DOPE).[1] This lipid nanotube self- only enable distance measurements with nanometer accuracy but also the assembly is unique, since it involves inverted hexagonal phase, where its low investigation of sample conformational heterogeneity. surface tension compared to the lamellar phase provides an advantage for form- ing tubes.[2] In our group, we have been attempting to explore possible appli- cations of these self-assembled lipid nanotubes in cell contractility Platform: Membrane Physical Chemistry I detection,[3] as a template for fabricating metal nanostructures,[4] etc. In this work, we present that these lipid nanotubes can be used as a model sys- 81-Plat tem for studying tunneling lipid nanotubes (TNT) between living cells.[5] Emerging Approaches to Fabricate Supported Lipid Bilayers: Moving TNTs are universal communication tools for many types of cells that have Beyond Vesicles diverse structures and functions, discovered in 2004. Currently, TNTs are be- Nam-Joon Cho. ing studied with either in-vivo or in-vitro cell models. However, such tradi- Materials Science and Engineering, Nanyang Technological University, tional biological approaches often suffer from the complexities and a lack Singapore, Singapore. of controllability in the connection. We demonstrated a simple approach to Controlled self-assembly of model lipid membranes at solid-liquid interfaces create a direct tubular connection between cells based on the self-assembled opens the door to a wide range of applications across membrane biophysics, lipid nanotubes. The technique requires only a micromanipulator and a fluo- biotechnology and medicine. Recently, we developed the solvent-assisted lipid rescence microscope for controlling the LNT positioning and the connection bilayer (SALB) method to form supported lipid bilayers at interfaces. A key to the cells. The diffusion of water-soluble dye was monitored from one feature of the SALB method is that the supported bilayers form in an energet- cell to the connected cells, demonstrating the cytosol connections between ically favored scenario, enabling bilayer fabrication on formerly intractable two cells. The scanning electron microscopy (SEM) images visualized the surfaces like gold. Moreover, the process does not require pre-formed precursor diameter of the lipid nanotubes after fusing to cell membranes, which matches vesicles allowing for arbitrary compositions. Aided by lipid-substrate interac- with the time scale of the dye transport we observed by fluorescence tions, surface-adsorbed lipids in organic solvent are rapidly converted into microscopy. lamellar phase, supported bilayer islands upon addition of aqueous buffer solu- [1]K. Sugihara, et al., ACS nano 6, 6626 (2012). tion. Lipid species in the aqueous solution may attach to the bilayer islands and [2]K. Sugihara, et al., Soft Matter 11, 2029 (2015). subsequently rupture to form a contiguous, supported lipid bilayer. Owing to [3]K. Sugihara, et al., Integrative biology 5, 423 (2013). the technically minimal requirements of solvent-assisted lipid self-assembly, [4]K. Jajcevic, M. Chami, and K. Sugihara, Small 12, 4830 (2016). we have also developed on-chip lipid microfluidics that take advantage of [5]A. Kozintsev and K. Sugihara, Rsc Adv 7, 20700 (2017). the SALB method to form miniaturized biomembranes with a rich complexity of components reminiscent of natural cell membranes and that can be utilized 84-Plat on a variety of substrates with different atomic compositions and nanostructure Outperforming Nature: Synthetic Enzyme Built from DNA Flips Lipids of morphologies. In addition to the SALB method, additional innovations have Biological Membranes at Record Rates 1 2 3 enabled streamlined fabrication of supported lipid bilayers in fully aqueous Alexander Ohmann , Chen-Yu Li , Christopher Maffeo , 1 1 1 3 conditions by utilizing mixtures of phospholipids with minimal preparation re- Kareem Al Nahas , Kevin N. Baumann , Kerstin Go¨pfrich , Jejoong Yoo , Ulrich F. Keyser1, Aleksei Aksimentiev4. quirements. In turn, all these capabilities should further enable academic inves- 1 tigations related to membrane biophysics and pharmaceutical drug Cavendish Laboratory, University of Cambridge, Cambridge, United Kingdom, 2Center for Biophysics and Computational Biology, University of development efforts towards high-throughput lipid membrane functional 3 assays. Illinois at Urbana-Champaign, Champaign, IL, USA, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Champaign, IL, 82-Plat USA, 4Department of Physics, University of Illinois at Urbana-Champaign, Lipid Sponge-Phase Nanoparticles as Carriers for Enzymes Champaign, IL, USA. Maria Valldeperas Badell1,2, Aleksandra Dabkowska1,2, Mimicking enzyme function and increasing performance of naturally evolved Polina Naidjonoka1, Rebecca Welbourn3, Gunnar K. Pa´lsson4,5, proteins is one of the most challenging and intriguing aims of nanoscience. Justas Barauskas6,7, Tommy Nylander1,2. Here, we employ DNA nanotechnology to design a synthetic enzyme that sub- 1Physical Chemistry, Lund University, Lund, Sweden, 2NanoLund, Lund stantially outperforms its biological archetypes. Consisting of only eight University, Lund, Sweden, 3ISIS Neutron and Muon Source, STFC, strands, our DNA nanostructure spontaneously inserts into biological mem- Rutherford Appleton Laboratory, Didcot, United Kingdom, 4Institut Laue branes by forming a toroidal pore that connects the membrane’s inner and outer Langevin, Grenoble, France, 5Physics, Uppsala University, Uppsala, Sweden, leaflets. The membrane insertion catalyzes spontaneous transport of lipid mol- 6Camurus AB, Lund, Sweden, 7Biomedical Science, Malmo¨ University, ecules between the bilayer leaflets, rapidly equilibrating the lipid composition. Malmo¨, Sweden. Through a combination of microscopic simulations and single-molecule exper- Immobilization of enzymes into different support materials has been widely iments we find the lipid transport rate catalyzed by the DNA nanostructure to studied as means to control their activity and stability. Here we will consider exceed 107 molecules per second, which is three orders of magnitude higher lipid liquid crystalline phases as enzyme carriers, as they have been demon- than the rate of lipid transport catalyzed by biological enzymes. Furthermore, strated to have a high potential in a range of applications such as drug delivery, we show that our DNA-based enzyme can control the composition of human protein encapsulation or crystallization thanks to the wide range of self- cell membranes, which opens new avenues for applications of membrane- assembly structures they can form, which have cavities of nano-scale dimen- interacting DNA systems in medicine.

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85-Plat University of Illinois at Urbana-Champaign, Urbana, IL, USA, 4Centre for Synthesis and Biophysical Characterization of the Chlorosulfolipids of Molecular Simulation, Department of Biological Sciences, University of Ochramonas danica Calgary, Calgary, AB, Canada, 5Department of Biochemistry and Molecular Grace M. McKenna, Frank R. Moss III, Matthew L. Landry, Noah Z. Burns, Biology, University of British Columbia, Vancouver, BC, Canada. Steven G. Boxer. Small interfering RNA (siRNA) which silences genes in gene therapies can be Chemistry, Stanford University, Stanford, CA, USA. delivered into target cells using specialized lipid nanoparticles (LNPs). LNPs The freshwater algae Ochramonas danica produces a range of polychlorinated designed to encapsulate siRNA are composed of several components: a phos- single-chain amphiphiles. Danicalipin A, a hexachlorosulfolipid makes up 90% pholipid, a cationic lipid, cholesterol, and a polyethylene glycol-lipid. Each of the polar lipid content of the flagellar membrane of O. danica. Its exotic of these constituents contributes to the successful delivery of the siRNA. The structure presents both synthetic challenges and raises questions about its func- LNP enters the target cell and delivers its contents via endocytosis. As the en- tion within the membrane. To address these questions, we have combined total dosome matures, the environment becomes more acidic and the cationic lipid synthesis and membrane biophysics to investigate the effects of structural ele- (DLin-KC2-DMA) of the LNP becomes protonated. This facilitates the release ments of chlorosulfolipids on their behavior in monolayers and bilayers. The of the siRNA into the cytoplasm via attractive electrostatic interactions be- discovery of a titanium-based catalytic, enantioselective dichlorination of tween the cationic lipid and an anionic lipid found in the endosomal membrane allylic alcohols enabled the eight-step synthesis of (þ)-Danicalipin A as a sin- (lysobisphosphatidic acid (LBPA)). These interactions are thought to disrupt gle stereoisomer in sufficient quantities for in vitro analysis. Nanoscale second- the membrane by inducing a phase change from bilayer to non-bilayer phases, ary ion mass spectrometry (NanoSIMS) confirmed that Danicalipin A is such as the inverted hexagonal or bicontinuous cubic phases. The inefficiency localized within plasma membrane of O. danica cells. Preliminary biophysical of this process is one of the barriers to the potency of siRNA drugs. Computer characterization of Danicalipin A has revealed that it alters the phase behavior simulations can provide valuable insights for optimizing the LNP composition and lateral organization in monolayers and bilayers of other lipids present in the to improve drug delivery efficiency. For the predictions to be successful it membrane of O. danica. Danicalipin A incorporates into monolayers of phos- is critical that the modelled lipids behave properly. In this work, we charac- pho- and glycolipids at the air-water interface and increases the surface pres- terize lipids found in LNPs and endosomal lipids using nuclear magnetic sure at which the liquid-expanded to liquid-compact phase transition occurs, resonance (NMR) and small angle X-ray scattering (SAXS). NMR allows us as well as increasing monolayer compressibility. Similarly, in giant unilamellar to determine the lipid phase(s) present and the order (mobility) of the lipid vesicles, Danicalipin A lowers the transition temperature of saturated phospho- chains. SAXS provides complementary information about the phases via and glycerolipids and causes phase separation. Natural and unnatural analogs to long-range correlations within the sample. These data are used to validate com- Danicalipin A are being synthesized to examine the effects of the stereochem- puter simulations of LNP component lipids and endosomal lipids leading to a istry, chlorination pattern, and sulfation on these biophysical properties. These stronger understanding of the behaviour of more complex systems including results may reveal how the membrane of O. danica accommodates high con- complete LNPs and ultimately enhancing the potency of these drug delivery centrations of chlorosulfolipids, which are toxic to other organisms. systems. 86-Plat 88-Plat Using Hyscore Spectroscopy of Nitroxides to Profile Water Content of Spectral STED Imaging of Cell Membranes Lipid Bilayers with 2 A˚ Spatial Resolution Erdinc Sezgin1, Falk Schneider1, Victoria Zilles1, Iztok Urbancic1, Melanie Chestnut, Sergey Milikisiyants, Amir Koolivand, Esther Garcia1, Dominic Waithe1, Andrey Klymchenko2, Maxim A. Voynov, Tatyana I. Smirnova, Alex I. Smirnov. Christian Eggeling1. Chemistry, NCSU, Raleigh, NC, USA. 1University of Oxford, Oxford, United Kingdom, 2University of Strasbourg, Mapping water molecules across lipid bilayers at high spatial resolution is Strasbourg, France. important for both understanding membrane biophysics and membrane protein The lateral organization of molecules in the cellular plasma membrane plays an biological function. Current biophysical methods to determine water concentra- important role in cellular signaling. A critical parameter for membrane molec- tion in biological systems have several limitations, the main one being insuffi- ular organization is how the membrane lipids are packed. Polarity sensitive cient spatial resolution. Water penetration profiles of lipid bilayers have been dyes are powerful tools to characterize such lipid membrane order. These measured using electron spin echo envelope modulation (ESEEM) of nitroxide dyes change their emission spectrum depending on the polarity of the environ- spin labels based on magnetic interactions of nitroxides with the matrix water ment which can be used to quantify the molecular ordering and to visualize molecules. However, the matrix ESEEM effect is caused by through-space lateral heterogeneity in membrane order of cellular membranes. These probes dipolar interactions, restricting spatial sensitivity to 10 A˚ , a distance which have been used in combination with confocal or multi-photon microscopy; spans a significant portion of the lipid bilayer. Overhauser DNP is a less direct however, the diffraction-limited spatial resolution of these techniques does method to determine local water concentration, via measuring bulk water polar- not allow observation and full characterization of nanodomains/clusters in ization induced by microwave irradiation of a spin label, and depends on com- the plasma membrane. The investigation of potential lipid nanodomains, how- plex spin dynamics inside the lipid bilayer. Hyperfine sublevel correlation ever, requires the use of super resolution microscopy. Here, we apply the polar- (HYSCORE) spectroscopy is a sensitive technique to detect hydrogen bonds ity sensitive membrane dyes in super-resolution STED microscopy. formed with paramagnetic centers. Here we demonstrate the use of HYSCORE Measurements on cell-derived membrane vesicles, in the plasma membrane spectroscopy to directly and accurately measure the fraction of water molecules of live cells, and on single virus particles show the high potential of these hydrogen bonded to the nitroxide oxygen atom. To convert HYSCORE data dyes for probing nanoscale membrane heterogeneity [Sezgin et al, Biophysical into local water concentration we employed a normalization factor for the H- Journal, 2017]. bonded deuteron signal that was taken as intensity of the ESEEM signal measured under identical experimental conditions and spectrometer tuning pa- rameters. We demonstrate that the water molecules hydrogen-bonded to trans- Platform: Cell Mechanics and Motility I membrane WALP peptides, which were spin-labeled at various positions with MTSL, can be accurately determined even in the hydrophobic region of the 89-Plat lipid membrane. A correlation between the observed H-bonded signals and Determination of 3D Amoeboid Migration Force through Utilization of local water concentration has been established using model systems containing Actuated Surface Attached Posts Jonathan E. Eicher1, Maryna Kapustina2, Michael Falvo3, mixtures of diglyme and CH3OD, as well as diglyme and deuterated water, with Tempol as the spin probe. Kenneth Jacobson2. 1Chemistry and Biology, Humboldt State University, Arcata, CA, USA, 2Cell 87-Plat Biology and Physiology, UNC Chapel Hill, Chapel Hill, NC, USA, 3Physics Characterization of Phases and Interactions Among Lipids Involved in and Astronomy, UNC Chapel Hill, Chapel Hill, NC, USA. Drug Delivery: An NMR and Small-Angle X-Ray Scattering Study Cell migration is essential for multicellular organization and survival but the Miranda L. Schmidt1, Bashe Y.M. Bashe1, Iulia Bodnariuc1, forces that govern 3D migration are still poorly understood. Methods that Joanne E. Mercer2, Sherry S.W. Leung1,3, Mohsen Ramezanpour4, measure force in 2D environments such as traction force microscopy and Yoav Atsmon-Raz4, Nandhitha Subramanian4, Pieter R. Cullis5, atomic force microscopy are not, in general, suited to measure forces during D. Peter Tieleman4, Jenifer L. Thewalt1,2. 3D migration, which comprises a majority of cell movement in complex tis- 1Department of Molecular Biology and Biochemistry, Simon Fraser sues. To address this need, an easily visualized, reproducible, method of University, Burnaby, BC, Canada, 2Department of Physics, Simon Fraser analyzing forces during cell migration through a 3D collagen matrix is being University, Burnaby, BC, Canada, 3Materials Science and Engineering, developed using actuated surface-attached posts (ASAP). ASAPs are arrays

BPJ 8532_8543 Sunday, February 18, 2018 17a of polydimethylsiloxane (PDMS) posts that can be calibrated to give force mea- 92-Plat surements based on deflection. The posts are embedded within a collagen ma- Vinculin Forms a Directionally Asymmetric Catch Bond with F-Actin trix to simulate a physiologically relevant environment and cells are then Derek L. Huang, Nicolas A. Bax, Craig D. Buckley, William I. Weis, induced to migrate through the collagen matrix, deflecting the posts. This Alexander R. Dunn. method allows for measurement of forces based on the Young’s modulus of Stanford University, Stanford, CA, USA. the post in question and the points of contact. The cell lines used were U937 Vinculin is an actin-binding protein thought to reinforce cell-cell and cell- leukocytes and Ref52 fibroblasts which both gave strikingly different results. matrix adhesions. However, how mechanical load affects the vinculin-F-actin U937 cells migrated through the collagen matrix through an amoeboid mech- bond is unclear. Using a single-molecule optical trap assay, we found that vin- anism which, combined with their small size, exerted little force on the environ- culin forms a force-dependent catch bond with F-actin through its tail domain, ment and prevented said force from being measured with the current properties but with lifetimes that depend strongly on the direction of the applied force. of ASAP. By contrast, mesenchymal Ref52 cells were able to bend the posts Force toward the pointed (-) end of the actin filament resulted in a bond that significantly which involved forming direct attachments with the post itself, was maximally stable at 8 pN, with a mean lifetime (12 sec) 10 times as consistent with the role of fibroblasts in remodelling the extracellular matrix. long as the mean lifetime when force was applied toward the barbed (þ) The maximum force measured was nearly 90 nN, consistent with traction anal- end. A computational model of actin dynamics near adhesions suggests that ysis for fibroblasts migrating on 2D flexible substrates. the directionality of the vinculin-F-actin bond can potentially establish long- range order in the actin cytoskeleton through a polarity-sorting effect, one 90-Plat that may reinforce persistent cell migration. We suggest that the recruitment Structure and Constriction Mechanism of the Actomyosin Ring of vinculin to cell-cell adherens junctions (AJs) may help the cell establish Lam T. Nguyen, Matthew Swulius, Grant J. Jensen. an actin organization conducive to AJ stability. This view is supported by Biology and Bioengineering, Caltech, Pasadena, CA, USA. recent results from the optical trap assay, which indicate that both the load- Cytokinesis is orchestrated by a contractile actomyosin ring, but its structure and direction-dependence of the bond between the cadherin-catenin complex and mechanism remain elusive. We visualized the 3D structure of the ring in and F-actin are dramatically altered by the presence of vinculin. frozen-hydrated dividing yeast cells by electron cryotomography (ECT). Detailed arrangements of actin filaments within the ring and with respect to 93-Plat the membrane were seen for the first time, providing a crucial spatial constraint 3D Matrix Architecture Regulates Cell Migration through Degradability for the constriction mechanism of the ring. Using the ECT data and input from Stephanie I. Fraley1, Daniel Ortiz2. the literature we then explored sixteen mechanistic models by coarse-grained 1University of California, San Diego, La Jolla, CA, USA, 2Bioengineering, simulations at the 3D molecular details, revealing plausible mechanisms for University of California, San Diego, La Jolla, CA, USA. preventing membrane distortion and protein aggregation. We found that, in Cells rely on four core biophysical processes to interact with and migrate the model that best fits experimental data, both bipolar and membrane- through 3D extracellular matrix: adhesion, contractility, matrix remodeling, attached unipolar myosins exist in the ring, reconciling two different views and cytoskeletal dynamics. Yet, the role of local matrix structure in modulating in the field regarding the myosin configuration. In this model, ring tension is these biophysical processes and migration phenotype is incompletely under- generated primarily by interactions between bipolar myosins and actin, and stood. Here, we used novel matrix engineering techniques to vary matrix archi- transmitted to the membrane via unipolar myosins. This model recapitulates tecture independently of stiffness and density and then characterized the a broad distribution of distances from actin filaments to the membrane observed coordination of the four key biophysical processes and resulting migration out- in our tomograms and separation of two different myosin isoforms into the comes. Processes were quantified in HT-1080 cells using: i) fluorescence recov- outer and inner subdomains of the ring reported in a previous fluorescence mi- ery after photobleaching (FRAP) to study actin dynamics in cellular protrusions croscopy study. Further, it rationalizes how bundles of actomyosin were able to labeled with LifeAct, ii) traction force microscopy (TFM) of matrix-embedded separate from the membrane in fluorescence microscopy experiments of the beads to study adhesion and contractility, and iii) fluorescent dye quenched same previous study. (DQ) collagen to study MMP activity. Cells embedded in matrices with small pores and short fibers produced smaller and shorter lived protrusions compared 91-Plat to cells embedded in matrices with longer, more bundled fibers. Actin FRAP The Arp2/3 Complex is Necessary for Migration of Glioblastoma Cells studies revealed that this difference in protrusion lifetime was not caused by on Compliant Substrates due to a Lamellipodia-Provided Mechanical differences in actin polymerization rates. TFM and protrusion-ECM displace- Advantage ment correlation showed that in short fiber architectures, cells fail to properly Devin B. Mair1, Jin Zhu2, Seth H. Weinberg3, Rong Li4. bind and pull on the surrounding matrix. Interestingly, DQ collagen degrada- 1Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA, 2 3 tion assays showed that cells in the short fiber architecture locally degrade Cell Biology, Johns Hopkins University, Baltimore, MD, USA, Biomedical collagen more than cells in longer fiber architectures. Preliminary results sug- Engineering, Virginia Commonwealth University, Richmond, VA, USA, 4 gest that this increased collagen degradation is accompanied by increased Cell Biology, Chemical and Biomolecular Engineering, Johns Hopkins collagen internalization. Gene expression analysis revealed that cells in this University, Baltimore, MD, USA. condition upregulate uPARAP, a mannose receptor responsible for internaliza- Glioblastomas (GBMs) are the most common and most lethal central nervous tion of collagen fragments. Subsequently, these cells switch into a collective system tumors, with a median survival time of approximately 15 months. This migration phenotype and form multicellular structures, whereas cells in long dismal prognosis has been attributed to the wide spread dissemination of fiber matrices migrate as single cells. Our results suggest that collagen architec- GBM tumor cells throughout the brain, which makes repeated recurrence tures with short fibers promote the upregulation of a collagen degradation and likely. The brain is one of the most compliant tissues in the body, with an internalization pathway that triggers changes in migration phenotype. elastic modulus of around 1 kPa. Migration and invasion on such soft sub- strates in 2D requires the wide, ruffled leading edge of the lamellipodia, cyto- 94-Plat skeletal structures formed through branched actin filament assembly, which is Tumor Invasion through Hyaluronic Acid Matrices is Mediated by CD44- mediated via nucleation by the Arp2/3 complex. While this behavior has been Dependent Microtentacles well-documented, the reasons for the phenomena have yet to be shown. Here, Kayla J. Wolf1,2, Sam Kenny3,KeXu3,4, Sanjay Kumar1,5. we show that Arp2/3-inhibited GBM cells transmit forces that result in sub- 1Bioengineering, University of California, Berkeley, Berkeley, CA, USA, strate deformation instead of locomotion. In contrast, Arp2/3-positive cells 2UC Berkeley-UCSF Graduate Program in Bioengineering, Berkeley, CA, form lamellipodia, which transmit forces over a wider area than the parallel USA, 3Chemistry, University of California, Berkeley, Berkeley, CA, USA, actin architecture that forms in the absence of Arp2/3, and thus successfully 4Division of Molecular Biophysics and Integrated Bioimaging, Lawrence transmit forces that lead to locomotion. On-going work will investigate this Berkeley National Laboratory, Berkeley, CA, USA, 5Chemical and mechanism of locomotion in a computational elastic-stochastic model of la- Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, mellipodia versus filopodia force transmission. The model will incorporate USA. focal adhesions formed along a surface with a defined curvature and contour Glioblastoma (GBM) is a highly malignant primary brain cancer that is chal- length that corresponds to the appropriate cytoskeletal structure. We expect lenging to treat due to resistant and diffusely invasive tumor cells. The trans- the model to illustrate the mechanical advantage of lamellipodia on compliant membrane receptor CD44 directly facilitates tumor cell invasion by engaging substrates via development of sustainable forces, while filopodia exhibit load- hyaluronic acid (HA) in brain matrix. Despite the acknowledged importance fail dynamics. In summary, our work provides insight into mechanisms of the of CD44 as a mechanistic driver and potential therapeutic target in GBM and migratory behavior of GBM tumor cells in the brain parenchyma and demon- other tumors, CD44-based motility is poorly understood. As a whole, little is strates a possible therapeutic target for GBM tumors. known about how cells navigate nonfibrillar 3D matrices such as the

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HA-rich brain parenchyma. Here, we show that continuous and patient-derived providing a paradigm for hierarchical mechano-regulation of receptor-ligand GBM tumor cells interacting with both 2D and 3D HA substrates exploit CD44- binding. based microtentacles (McTNs) to support cell migration. McTNs are highly dy- namic protrusions that can reach lengths of over 40 mm and stain positive for 97-Plat tubulin and actin. Super-resolution microscopy reveals that McTNs are tubular Compressive Stress Stalls Growth and Decrease Cytoplasmic Diffusion Morgan Delarue1, Liam Holt2. structures approximately 150-200 nm in diameter and are covered with 1 2 membrane-localized CD44 that contain actin filaments and microtubules. LAAS, Toulouse, France, Institute for Systems Genetics, NYULMC, New McTNs collapse in response to microtubule depolymerizing drugs, but grow York, NY, USA. when actin is depolymerized. Microtubule depolymerization leads to reduced Any cell population growing in a limited space can generate mechanical migration speed and reduced adhesion on HA substrates, while depolymeriza- compressive stresses. Tumors growing within tissues and microbes that are tion of actin results in an increase in adhesion to HA substrates and decreased naturally confined by their environment both build up growth-induced pressure. migration speed. The growth of McTNs is mediated by the expression of CD44, While the effects of tensile mechanical stresses have been widely studied, much with shRNA knockdown of CD44 shortening and reducing the number of less is known about the effects of compressive mechanical stresses on cell phys- McTNs. McTNs in GBM cells are reminiscent of McTNs recently observed iology. We developed a microfluidic device that enables precise temporal con- in metastatic breast cancer as well as microtubes and protrusions described trol of mechanical and chemical conditions. We found that rate of cell growth is for GBMs in vivo. Together, these results suggest that CD44 promotes affected by compressive stress: Cell growth decreased roughly exponentially McTN growth and that cytoskeletal interplay involved in McTN formation en- with pressure. In order to investigate the molecular origin for such a growth ables motility on HA. Additionally, these findings provide new mechanistic decrease, we developed genetically encoded multimeric nanoparticles insight into migration on nonfibrillar matrices. (GEMs) to assess the effects of a mechanical compressive stress on cell micro- rheology. We observe that the motion of GEMs decreases exponentially as cells 95-Plat experience increasing compressive stress. We developed a model of cyto- FLNa and FilGAP Interactions Regulate the Contractility of Cells in Shear plasmic crowding regulation, which predicts how the motion of protein com- Stress plexes should decrease under pressure. We found that the expression of a Rosa Kaviani, Chris Sitaras, Haruka Yoshie, Allen Ehrlicher. reporter gene decreases with the same specific pressure as the growth rate Bioengineering, McGill University, Montreal, QC, Canada. decay. We hypothesize that macromolecular diffusion becomes rate limiting Force-sensing and mechanical adaptation can be seen in nearly every aspect of for growth under compressive stress, thus globally decreasing the rates of our physiology, including the adaptation of vascular cells to shear flow, differ- biochemical reactions. We speculate that compressive mechanical stresses entiation of stem cells according to the stiffness of their environment, or in dis- could regulate cell physiology through increased molecular crowding. eases such as cancer changes in cell traction forces and stiffness with respect to Different pathways will be differentially affected depending on the typical their invasiveness. However, despite many landmark findings in this area, the size of molecules involved. Therefore, we provide a possible mechanism for molecular mechanism responsible for mechanosensing remain elusive. the effects of compressive stress on multiple aspects of cell biology, from meta- Recent studies on reconstructed networks suggest that conformational changes bolism to signaling and differentiation. in Filamin A (FLNa) structure in response to external mechanical signals and subsequently changes in the binding affinity of this protein for signalling pro- 98-Plat teins such as FilGAP (a RhoGTPase-activating protein) can be the underlying Structural Features and Molecular Bases Underlying the Ion Permeation molecular mechanism for force sensing in shear. and Mechanogating of the Mechanosensitive Piezo Channels In this study, we have evaluated the effect of FLNa-FilGAP interaction in cell Bailong Xiao. mechanotransduction in shear. We have combined microfluidics with traction School of Pharmaceutical Sciences; Tsinghua-Peking Center for Life force microscopy to be able to detect changes in contractile forces of cells in Sciences; IDG/McGovern Institute, Tsinghua University, Beijing, China. response to shear stress. FLNa deficient human melanoma cells (M2) have The evolutionarily conserved Piezo family of proteins, including Piezo1 and been transfected with wt FLNa and FLNa with M2474E mutation which creates Piezo2, forms the long-sought-after bona fide mechanosensitive cation chan- a FLNa that is unable to bind to FilGAP. Changes in the contractile forces of nels in mammals, and plays critical roles in various mechanotransduction pro- cells from all three groups (FLNa-Null, wt FLNa and M2474E FLNa) were cesses such as touch, pain, proprioception, and blood pressure regulation. measured during time. Our results show that in absence of FLNa, contractile Mammalian Piezo proteins contain over 2500 amino acids with 30-40 predicted forces are irresponsive to shear stress magnitude while in FLNa expressing transmembrane segments (TM), and do not bear sequence homology with any cells, there is an increase in contractile forces in a time and magnitude depen- known class of ion channels. Thus, it is imperative, but challenging, to under- dent manner. If FLNa-FilGAP binding is inhibited, a phenotype unresponsive stand how they serve as effective mechanotransducers for converting mechan- to shear stress is created. ical force into electrochemical signals. Taking a multidisciplinary approach The results of this study helps describe the role of FLNa expression and FLNa- combining protein engineering and purification, cryo-EM, mutagenesis and FilGAP binding interaction in mechanosensory response of cells, providing electrophysiology, we have first determined the three-bladed, propeller-shaped new insight on how cells perceive and sense the mechanical signals. structure of Piezo1, and then functionally identified the bona-fide ion-con- ducting pore, key pore-property-determining residues, and mechanotransduc- tion components. Our studies suggest that Piezo1 can be deduced into two Platform: Mechanosensation functional modules, including the central pore module formed by the last- two-TM-containing C-terminal region of 350 residues, and the mechano- 96-Plat transduction module formed by the rest of the N-terminal region of 2200 res- Stretching and Activation of Single Protein Molecules by Flow Reveals the idues. Furthermore, we propose that Piezo1 employs the featured 80 A˚ -long Mechanism of von Willebrand Factor Adhesion intracellular beam structures, which bridge the peripheral blades to the pore, to 1,2 3 1,2 4 Yan Jiang , Hongxia Fu , Darren Yang , Friedrich Scheiflinger , form a lever-like apparatus for long-distance mechanogating. Taken together, 1,2 1,5 Timothy A. Springer , Wesley P. Wong . our studies provide important insights into the structure, ion permeation and 1Harvard Medical School, Boston, MA, USA, 2Boston Children’s Hospital, 3 4 mechanogating of this novel and distinct class of mechanosensitive ion Boston, MA, USA, University of Washington, Seattle, WA, USA, Shire, channels. Vienna, Austria, 5Boston Children’s Hospitcal, Boston, MA, USA. Von Willebrand factor (VWF), an ultralarge concatemeric blood protein, must 99-Plat bind to platelet GPIba during bleeding to mediate hemostasis, but not in the The Dynamics of Somatosensory Mechanotransduction in C. elegans normal circulation to avoid thrombosis. VWF is proposed to be mechanically Touch Receptor Neurons activated by flow, but the mechanism remains unclear. Using microfluidics Samata Katta1, Valeria Va´squez1,2, Miriam B. Goodman1. with single-molecule imaging, we simultaneously monitored reversible VWF 1Stanford University, Stanford, CA, USA, 2University of Tennessee Health extension and binding to GPIba under flow. We show that VWF is activated Sciences Center, Memphis, TN, USA. through a two-step conformational transition: first, elongation from compact Mechanosensitive DEG/ENaC/ASIC channels in C. elegans touch receptor to linear form, and subsequently, a tension-dependent local transition to a state neurons (TRNs) respond at the onset and offset of a stimulus. The rapidly with high affinity for GPIba. The elongation is regulated by divalent salts and adapting and nearly symmetric mechanoreceptor currents (MRCs) in TRNs independent of sodium concentration. High-affinity sites develop only in up- resemble those believed to underpin receptor potentials in mammalian Pacinian stream regions of VWF where tension exceeds 21 pN and depend upon elec- corpuscles. Though TRN anatomy differs from that of Pacinian corpuscles, the trostatic interactions. This mechanism enables VWF to be locally activated by TRNs, like other touch-sensitive somatosensory neurons, are embedded in the hydrodynamic force in hemorrhage and rapidly deactivated downstream, animal’s skin and rely on that skin to filter and transfer mechanical stress. The

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C. elegans TRNs provide a system in which we not only know the identity of conducted metabolism inhibition studies to confirm the shift from OXPHOS the mechano-electrical transduction channel and many of its interacting part- to glycolysis and vise-versa on the phasor plot. These results show that the pha- ners, but can also explore how MRCs are shaped by the mechanical microen- sor/FLIM approach is a powerful method in monitoring metabolism and me- vironment and surrounding tissues. We use in vivo whole-cell patch-clamp in chanics that may improve our understanding in the potential roles it has in combination with a piezoelectric stack-based stimulator and photodiode- cell invasion. This work is supported by the National Institutes of Health grant based motion detector (Peng et al., Neuron, 2013) to probe this system at P41-GM103540. high (2.5kHz) bandwidth. Consistent with prior work, we find that MRC ampli- tude increases with displacement and decreases with distance from the cell 102-Plat body. The amplitude and kinetics of MRCs increase with stimulus rate, satu- Cellular Mechanotransduction via Ion Channels at the Cell-Substrate rating above a speed of 6mm/s. With sinusoidal stimuli, we confirm that Interface MRC activation is frequency dependent. Lastly, we find that MRCs adapt Navid Bavi, Jessica Richardson, Kate Poole. both to a constant pre-indentation and during continuous sinusoidal stimuli. Department of Physiology, School of Medical Sciences, The University of We are developing a model that integrates the biomechanics of the worm New South Wales, Sydney, Australia. body, the distribution of DEG/ENaC/ASIC channels, and the channels’ depen- Mechanosensitive (MS) ion channels are the fastest mechanotransducers in dence on both stimulus indentation and velocity to understand these biophysi- living cells as they respond to mechanical stimuli on a less than millisecond cal phenomena. We hope this model will guide genetic dissection of the time scale. They may transduce the mechanical force directly into electrochem- molecular basis of frequency dependence and both time- and indentation- ical signals. This pathway underlies several physiological processes such as dependent adaptation. gentle touch and hearing sensation. A number of tools have been developed to Work was supported by F31NS093825 to SK and R01NS047715 to MBG. We measure ionic currents mediated by MS ion channels across a wide variety of thank A. Ricci and A. Peng for assistance in building the stimulator/photodiode cell types. These channels have traditionally been studied by membrane stretch motion detector. (using high-speed pressure clamp), shear stress (flow) or cellular indentation (us- ing a glass probe). More recently we have established elastomeric pillar arrays as 100-Plat force transducers to apply fine mechanical stimuli directly at the interface be- The Integration of Mechanical and Chemical Signalling in the Developing tween cells and their substrate. One advantage of this technique is that the Brain cell-matrix interface is the proposed site of mechanotransduction in many MS Kristian Franze. cells, such as sensory neurons and chondrocytes. MS channels such as PIEZO1 University of Cambridge, Cambridge, United Kingdom. and PIEZO2 can be activated by membrane stretch, cell indentation and pillar- During development and pathological processes, cells in the central nervous deflection. In contrast, TRPV4 is poorly responsive to membrane stretch, non- system (CNS) are highly motile. Despite the fact that cell motion is driven responsive to indentation but robustly activated by pillar-deflection. What has by forces, our current understanding of the mechanical interactions between not been clear is i) how pillar deflection leads to channel activation and ii) if CNS cells and their environment is very limited. We here show how nanometer the mechanism of deflection-mediated channel activation is distinct from deformations of CNS tissue caused by piconewton forces exerted by cells stretch-activation. We are currently utilising empirical analysis of an array of contribute to regulating CNS development and pathologies. In vitro, growth MS channels combined with light microscopy and finite element modelling. and migration velocities, directionality, cellular forces as well as neuronal This will enable us to characterize whether MS channels are activated in the fasciculation and maturation all significantly depended on substrate stiffness. cell-substrate interface by changes in membrane tension, and how these mem- Moreover, when grown on substrates incorporating linear stiffness gradients, brane tensions relate to those applied using high-speed pressure clamp. glial cells migrated towards stiffer, while axon bundles turned towards softer 103-Plat substrates. In vivo atomic force microscopy revealed stiffness gradients in Substrate Rigidity Modulates the Composition in Cell-Matrix Adhesions developing brain tissue, which axons followed as well towards soft. Interfering Thomas Schmidt, Hayri E. Balcioglu, Rolf Harkes, Erik H.J. Danen. with brain stiffness and mechanosensitive ion channels in vivo both led to Leiden University, Leiden, Netherlands. similar aberrant neuronal growth patterns with reduced fasciculation and path- In cell matrix adhesions, integrin receptors and associated proteins provide a finding errors. Importantly, CNS tissue significantly softened after traumatic in- dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. juries. Ultimately, mechanical signals not only directly impacted neuronal This allows bidirectional transmission of forces between the ECM and the cyto- growth but also indirectly by regulating neuronal responses to chemical guid- skeleton, which tunes intracellular signaling cascades that control survival, pro- ance cues, strongly suggesting that neuronal growth is not only controlled by liferation, differentiation, and motility. The quantitative relationships between chemical signals – as it is currently widely assumed – but also by the tissue’s recruitment of distinct cell matrix adhesion proteins and local cellular traction local physical properties. forces are not known. Here, we applied quantitative superresolution micro- scopy to cell matrix adhesions formed on fibronectin-stamped elastomeric pil- 101-Plat lars and developed an approach to relate the number of talin, vinculin, paxillin, Metabolism Modulation of Cancer Cells on Varying Substrate Stiffnesses and focal adhesion kinase (FAK) molecules to the local cellular traction force. Emma J. Mah1, Albert F. Yee1, Michelle A. Digman2. 1 We find that FAK recruitment does not show an association with traction-force Chemical Engineering and Materials Science, University of California, 2 application whereas a 60 pN force increase is associated with the recruitment Irvine, Irvine, CA, USA, Biomedical Engineering, University of California, of one talin, two vinculin, and two paxillin molecules on a substrate of effective Irvine, Irvine, CA, USA. stiffness of 47 kPa. On a substrate with a four-fold lower effective stiffness the Cancer cells sense their microenviroment and respond to biophysical cues that stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same 60 pN activate signaling networks through ion channels and integrin proteins. This al- traction force. The marked relative change in force-related vinculin recruitment lows cells to adapt by adjusting their cellular shape and tension that remodels indicates a stiffness-dependent switch in vinculin function in cell matrix adhe- the extracellular matrix (ECM). New evidence shows that these ECM signaling sions. Taken together, our results reveal a substrate-stiffness-dependent modu- cascades may increase glucose uptake leading to altered metabolic states. The lation of the relation between traction-force and molecular composition of cell- hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in matrix adhesions. cancer cells together with atypical ECM structure may be responsible for tumor cell aggressiveness and drug resistance. While it is known that tumor cells stiffen the ECM as the tumor progression occurs, a direct relationship between Platform: Sensing In Vivo and In Vitro ECM stiffness and altered metabolism has not been explicitly measured. Here we apply the phasor approach to fluorescence lifetime imaging microscopy 104-Plat (FLIM) as a method to measure metabolic as a function of ECM mechanics. Nanofluidic Sensor for Antigen-Antibody Binding Detection We imaged two breast cancer cell lines of high and low aggressiveness Denise Pezzuoli1, Alessia Cazzulo1, Elena Angeli1, Francesca Ferrera2, (MDA-MB231 and MCF-7, respectively) and compared them to non- Giuseppe Firpo1, Patrizia Guida1, Roberto Lo Savio1, Diego Repetto1, cancerous cells. Cells were plated on varying collagen density as measured Luca Repetto1, Ugo Valbusa1. by image correlation microscopy. Our results show that MDA-MB231 exhibit 1Physics, University of Genova, Genova, Italy, 2Centre of Excellence for a decreased fraction of bound NADH (indicative of glycolysis) with increasing Biomedical Research, University of Genova, Genova, Italy. substrate stiffness. Inhibition of cell contractility with Y27632 or blebbistatin Nanofluidics is a well established tool for conducting fundamental studies of showed shifts towards a higher free NADH fractional contribution (indicative molecular-scale phenomena. Both the structures nanometric dimension and of oxidative phosphorylation, OXPHOS). All other cell lines showed little the size of biomolecules such as DNA or proteins, added to the excellent con- change in fraction bound NADH on the varying collagen stiffnesses. We also trol on the geometry, give unique features to nanofluidic devices. In particular,

BPJ 8532_8543 20a Sunday, February 18, 2018 nanopores and nanochannels are used for many applications, such as separa- 107-Plat tion, detection and manipulation of biomolecules [Abgrall P. (2008); Fanzio Live Cell Surface Conjugation Methods for Imaging, Sensing and Therapy P. et al.(2012)]. Nanostructures are directly created on the surface of a Joydeb Majumder, Gaurav Chopra. micro-machined silicon mould by using a FIB, and replicated using Poly(Di- Chemistry, Purdue University, West Lafayette, IN, USA. MethylSiloxane) PDMS with REplica Moulding (REM) techniques, reducing Surface modification of live cells has many biological applications including the fabrication costs and allowing high-throughput production of nanofluidic imaging, control of cell surface interactions, tracking and sensing biological en- devices [Angeli E. et al. (2011)]. vironments in vitro and in vivo. Over the years, several methods such as chemo- Combining accumulation/pre-concentration of biomolecules, governed by selective conjugation, PEGylation etc. have been extensively explored to electro-hydrokinetic phenomena, that occurs applying high voltage across the modify cell surfaces with various cargos/therapeutic agents via non-covalent device, and an appropriate functionalization of nanochannel polymeric surface interactions (between positively-charged polyelectrolytes and negatively- with antibodies linked by using APTES (3-Aminopropyl)triethoxysilane and charged cell surfaces) and covalent interactions (bonding between cargos GA (Glutaraldheyde)[Dohyun K. et al.(2013)], we are able to decrease biomol- and the functional groups on cell surfaces). However, problems remain for ecules’ sensing limit for the detection of one or several targeted antigens for both non-covalent interaction based conjugation of cell surface modification, clinical diagnostics. as well as, covalent conjugation of surfaces based on unnatural chemical reac- Here, we identify through fluorescence optical microscopy and electric mea- tions. Conjugates of cell surfaces via non covalent interactions show limited surements, the uptake of a specific antigen, diluted in solution (from 125 pg/ time stability. On the other hand, covalent conjugation via unnatural chemical ml to 10 pg/ml), to the nanochannel surface functionalized with antibodies. reactions provides longer stability, but these reactions are not cytocompatible So, in this condition, we successfully detected antigen-antibody binding on with mammalian cells due to toxicity from metal catalysed chemical modifica- nanostructure surface, a promising step for realizing high-sensitivity nanoflui- tion of membrane proteins. Recently, cell membranes were conjugated with dic immuno-assay sensor. well-engineered therapeutic loaded nanoparticles but they cannot be used in vivo due to toxicity resulting from entrapment in the reticuloendothelial system 105-Plat of the liver and spleen. We have these addressed two challenges, namely, cy- Structure and Dynamics of the MUC1-Binding Aptamer Attached to a tocompatible with mammalian cell membranes and stable conjugation for Biosensor Surface long time in live cells. We have designed a cargo molecule with a cationic Iman Jeddi, Leonor Saiz. side chain which will form non-covalent bond with the negatively charged Biomedical Engineering, University of California, Davis, Davis, CA, USA. cell surface and a phosphoric acid containing ligand which will facilitate Aptamers are short oligonucleotides that are selected for affinity binding to a phosphor-ester covalent bonding with the cell membrane phosphate function- wide range of targets and provide a number of advantages over antibodies ality as separate bio-orthogonal reactions. Infact, conjugation of our well de- including robustness, low cost, and reusability [1,2]. The robustness and signed cargo with Jurkat T-cells show cytocompatibility and stability of the simplicity of aptamers has allowed for multiple uses of aptamer-based biosen- surface conjugation for a long period of time in varying pH (5.5 to 8.5) condi- sors. However, in order for commercialization of these devices to become tions. We believe our dual-conjugation approach will provide a stable technol- feasible, significant improvements in optimization for consistency and repro- ogy for drug delivery, imaging and cellular sensing with live cells. ducibility must be done. Overcoming these challenges has been hampered by 108-Plat a lack of complete understanding of the molecular level biophysics involved Non-Invasive Monitoring of Mitochondrial Oxygen Consumption and [3,4]. In this regard, computational studies can complement experimental Intracellular Distribution of [O ] studies in improving our understanding about the structure, molecular-level in- 2 Rozhin Penjweini1, Alessio Andreoni1, Dan L. Sackett2, Jay R. Knutson1. teractions, dynamics, and solvent effects of biomolecular complexes [5,6]. Here, 1National Heart, Lung and Blood Institute, National Institute of Health, we present the results of molecular dynamics simulations on the structure and Bethesda, MD, USA, 2Eunice Kennedy Shriver National Institute of Child dynamics of the MUC1-binding DNA aptamer attached to a biosensor surface. Health and Human Development, National Institutes of Health, Bethesda, References: MD, USA. [1] Ellington AD & Szostak JW (1990) In vitro selection of RNA moleculesthat Mitochondria are the primary consumers of oxygen (O ) in cells, to allow oxida- bind specific ligands. Nature 346: 818-822. 2 tive phosphorylation, which is the primary metabolic pathway for adenosine [2] Liu Y et al. (2010) Aptamer-based electrochemical biosensor for interferon- triphosphate (ATP) and adenosine diphosphate (ADP) production. Altered mito- gamma detection. Analytical chemistry 82: 8131-8136. chondrial oxygen concentration ([O ]) is suggested to be a key issue in traumatic [3] Zhou W et al. (2014) Aptamer-based biosensors for biomedical diagnos- 2 brain injury (TBI) and many human diseases such as age-related pathologies, tics.The Analyst 139: 2627-2640. cancer, and type 2 diabetes mellitus. Whereas some authors report decreased [4] I. Jeddi and L. Saiz, Three-dimensional modeling of single stranded DNA mitochondrial [O ] in some of these diseases, others found enhanced [O ] under hairpins for aptamer-based biosensors, Scientific Reports, 7, 1178 (2017). 2 2 similar circumstances. Most knowledge on mitochondrial dysfunction is derived [5] Saiz L (2012) The physics of protein-DNA interaction networks in the con- from isolated mitochondria from cells or tissue biopsies, providing detailed trolof gene expression. Journal of Physics: Condensed Matter 24: 193102. insight into respiratory chain function. The possible loss of essential metabolites [6] Sinha SK & Saiz L (2014) Determinants of protein-ligand complex forma- during mitochondrial isolation and disruption of the normal interactions of the tionin the thyroid hormone receptor a: a Molecular Dynamics simulationstudy. organelle with the cytoskeleton may cause these data to misrepresent intact cells. Computational and Theoretical Chemistry 1038, 57-66. Such ex vivo measurements pose experimental limitations that may partly explain the controversial reports on the relationship between mitochondrial 106-Plat dysfunction and [O ]. In this study, we introduce a non-invasive and accurate Detection of Bacillus thuringiensis HD-73 Spores using Protein Nanopores 2 method for the monitoring of the mitochondrial [O2] consumption and gradients and Complementary Aptamers with DNA Hairpin Probes (if any) within intracellular environments based on fluorescence lifetime imag- Hyunil Ryu. ing (two-photon FLIM) of a Fo¨rster Resonance Transfer (FRET)-based probe. Inha University, Incheon, Republic of Korea. The [O2] probe was developed by coupling Myoglobin, a high affinity O2 bind- The aptamers, the short oligonucleotides which selectively bind to specific ana- ing heme protein, with the red fluorescent protein mCherry (Mb-mCherry). lytes, are able to make distinct electrical signal differences between bound and A549 non-small cell lung cancer cells and their mitochondrial DNA-depleted unbound analytes when it passes through alpha-hemolysin nanopore. Therefore (mtDNA) r0 counterparts (that are incapable of aerobic respiration due to the they have been used as an intermediary for electrical stochastic sensor. However, lack of key respiratory chain components) provided different consumption im- most aptamers with high affinity are hard to be designed to have discriminable ages in response to external [O ] variation. signal, simultaneously. Due to the fact the aptamers are selected among randomly 2 synthesized oligonucleotide library. Consequently, we designed the DNA hairpin 109-Plat that has complemental region to the aptamer and shows discriminable signal. Novel Conformation Selective Molecular Sensors for Amyloid Aggregates Then, the spore and aptamer are mixed and only spore-aptamer complex was Eva Y. Chi1, Florencia A. Monge2, Patrick L. Donabedian3, collected by centrifugation. Repeatedly, the designed hairpin was mixed with Adeline M. Fanni2, Nicole M. Maphis4, Kiran Bhaskar4, David G. Whitten1. spore-aptamer complex and then remaining hairpin was collected. By measuring 1Chemical and Biological Engineering, University of New Mexico, unbound hairpin concentration through alpha hemolysin based electrical mea- Albuquerque, NM, USA, 2Biomedical Engineering Graduate Program, surement system, concentration of Bacillus thuringeinsis HD-73 spores was University of New Mexico, Albuquerque, NM, USA, 3Nanoscience and calculated, resulting in the detection limit as low as 1.2 101 CFU/mL. This pro- Microsystems Engineering Graduate Program, University of New Mexico, cess only require less than 1 hour, and it is accurate and adaptable since the hairpin Albuquerque, NM, USA, 4Department of Molecular Genetics/Microbiology, DNA structure can be modified depending on target aptamers. University of New Mexico, Albuquerque, NM, USA.

BPJ 8532_8543 Sunday, February 18, 2018 21a

Misfolding and aggregation of proteins is a central pathogenic event in a three useful tools: FRET-based biosensor, which could be used to visualize myriad of neurodegenerative disorders including Alzheimer’s and Parkinson’s the activation of SFKs at micron-scale; ‘Photo-uncaging’ of SspB could be diseases. Importantly, as protein aggregation is believed to initiate a cascade of used to photo-activate the kinase; The docking of fluorescent SspB to the acti- events culminating in neurodegeneration and cognitive decline decades before vated conformation of SsrA-tagged kinase could be used to reveal activated the onset of clinical symptoms, protein aggregates are ideal biomarkers for SFKs during high-speed single molecule tracking (SMT) at nano-scale. SspB early disease detection and therapeutic intervention. Currently, there lacks and SsrA affinities were appropriately optimized for each technique. For diagnostically useful sensors for detecting protein aggregates involved in dis- SMT of kinase activation, we adopted TIRF or oblique illumination. SMT ease etiology. To meet this critical need, we are developing novel oligo(p-phe- tracks of the Binder revealed the distribution, diffusion and activation state nylene ethynylene) electrolytes (OPEs) for the direct sensing of amyloid of SFKs relative to focal adhesion (FA) dynamics during cell migration and ef- aggregates and have identified an effective selective sensor, OPE1, for the am- fects of altering adhesion proteins. yloid conformation of model proteins (lysozyme and insulin) and disease- associate proteins (amyloid-b, tau, and a-synuclein). On the basis of protein binding and photophysical changes observed, keys to the selective detection Platform: Membrane Proteins: Structure and of the amyloid protein conformation include moderate size, negative charge, Folding and substituents that provide high microenvironment sensitivity to the fluores- cence yield. We have further validated the use of OPEs for ex vivo detection of 112-Plat amyloid pathology in brain tissue sections. Confocal fluorescence microscopy Integrin-Based Mechanosensing is Mediated by Conformational Activa- combined with co-localization analysis using tau specific antibody AT180 and tion existing ‘‘gold-standard’’ amyloid dye Thioflavin-T (ThT) showed turn-on Tamara C. Bidone1, Tristan Driscoll2, Martin A. Schwartz2, OPE1 fluorescence when bound to tau amyloid deposits in transgenic mice Gregory A. Voth1. and human frontotemporal dementia brain sections with little to no non- 1University of Chicago, Chicago, IL, USA, 2Yale University, New Haven, specific binding. Importantly, both in vitro and in vivo studies demonstrated CT, USA. that OPEs are superior sensors compared to the conventional dye ThT, exhibit- Conversion of integrins from low affinity to high affinity states, termed acti- ing lower detection limits and higher fluorescence signal. Combined with vation or inside-out signaling, is important in immunity, hemostasis, angio- OPE’s versatile and highly tailorable structural and chemical properties, these genesis and embryonic development. It has been well documented that facile probes have the potential to simultaneously and dynamically track the integrin activation is regulated by large-scale conformational transitions protein misfolding and aggregation process for both in vitro and in vivo sys- from closed, low affinity states to open, high affinity states. However, how tems, facilitating research into the cause, diagnosis, and treatment of major this structural transition is related to rigidity sensing is poorly understood. neurodegenerative disorders. In this study, we demonstrate that integrin conformational activation deter- mines mechanosensing. We first build molecular and coarse-grained compu- 110-Plat D D tational models of integrin avb3 to demonstrate that, contrary to established Organelle-Targeting of Apollo-NADP Makes Tracking NADPH/NADP hypotheses, integrin activation can be weakly cooperative; rather it can be Redox Possible across Multiple Organelles regulated by local atomistic motional correlations. In the absence of tension, 1 1,2 William D. Cameron , Jonathan Rocheleau . integrin intermediates emerge in the simulations, without persistence of short- 1Institute of Biomaterials and Biomedical Engineering, University of 2 range motional correlations across the entire receptor. In these intermediates, Toronto, Toronto, ON, Canada, Toronto General Research Institute, headpiece extension is not coupled to separation of the transmembrane legs, University Health Network, Toronto, ON, Canada. consistent with recent cryoEM reconstructions of the closely related integrin, We recently developed a spectral family of genetically encoded homoFRET þ aIIbb3. Second, we use coarse-grained models of avb3 integrin to show that sensors to measure NADPH/NADP redox state based on changes in anisot- different mutants respond differently to tension. Mutants that present in the ropy/polarization due to oligomerization of glucose-6-phosphate dehydroge- molecular dynamics an enhanced fraction of local motional correlations, rela- nase (Apollo-NADPþ). In cells such as insulin-secreting beta-cells, the þ tive to the total number of dynamic correlations across the receptor, extend NADPH/NADP redox state supports the scavenging of the reactive oxygen more easily under tension. Experimental measurements of ligand binding species H2O2 by the glutathione/thioredoxin antioxidant pathway. A loss of show that integrin b3 mutations that destabilize the closed conformation beta-cell mass due to oxidative stress leads to type 2 diabetes. A major source induce activation. Traction force measurements in cells on substrates with of H2O2 production is the mitochondrial electron transport chain (ETC), with different rigidities reveal that activating mutations alter stiffness sensing, al- overproduction a characteristic of many metabolic disorders including type 2 lowing cells to spread and exert high tension on soft substrates that correlate diabetes. Although H2O2 can cross cell membranes through facilitated diffu- well with results from computational models. Together, our experimental and sion, the NADPH/NADPþ redox is compartmentalized within organelles. We þ computational results support the notion that stabilization of the activated, therefore explored targeting Apollo-NADP to various organelles including: high affinity state by tension is crucial for cellular mechanosensing and that the mitochondria, nucleus, plasma membrane, peroxisomes, Golgi apparatus, this stabilization is mediated by local, correlated molecular fluctuations in and endoplasmic reticulum. Unlike the cytoplasm, however, the pH of organ- the receptor. elles such as the mitochondria are dynamic and therefore require pH- independent sensors. Here we found that pH significantly affects the anisotropy 113-Plat of fluorescent proteins with high pKa (ex. Venus), while fluorescent proteins Molecular Simulations Provide Insite on Lysine Snorkeling Modulation of with low pKa values (ex. Cerulean3, Turquoise2) maintained stable anisotropy the Integrin Transmembrane Domain values across a wider range of pH values (4.0-8.0). We also found that dimeric Melanie Muller, Emad Tajkhorshid. þ Turquoise2-tagged Apollo-NADP was stable from pH = 5.0-8.0, making it University of Illinois, Urbana, IL, USA. suitable for use in most cellular compartments. We are now investigating the Integrins are signaling proteins involved in many critical cellular functions, þ ability to simultaneously measure the NADPH/NADP redox state in various including cell adhesion and motility. Their activation process is triggered by organelles of beta-cells as well as using these sensors in high throughput the dissociation of a and b transmembrane domains. Integrin aIIbb3is screening. Overall, this project demonstrates how homoFRET-based sensors involved in aggregation of platelets during blood clotting. Mutagenesis of a sin- may be adapted for specific organelles while revealing a novel use of the gle lysine, K716, on the b3 transmembrane domain has been shown to lead to intensity-independence property of homoFRET towards simultaneous single- spontaneous integrin aIIbb3 activation, although the mechanism of this activa- colour measurements and high-throughput assays. tion has yet to be fully elucidated. Atomic-level molecular dynamics simula- tions have been performed on wild-type b3 as well as K716E and K716A 111-Plat mutants inserted into a phospholipid bilayer, allowing for detailed characteriza- Probe the Conformational Changes of Individual Molecules in Living Cells tion of the lipid-protein interactions though which K716 modulation of aIIbb3 Bei Liu, Orrin Stone, Onur Dagliyan, Klaus Hahn. activation occurs. Use of a specialized membrane model, the highly mobile University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. membrane mimetic (HMMM), in which the membrane core is replaced with We present a versatile approach to sense and control the conformations of auto- solvent molecules and the lipid head groups are retained at atomic resolution, inhibitory proteins by harnessing a specific, high-affinity interaction between has allowed for enhanced sampling of critical protein-lipid interactions. Fifteen the 18 kDa bacterial protein SspB (Binder) and the seven-residue peptide total simulations of 50 ns each have been performed, five for each of the wild SsrA (Tag). Taking Src family kinases (SFKs) as an example, the small Tag type and mutant structures. Each HMMM simulation was then converted to full was inserted into the C-terminus of kinases, where it would be exposed when membrane and simulated for an additional 50 ns. The wild type b3 domains the kinases were in the activated conformation. This one procedure produces converged to a larger angle to the membrane than did the mutant structures,

BPJ 8532_8543 22a Sunday, February 18, 2018 a finding consistent with the results of experimental mutagenesis studies. It was 116-Plat found that lysine snorkeling allows for critical interactions which control tilt of Cryo-EM structure of the Mechanotransduction Channel NOMPC the domain in the membrane. This study has allowed for detailed characteriza- Peng Jin1, David Bulkley2, Yanmeng Guo1, Wei Zhang1, Zhenhao Guo1, tion of the protein-lipid interactions involved in modulating b3 tilt to the mem- Walter Huynh3, Shenping Wu2, Shan Meltzer1, Tong Cheng1,4, Lily Yeh brane and, ultimately, aIIbb3 activation. Jan1,4, Yuh-Nung Jan1,4, Yifan Cheng2,4. 1Physiology, UCSF, San Francisco, CA, USA, 2Biochemistry and 114-Plat Biophysics, UCSF, San Francisco, CA, USA, 3Cellular and Molecular Structural Characterization of the N-Terminus of CRGA: An Intrinsically Pharmacology, UCSF, San Francisco, CA, USA, 4Howard Hughes Medical b Disordered Region and Short Strands to Stabilize Dimerization Institute, San Francisco, CA, USA. Yiseul Shin1,2, Riqiang Fu1, Huajun Qin1, Joshua Taylor1, Mechanosensory transduction for senses such as touch, hearing, proprioception Malini R. Rajagopalan3, Timothy A. Cross1,2. 1 and balance rely on the conversion of mechanical force into nerve impulses by NMR, The National High Field Magnet Laboratory, Tallahassee, FL, USA, mechanotransduction ion channels. How force is trasmitted to the pore-forming 2Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 3 domain of these channels and switch the channels between open and closed USA, Biomedical Research Branch, The University of Texas Health Science states remians a key open question in this field, for which two major models Center at Tyler, Tyler, TX, USA. have been proposed. One is the membrane-tension model: force applied to the CrgA is a transmembrane (TM) protein from Mycobacterium Tuberculosis.It membrane generates a change in membrane tension that is sufficient to gate consists of 93 residues, but this small helical protein is known to interact with the channel, as in the case of bacterial MscL channel and certain eukaryotic at least five different proteins in the divisome, a cell division apparatus [1]. K2P channels. The other is the tether model: force is transmitted via a tether to The structure of CrgA provides important clues to understand its own function gate the channel. NOMPC, the founding member of the TRPN sub-family of during Mtb cell division. Previously, the structure of CrgA in the TM domain Transient Receptor Potential (TRP) channels, mediates a variety of has been characterized [2]. However, further studies of the N-terminus are mechanosensation-related behaviors such as locomotion, touch and sound needed by using solid state NMR, in that it is exposed to the different envi- sensation across different species including worm, fly and fish. Recent studies ronment from the TM domain. Specifically, they are located in the cyto- suggest NOMPC is a mechanotransduction channel gated by the tether mecha- plasmic domain, and therefore they are very dynamic and also able to nism: force transduction to the channel requires its 29 ankyrin repeats (ARs) interact with the surface of lipid bilayers. To describe the dynamics and the domain tethered to the microtubule cytoskeleton. But how NOMPC, and in structure of the N-terminus, magic angle spinning solid state NMR is used particular its ARs domain, is organized to transduce mechanical displacements with two different pulse sequences: Dipolar Assisted Rotational Resonance of microtubules into opening of the channel pore has remained unresolved. Here (DARR) and Insensitive Nuclei Enhanced by Polarization Transfer (INEPT). ˚ 3þ we present the structure of NOMPC at 3.6 A resolution, determined by single Additionally, 16:0 PE-DTPA (Gd ) was added to a proteoliposome sample particle cryo-EM. The ARs domains of the tetrameric channel converge into a in order to utilize the Paramagnetic Relaxation Enhancement (PRE) effect quadruple structure with each domain resembling a helical spring. The confor- to observe the changes of signals from the N-terminus in NMR spectra. These mational fluctuation of ARs domain is coupled to the channel pore through a NMR results support our new dimer model that the 5 residues from the latter well-ordered linker domain. This architecture underscores the basis of trans- N-terminus of each monomer generate a pair of short juxtamembrane b lating mechanical displacement of the ARs into opening of the pore. strands to benefit dimerization forming a hydrophobic pocket in between two monomers. Also, mutagenesis is performed, and G44V forms a stronger 117-Plat dimer. However, the deletion of the N-terminus causes a disruption of the Structural Basis for Anion Conduction and Gating in the Calcium- dimer formation for both the wild type CrgA and the G44V mutant. This in- Activated Chloride-Channel TMEM16A dicates that the N-terminus plays a crucial role in dimerization. [1] Kieser et. Cristina Paulino1,2, Valeria Kalienkova2, Andy K.M. Lam2, al., Nat Rev Microbiol 12(8):550-562 (2014) [2] Das et. al.,PNAS 112:E119- Yvonne Neldner2, Raimund Dutzler2. E126 (2014) 1Department of Structural Biology, University of Groningen, Groningen, Netherlands, 2Department of Biochemistry, University of Zurich, Zurich, 115-Plat Switzerland. Dynamics and Ligand Binding of the GHS G Protein-Coupled Receptor in The TMEM16 family of eukaryotic membrane proteins, also known as anocta- Lipid Membranes mins, plays a key role in a variety of physiological processes. Despite their close Daniel Huster1, Gerrit Vortmeier1, Stefanie Schrottke1, Sylvia Els-Heindl2, relationship, TMEM16 members feature a striking functional diversity, as some Stephanie DeLuca3, Brian Bender3, Annette Beck-Sickinger2, Jens Meiler3. 1 work as calcium-activated ion channels and others as lipid scramblases. Although Institute of Medical Physics and Biophysics, University of Leipzig, Leipzig, the structure of the nhTMEM16 scramblase revealed the general architecture of Germany, 2Institute of Biochemistry, University of Leipzig, Leipzig, 3 the protein family it remained unclear how a channel has adapted to cope with Germany, Vanderbilt University, Nashville, TN, USA. its distinct functional properties. We have addressed this question by determining The molecular dynamics of the human growth hormone secretagogue (GHS) the cryo-EM structure of the mouse TMEM16A channel in presence and absence receptor in membranes was studied. The receptor was expressed in E. coli, re- ˚ ˚ 15 of calcium at 3.75A and 4.06A, respectively. The protein shows a similar organi- folded, and reconstituted. Static N NMR spectra are indicative of axially zation to nhTMEM16, except for changes at the site of catalysis. Here, the confor- symmetric rotational diffusion of the receptor in the membrane. About 25% 15 1 13 mation of transmembrane helices, which line a membrane-spanning furrow in of the N sites undergo large amplitude motions. H- C dipolar coupling scramblases, has changed to form an enclosed aqueous pore that is largely values, which are scaled by molecular motions, were determined. From these shielded from the membrane. The pore adopts an hourglass shape, composed of values, order parameters, reporting the motional amplitudes of the individual a narrow amphiphilic central neck and two positively charged intracellular and receptor segments were derived. Backbone order parameters were determined extracellular vestibules. Pronounced differences between the calcium-bound ranging between 0.51 and 0.69, corresponding to motional amplitudes of 40- and calcium-free mTMEM16A structures are confined to the inner half of 50 of these segments. Binding of the agonist ghrelin only insignificantly TMH6, which acts a gating element during activation. In the open state of the influenced the average molecular dynamics of the GHS receptor. Ghrelin is channel TMH6 lines the pore and it provides coordination partners for calcium- acylated at Ser3 and represents the only peptide hormone in the human ions. In contrast, in the closed state it has detached from the vacant calcium- body that is lipid-modified. We carried out NMR studies of ghrelin in lipid binding site, which is now accessible from the intracellular side. Small conforma- vesicles, followed by computational modeling using Rosetta. Chemical shift tional changes in the neck and an increase of the electrostatic barrier at the pore data of isotopically labeled ghrelin provide information about the peptide’s entrance might suffice to prevent anion permeation. Our studies, thus, reveal secondary structure. Spin diffusion experiments indicate that ghrelin binds the structural basis of anion conduction and gating in TMEM16A and define to membranes via its lipidated Ser3, Phe4, as well as electrostatics involving the foundation for the diverse functional behavior in the TMEM16 family. the peptide’s positively charged residues and lipid headgroups. Ghrelin is highly flexible and mobile at the membrane surface. This observation is sup- 118-Plat ported by our predicted model ensemble, which is in good agreement with Magnetically Oriented Phospholipid Bilayer Discs for Membrane Protein experimentally determined chemical shifts. In the final ensemble of models, NMR ghrelin is largely unstructured with some tendency to form helical conforma- Sang Ho Park, Jasmina Radoicic, Stanley J. Opella. tion. These helices appear to assist the peptide in forming an amphipathic Chemistry and Biochemistry, University of California, San Diego, La Jolla, conformation so that it can bind to the membrane. First results on the changes CA, USA. of the isotropic chemical shifts of ghrelin upon binding to the GHS receptor It is important to utilize samples containing phospholipid bilayers that are will be reported. compatible with native structures, dynamics, and functions of membrane

BPJ 8532_8543 Sunday, February 18, 2018 23a proteins for biophysical studies by NMR and other spectroscopies. Bicelles and genesis, and ending with demonstration of how we have leveraged our under- nanodiscs have been successfully used in many cases. We will describe recent standing of the importance of mechanical forces as bioregulators to develop progress in developing new types of phospholipid bilayer systems for NMR new medical devices and therapeutics. Our work began with the hypothesis studies, such as large 30-50 nm magnetically alignable macrodiscs that consist the mechanical forces are as important biological regulators as chemicals of phospholipid bilayers enclosed by proteins or synthetic polymers and genes, and that cells use tensegrity architecture to control their form (SMALPs). The spectroscopic properties of membrane proteins with one and and function. Testing this theory required development of new analytical multiple transmembrane helices will be compared. tools, including magnetic cytometry, femtosecond laser nanosurgery, micro- contact printing, and microfluidic culture systems. These studies led to 119-Plat confirmation of the critical role that physical forces play in developmental Lipids as Determinants of Membrane Protein Structure control, experimental confirmation of the cellular tensegrity model, and dis- William Dowhan, Mikhail Bogdanov, Heidi Vitrac. covery that transmembrane integrins and cytoskeletal prestress mediate Biochemistry and Molecular Biology, McGovern Medical School, Houston, mechanotransduction and developmental control. Importantly, we also lever- TX, USA. aged these insights to develop new engineering innovations that are being We have focused on the role of lipid-protein interactions as a determinant of translated into the clinic or commercial marketplace. Examples include: membrane protein structure. Orientation of transmembrane domains (TMDs) angiogenesis inhibitors for cancer therapy identified based on cell shape is generally accepted to be determined at the time of initial membrane insertion changes; ‘shrink wrap’-like polymer gels that trigger tissue and organ forma- by interactions between the membrane insertion machinery and topogenic sig- tion by physically inducing mesenchymal condensation; mechanotherapeu- nals within the protein as governed by the Positive Inside Rule. However, we tics that prevent pulmonary edema by inhibiting integrin-mediated established that post-translational lipid-protein interactions influence final mechanochemical signal transduction; shear stress-targeted drug delivery TMD orientation at the time of and dynamically after initial folding (Charge systems that are directed to sites of vascular occlusion and dissolve blood Balance Rule). Herein, we provide evidence that the presence of net neutral clots without systemic toxicity; and microfluidic cell culture devices lined lipids such as phosphatidylethanolamine (PE), which dilutes the membrane by living human cells, known as ‘Organs-on-Chips’, which recapitulate negative surface change density contributed by anionic lipids, directly reduces organ-level structure and functions by providing appropriate mechanical the effective negative charge of acidic amino acids in extramembrane domains cues as a way to replace animal testing for drug development, mechanistic (EMDs) thus favoring the potential of unaffected basic amino acids to orient discovery, and personalized medicine. EMDs towards the cytoplasm. We demonstrate that the free energy of insertion (DG) into a lipid bilayer of acidic amino acids, when placed centrally in a test 122-Symp TMD, is more positive in the absence of PE than in the presence of PE; the Role of Matrix Proteins in Balancing Tissue Stiffness and Inflammation in positive DG for bilayer insertion of basic amino acids, which remain proton- Fibrosis ated, is unaffected by PE. The potential for transmembrane movement of an Shyni Varghese. EMD containing acidic amino acids is reduced by the presence of PE. These Duke University, Durham, NC, USA. results explain why: basic amino acids display a higher potential for cyto- Fibrosis, the accumulation of excessive and disordered extracellular matrices plasmic retention of EMDs than acidic residues display for membrane translo- (ECMs), is a pathological feature of many diseases leading to organ scarring cation of EMDs; EMDs containing a mixture of acidic residues in excess over and failure. Fibrosis affects most organs and contributes to approximately one basic residues are not generally translocated across the membrane; and post- third of natural deaths worldwide. Fibrosis appears to usurp a normal wound assembly changes in lipid environment can alter protein topology. Thus, the healing response in which fibroblast activation and deposition of a controlled membrane protein insertion machinery is responsible for delivery of proteins amount of ECM and its remodeling are required for the proper repair of to the membrane while lipid-protein interactions are responsible for deter- damaged tissues. An altered wound healing response, characterized by aber- mining final protein folding. Supported in part by NIGMS grant GM121493. rant and sustained activation of fibroblasts, often leads to fibrosis. Although the underlying mechanisms are mostly unknown, altered physicochemical Symposium: Translational Biophysics properties of the ECM has been considered as a contributor to fibrosis, rather than merely a manifestation of the disease. Here, we show that the 120-Symp loss of Fibulin-5, an elastic fiber component, not only decreases mechanical Agents to Block the Neurite Outgrowth Inhibitor (Nogo, RTN4) Inspired properties of the tissue, but also diminishes the inflammatory response and ab- by the Structure rogates the fibrotic phenotype in a mouse model of cutaneous fibrosis. Melanie J. Cocco. Increasing matrix stiffness raises the inflammatory response above a Dept Mol Biol and Biochem, University of California, Irvine, CA, USA. threshold level, independent of TGF-b, to stimulate further ECM secretion Compelling evidence indicates that repair of damage to the central nervous sys- from fibroblasts and advance the progression of fibrosis. Our studies further tem (CNS) is inhibited by the presence of protein factors within myelin that pre- show that Fibulin- 5 could be an effective therapeutic target to short- vent axonal regrowth. Myelin growth inhibitors and their common receptor circuit this self-sustaining, profibrotic feedback loop, and thereby resolving have been identified as targets in the treatment of spinal cord injury and stroke. fibrogenesis. We determined the NMR structure of one of the myelin growth inhibitors, the neurite outgrowth inhibitor (Nogo). We studied the structure of this protein 123-Symp alone and in the presence of dodecylphosphocholine micelles to mimic the nat- Mechanisms Defining the Neuronal State Space ural cell membrane environment. Using several paramagnetic probes, we have Shankar Subramaniam, Andrew Caldwell, Vipul Bhargava, defined portions of the growth inhibitor that are accessible to solvent (and Dinorah Friedman-Morvinski, Qing Lu, Shauna Yuan, Douglas Galasko, consequently the Nogo receptor). Mutagenesis probed through phage-display Inder Verma, Steven Wagner. confirms that the positions predicted to be extra-cellular are sensitive to recep- University of California, San Diego, La Jolla, CA, USA. tor binding. Using computational docking methods, NMR data and mutagen- During neurogenesis in all animals, neurons are generated from neural stem esis results, we calculated the optimal protein-protein interface between our cells and progenitor cells. The molecular and genetic factors influencing neu- structure of Nogo and the Nogo receptor. With these data we predict that res- rogenesis have been elucidated in detail and notably involve activation of key idues (28-58) are available to bind the Nogo receptor which is entirely consis- transcription factors concomitantly with repression of other factors including, tent with functional assays. Moreover, the conformations and relative positions primarily, the REST protein. All neuronsareunderstoodtobe‘‘post- of side chains recognized by the receptor are now defined and may be useful in mitotic’’. The central question that arises in neuronal pathologies is what hap- antagonist design. Using this structure, we developed a library of compounds pens to these neurons? Through a combination of stem cells derived from capable of binding to the surface of Nogo. Functional assays show these com- subjects with distinct diseases of the brain including Alzheimer’s disease pounds to be active and non-toxic. and glioblastoma, high throughput transcriptomic measurements, and a host of phenotypic measurements, we have uncovered surprising and inter- 121-Symp esting mechanistic origins underlying several neural pathologies. De- Biophysics-Enabled Translational Medicine differentiation and reintroduction of cell cycle appear as common mecha- Donald Ingber. nisms that affect neurons triggered by changes in chromatin topologies. In Wyss Institute at Harvard University, Boston, MA, USA. this talk, I will discuss our results and provide insights into the question of I will review work from my laboratory beginning with mechanistic studies what happens to neurons in these diseases and explore strategies that can relating to the biophysical basis of cell shape determination and tissue morpho- be used to reverse these altered causal mechanisms. Supported by: NIH

BPJ 8532_8543 24a Sunday, February 18, 2018 grants, U01 DK097430, U01 CA200147, U01 CA198941, U19 AI090023, arise from multi-phase coexistence, which may have important consequences R01 HL106579, R01HL108735, R01 HD084633, R01 DK109365, NSF grant for sequential RNA processing. I will also discuss our new ‘‘Optodroplet’’ ap- CCF-0939370. proaches, which use light to enable spatiotemporal control of phase transitions within living cells. We are now using Optodroplets to quantitatively map intra- cellular phase diagrams. This approach has begun to yield rich insights into the Symposium: Protein and RNA Phase Separation link between intracellular liquids, gels, and the onset of pathological protein aggregation. 124-Symp Organizing Living Matter: The Role of Phase Transitions in Cell Biology 127-Symp and Disease Physical Mechanisms of Cell Organization on Micron Length Scales Simon Alberti. Michael K. Rosen. Max Planck Institute, Germany, Dresden, Germany. Biophysics, UT Southwestern Medical Center and Howard Hughes Medical Cells that are exposed to environmental fluctuations undergo changes on mul- Institute, Dallas, TX, USA. tiple levels to alter their physiology, metabolism and architecture. Our recent Cells are organized on length scales from Angstroms to microns. But the work shows that many of these changes happen in a controlled manner and mechanisms by which Angstrom-scale molecular properties are translated involve a reorganization of the cytoplasm and the formation of membraneless to micron-scale macroscopic properties are not well understood. We have compartments via a process known as phase separation. This challenges an es- shown that interactions between multivalent proteins and multivalent ligands tablished paradigm in cell biology, which posits that intracellular compartmen- can cause oligomerization and concomitant liquid-liquid phase transitions, talization requires membranes. Our very recent findings show that the material resulting in formation of micron-sized liquid droplets in aqueous solution properties of these membrane-less compartments are widely adjustable and can and micron-sized puncta on membranes. Through this idea of be modified along a continuum of physical states from liquid to gel to solid. multivalency-driven phase transitions we have explained behaviors of multi- Such changes in the material state endow cells with control over diffusion- domain proteins, intrinsically disordered proteins and nucleic acids. I will limited biochemical processes. Most importantly, we recently discovered that discuss how such transitions may control the spatial organization and the initially beneficial ability to form membrane-less compartments becomes biochemical activity of actin regulatory signaling pathways, and contribute detrimental with increasing age. This is because many compartment-forming to formation and regulation of biomolecular condensates such as PML nu- proteins are hypersensitive to changing conditions and have a tendency to clear bodies and P bodies. Our data suggest a general mechanism by which form aberrant structures that cause aging-associated diseases. Thus, we propose cells may achieve micron-scale organization based on interactions between a new model for many age-related neurodegenerative diseases, where we link multivalent macromolecules. the physiological function of compartment-forming proteins with their role in disease. Platform: Ligand-gated Channels 125-Symp Dysregulation of Phase Separation in Cancer 128-Plat Tanja Mittag. Ion Selectivity in Acid-Sensing Ion Channels and Epithelial Sodium Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, Channels USA. Zeshan P. Sheikh, Timothy P. Lynagh, Stephan A. Pless. Liquid-liquid phase separation leads to demixing of proteins from solution and Department of Drug Design and Pharmacology, University of Copenhagen, results in a dense, protein-rich phase, which co-exists with a light phase Copenhagen NV, Denmark. depleted of protein. Recent findings support a model in which phase separation Members of the epithelial Naþ channel/degenerin (ENaC/DEG) superfamily is the biophysical driving force for the formation of membrane-less organelles are Naþ-selective channels with a common overall structure. Each of the in the cell, such as stress granules, nucleoli and nuclear speckles. Current open three subunits within these trimeric channels has two transmembrane do- questions are: (i) How is phase separation propensity encoded in the protein mains (M1 and M2), of which M2 forms the pore. Included in this family sequence, (ii) are dense liquid droplets used as reaction compartments in the are the proton-gated acid-sensing ion channels (ASICs), which are formed cell, and (iii) is physiological phase separation disrupted in disease states? by identical or homologous subunits. ASICs mediate excitatory Naþ currents To address them, we study the interaction of the tumor suppressor Speckle- (relative Naþ/Kþ permeability of 10/1)andplaykeyrolesinmanyphysi- type POZ protein (SPOP), a substrate adaptor of a ubiquitin ligase, with its sub- ological processes, including nociception and cell death following ischemic strates. SPOP localizes to different liquid membrane-less organelles in the cell stroke. ENaCs are obligate heterotrimers comprised of the principal a sub- nucleus, where it encounters its substrates, but it is never found diffuse in the units, as well as b and g subunits and display significantly higher Naþ selec- cell. However, its recruitment mechanism to these organelles is not understood. tivity than ASICs (relative Naþ/Kþ permeability of 100/1). Previous work Here, we show for the first time that SPOP undergoes liquid-liquid phase sep- had indicated that the selectivity filter in both ENaCs and ASICs is formed by aration with substrate proteins, and that this mechanism underlies its recruit- a conserved G-X-S motif in the pore. However, we have recently shown the ment to membrane-less organelles. Multivalency of SPOP and substrate for mASIC1a selectivity filter to be composed of two carboxylate pairs in the each other drive their ability to phase separate. Moreover, we present strong ev- lower part of the pore, namely M2 E18’ and D21’. As both the G-X-S motif idence that the SPOP/substrate assemblies are active ubiquitination compart- and the carboxylates in 18’ and 21’ are conserved between ASICs and ENaCs, ments in vitro and in cells. SPOP cancer mutations reduce the propensity for there are likely other factors contributing to the stark differences in Naþ phase separation and tune the material properties of mesoscale assemblies. In selectivity. We have thus probed the contribution of residues in M1 to ion the cell, cancer mutants fail to localize to the proper organelles and to recruit selectivity in ASICs and ENaCs. By introducing point-mutations, bulky substrate. We propose that SPOP has evolved a propensity for phase separation side chains of the ENaC M1 were replaced by less bulky side chains, and in order to target substrates localized in membrane-less compartments. Our re- the converse was performed in ASIC1a. We show that the bulky residues sults provide mechanistic insights into the contributions of structured and disor- in M1 are important for Naþ selectivity, conceivably by influencing the dered domains to phase separate, enzymatic activity inside liquid organelles, size of the pore. and disruption of phase separation by cancer mutations. 129-Plat 126-Symp Mechanism of NMDA Receptor Channel Block by MK-801 and Memantine Lighting up Intracellular Phase Space Xianqiang Song1, Morten Ø. Jensen2, Vishwanath Jogini2, Richard A. Stein3, Clifford P. Brangwynne. Chia-Hsueh Lee4, Hassane S. Mchaourab3, David E. Shaw5, Eric Gouaux1. Department of Chemical and Biological Engineering, Princeton University, 1Vollum Institute, Oregon Health & Science University, Portland, OR, USA, Princeton, NJ, USA. 2D. E. Shaw Research, New YorK, NY, USA, 3Department of Molecular In this talk I will discuss our work showing that phase transitions play an impor- Physiology and Biophysics, Vanderbilt University, Nashville, TN, USA, tant role in organizing the contents of living cells. We focus on a class of 4Rockefeller University, New YorK, NY, USA, 5Department of Biochemistry membrane-less RNA and protein rich condensates, known as RNP bodies, and Molecular Biophysics, Columbia University, New YorK, NY, USA. which help control the flow of genetic information within cells. The nucleolus The N-methyl-D-aspartate (NMDA) receptors are neurotransmitter receptors is one such nuclear RNP body, which is important for cell growth and size ho- and calcium-permeable ion channels that transduce synaptic signals initiated meostasis. We’ve shown that a phase transition model explains many features by binding of glutamate and glycine to the opening of the ion channels. Their of nucleolar assembly, and that the internal subcompartments of the nucleolus activities play important roles in physiological and pathological conditions.

BPJ 8532_8543 Sunday, February 18, 2018 25a

Ion channel blockers, such as the ionic small molecules MK-801 and meman- 132-Plat tine—a drug approved for the treatment of Alzheimer’s disease—are able to Identification of Neurosteroid Binding Sites on GABAA Receptors using attenuate the channel activity and thus are of long standing biophysical and Photolabeling with Mass Spectrometry biomedical importance. Because the NMDA receptor has proven recalcitrant Zi-Wei Chen, John Bracamontes, Wayland W.L. Cheng, Melissa Budelier, to the determination of a high resolution structure by either x-ray crystallog- Krishnan Kathiresan, Mingxing Qian, Douglas F. Covey, Alex S. Evers. raphy or cryo-electron microscopy (cryoEM), the mechanism by which ion Washington University in St. Louis, St. Louis, MO, USA. channel blockers occlude ion permeation is not well understood. Here we Neurosteroids are endogenous modulators for neuronal excitability and nervous show that removal of the amino terminal domains (ATDs) from the GluN1/ system development and are being therapeutically applied as anesthetic agents GluN2B NMDA receptor (DATD) yields a functional receptor and well dif- and treatments for psychiatric disease. While GABAA receptors are the prime fracting crystals, allowing us to map the binding site of MK-801. Together molecular targets of neurosteroid action, the molecular details of neurosteroid with long-timescale molecular dynamics simulations, we illustrate how binding to these proteins are not defined. Accumulated evidence suggests that MK-801 and memantine bind within the transmembrane domain (TMD) neurosteroids may modulate GABAA receptor via multiple binding sites. In this channel vestibule in different modes. Both MK-801 and memantine lodge be- study, we synthesized neurosteroid analogue photolabeling reagents in which tween the M3 helix bundle crossing and the M2 pore loops, and elicit NMDA diazirine or trifluromethylphenyldiazirine photolabeling groups were placed receptor antagonism by promoting closure of the ion channel gate and phys- at the 3, 6, 17 or 20-positions of the neurosteroid, allowing us to identify bind- ically blocking ion permeation. ing sites and orientations of neurosteroids in these sites. A stable HEK cell line expressing a1FLAG-HISb3 GABAA receptors was generated and membranes 130-Plat from these cells (expressing 25 pmol of receptor/mg protein), were used Measuring Conformational Dynamics of AMPA Receptor-TARP Com- for photolabeling. The photolabeled proteins were detergent-solubilized and af- plexes using FRET finity purified on anti-FLAG beads. The purified receptors were digested with Ljudmila Katchan1,2, Yuchen Hao1,3, Andrew J. Plested1,3. trypsin and the resulting transmembrane domain (TMD) peptides were 1 € 2 Leibniz Institut fur Molekulare Pharmakologie, Berlin, Germany, UC analyzed using middle-down mass spectrometry. Three clusters of photola- 3 € Berkeley, Berkeley, CA, USA, Cellular Biophysics, Humboldt Universitat beled residues were identified consistent with three distinct neurosteroid bind- zu Berlin, Berlin, Germany. ing sites. One site was located in the interface between b3-TM3 and a1-TM1, in AMPA receptors (AMPARs) are glutamate-gated ion channels that mediate the intracellular end of the TMDs; this inter-subunit site is consistent with our fast excitatory neurotransmission in the central nervous system. Native previous identification of F301 as a neurosteroid binding site in b3 homomeric AMPA receptors form complexes with transmembrane AMPA-Receptor Reg- GABAA receptors. Intra-subunit sites were located in both the a1 and b3 sub- ulatory Proteins or TARPs. Crystal structures and single particle EM recon- units, with labeling at the extracellular end of TM1 and TM4. Docking studies structions of full-length receptors alone and in complex with auxiliary performed using AutoDock and a structural model based on the X-ray structure subunits such as Stargazin have revealed rich set of geometries, from compact of the b3 homomer were consistent with neurosteroid binding at the sites iden- to dispersed, but which of these are adopted during normal gating and desensi- tified by photolabeling. Further mutagenesis and computational simulation tization remains unclear. studies will be pursued to confirm these two photolabeling sites. In present study, we have explored fusions of green fluorescent protein variants at extracellular sites of AMPARs to enable measurement of conformational 133-Plat changes using Fluorescence Resonance Energy Transfer (FRET) in live cells. ‘‘DSPER’’ - The Depolarizing Protein of Human Sperm Conformational rearrangements accompanying receptor activity were Nadine Mundt, Polina Lishko. measured in live HEK 293 cells in real time using a patch clamp fluorometry Department of Molecular and Cellular Biology, University of California, setup. To explore conformational dynamics in an AMPAR and TARP complex, Berkeley, Berkeley, CA, USA. we engineered fusions between Stargazin and fluorescent proteins, with the Ion channels control the sperm ability to successfully fertilize the egg by trig- latter acting as either FRET donors or acceptors. Fluorescence Lifetime Imag- gering sperm hyperactivation, chemotaxis and the acrosome reaction. The deli- ing showed ligand-dependent FRET efficiency within the complexes in both cate regulation of these processes occurs on many levels. However, membrane HEK cells and neurons. Concurrent electrical and optical recordings of potential, intracellular pH and cytosolic calcium levels are of fundamental AMPA-TARP complexes activated by glutamate showed that FRET between importance. Previous work has unraveled three essential ion channels that the receptor and Stargazin is both state and position dependent, suggesting work in a concerted manner to regulate these parameters in human spermato- several conformational rearrangements within the extracellular domains occur zoa: 1) calcium channel CatSper, 2) potassium channel KSper, and 3) proton during receptor activation. These measurements suggest that direct detection of channel Hv1. However, the molecular identity of the sperm’s sodium conduc- dynamic conformational changes in AMPAR complexes at synapses is tance that is required for initial membrane depolarization and triggers the un- feasible. derlying signaling cascades is yet to be defined. Here, we shed light on the identity of ‘‘DSper’’, the depolarizing protein of human sperm as well as its un- 131-Plat derlying activation mechanisms. The combination of sperm electrophysiology Molecular Mechanisms of NMDA Receptor Function and Regulation and calcium imaging allowed us to characterize DSper in a deeply functional Nami Tajima. manner and investigate its impact on CatSper activity. DSper activity would Keck Structural Biology, Cold Spring Harbor Laboratory, Cold Spring provide positive net charge influx resulting in depolarization, consequent acti- Harbor, NY, USA. vation of Hv1/CatSper and sperm hyperactivation. N-methyl-D-aspartate receptors (NMDARs) belong to a class of ionotropic glutamate receptors that are crucially involved in brain development and func- 134-Plat tion, and NMDAR dysfunction is implicated in various neurological diseases. Visualizing Adenine Nucleotide Regulation of the KATP Channel The transmembrane ion channel opens upon membrane depolarization and Samuel G. Usher, Natascia Vedovato, Michael C. Puljung, agonist binding to the extracellular ligand-binding domain, while the extracel- Frances M. Ashcroft. lular amino terminal domain (ATD) tightly regulates functional properties. Department of Physiology, Anatomy and Genetics, University of Oxford, Although NMDAR structures representing inhibited states are available, there Oxford, United Kingdom. is no clear understanding of how conformational alteration in the extracellular The ATP-sensitive potassium (KATP) channel of the pancreatic b-cell is domains regulate NMDAR activity. We will describe the first structural evi- composed of four pore-forming (Kir6.2) subunits and four modulatory sulpho- dence for conformational alteration of the NMDARs and how the NMDARs nylurea receptor (SUR1) subunits. KATP is regulated by three distinct classes of are activated and inhibited. To understand the regulation mechanisms above, intracellular adenine nucleotide-binding site - one inhibitory site on each Kir6.2 we conducted structural and functional studies. First we present the first struc- subunit and two stimulatory sites on each SUR1 subunit. To study the interplay tural evidence for conformational alteration in the NMDAR ATD wherein the between these sites, we developed a novel approach to probe each class of bind- bilobed structure of the ATD opens and closes. On the basis of structure-based ing site with high spatial and temporal resolution. This method utilizes FRET mutagenesis coupled to electrophysiology, we show that stabilization of open between channels site-specifically labelled with the fluorescent amino acid L-3- and closed cleft conformations leads to activation and allosteric inhibition, (6-acetylnaphthalen-2-ylamino)-2-aminopropionic acid (ANAP) and trinitro- respectively. In order to understand the conformational change in the context phenyl (TNP) ATP. We used amber codon suppression to label two positions of full length, we obtained the intact NMDAR structure in an active conforma- close to the inhibitory ATP binding site on Kir6.2 with ANAP (either tion by cryo-electron microscopy in the absence of inhibitors. These studies I182ANAP or F183ANAP). Both constructs produce full length, ANAP incor- allow us to uncover the conformational change in multiple domains and molec- porating channels that are expressed on the surface of HEK293T cells. ular mechanisms. Adherent cell membranes were ‘‘unroofed’’ by sonication to expose the

BPJ 8532_8543 26a Sunday, February 18, 2018 intracellular nucleotide binding sites. Fluorescence spectra of ANAP labelled led to both cleft and cap closing observable by SAXS. We also present a tool Kir6.2 subunits were acquired after exposure to increasing concentrations of for high throughput comparison of SAXS profiles. TNP-ATP. Binding of TNP-ATP was measured as quenching of the ANAP From these results, it is evident that when SAXS experiments are combined fluorescence at 470 nm and could be competed off with addition of unlabelled with atomic models, relatively small conformations can be captured. This ex- ATP. Both Kir6.2-I182ANAP and F183ANAP co-expressed with SUR1 bound tends SAXS capabilities beyond its current routine use of generating low res- TNP-ATP in the low mM range in the absence of Mg2þ, comparable to the olution molecular envelopes. apparent affinity for inhibition of wild-type Kir6.2/SUR1 by TNP-ATP. Similar apparent affinities were obtained from C-terminally truncated or GFP-tagged 137-Plat Kir6.2-F183 constructs expressed without SUR1. A mutation (G334D) in the From Disordered Polypeptide to Functional Regulator: A Structural View of WASp-Interacting Protein and its Complex with WASp in Human ATP binding site of Kir6.2 that does not affect intrinsic KATP gating greatly decreased the apparent nucleotide affinity. Similar effects on TNP-ATP binding T-Cells 1 1 1 were observed with the introduction of the C166S gating mutation (which in- Adi Halle-Bikovski , Eva Rozentur-Shkop , Hadassa Shaked , Mira Barda-Saad2, Jordan H. Chill1. creases open probability), suggesting that conformational changes at the pore 1 2 of Kir6.2 can influence nucleotide binding. Chemistry, Bar Ilan University, Ramat Gan, Israel, Life Sciences, Bar Ilan University, Ramat Gan, Israel. 135-Plat Wiskott-Aldrich syndrome protein (WASp) is responsible for actin-dependent Mechanism of Anion Conduction in the Calcium-Activated Chloride processes in hematopoietic cells, including cellular activation, migration and Channel TMEM16A invasiveness. A key regulator of WASp is WASp-Interacting Protein (WIP), Andy Lam, Raimund Dutzler. whose mostly disordered C-terminal domain (WIPC) physically shields University of Zurich, Zurich, Switzerland. WASp from degradation in a phosphorylation dependent manner. Indeed, The calcium-activated chloride channel TMEM16A mediates selective anion WASp mutations in its N-terminal EVH1 domain (WASpEVH1) that abolish conduction upon activation by intracellular calcium. Using the cryo-EM struc- WIP affinity lead to the primary immunodeficiencies Wiskott-Aldrich syn- ture of mouse TMEM16A as a template, we have investigated the mechanism drome (WAS) and X-linked thrombocytopenia (XLT). Employing a structural of anion conduction by patch-clamp electrophysiology. In the structure, an NMR approach, we have uncovered the molecular determinants governing the hourglass-shaped, protein-enclosed aqueous conduit populated by basic resi- transition of WIP from an unstructured peptide to the functional regulator of dues is found in each subunit in a homodimer. We analysed the conduction WASp degradation. A native recombinant WIPC-WASpEVH1 complex was suc- characteristics of alanine mutants of these basic residues and found a cessfully formed by co-expression, enabling NMR distance and dihedral mea- position-dependent effect on ion conduction that is manifested in the rectifica- surements to reveal a typical 7-stranded two-winged pleckstrin homology tion of current-voltage relationships. This underlines their importance for the domain fold for WASpEVH1. In contrast, WIPC is a flexible disordered peptide electrostatics in the narrow neck region of the pore creating a favourable envi- in its free form, but upon binding spools around WASpEVH1 to form an exten- ronment for anion conduction. The compromised conduction characteristics of sive binding surface. Mutations along the WIPC reveal four binding epitopes at the cysteine mutant of a basic residue located on a-helix 5 at the intracellular the complex interface, including a previously unknown helical motif packing end of the narrow neck can be reverted to the wild-type phenotype upon reac- against the N-terminal EVH1 b-sheet. The importance of these epitopes was tion with the small, positively charged MTSEA, indicating the intracellular confirmed in the cellular setting by FRET-based in vivo molecular imaging, accessibility of the site and reinforcing the role of the positive potential for demonstrating that the 454-456 epitope is the major contributor to WASp affin- anion conduction. The equivalent mutation of a residue located one helix ity, whereas the novel 449-451 epitope is most important for inhibiting WASp turn towards the extracellular side is inaccessible, consistent with its location ubiquitylation and degradation. Further mutagenesis results hint to the phos- in the constricted part of the pore. When analysed in the context of a phenom- phorylation event that induces WIP-dissociation. Our complementary combina- enological model of ion permeation, it appears that the positively charged res- tion of technologies provides new insights into the nature of the WIP-WASp idues in the aqueous pore facilitate anion conduction by lowering local energy complex, which will be important in future efforts to develop new therapeutic barrier(s) according to their specific position in the pore. Because the major en- approaches to WAS and XLT. ergy barriers are located at the inner and outer ends of the pore, it is plausible that anions dehydrate as they enter the narrow conduit for conduction, a process 138-Plat that might be facilitated by the resident basic residues. Investigating the Conformational Transitions of Human Adipocyte Fatty Acid Binding Protein Upon Binding Leukotriene B4 by Solution-State NMR Spectroscopy Platform: Protein Structure and Conformation I Kim N. Ha1, Youlin Xia2, Yenchi Tran1, Gianluigi Veglia3, David A. Bernlohr4. 136-Plat 1Chemistry and Biochemistry, St. Catherine University, St. Paul, MN, USA, Characterizing E. coli Phosphoenolpyruvate Carboxykinase Conforma- 2Minnesota NMR Center, University of Minnesota, Minneapolis, MN, USA, tional States through Small Angle X-Ray Scattering 3Biochemistry, Molecular Biology, and Biophysics and Chemistry, Greg L. Hura, Henry Y.H. Tang, John A. Tainer. University of Minnesota, Minneapolis, MN, USA, 4Biochemistry, Molecular Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA. National Laboratory, Berkeley, CA, USA. Adipocyte fatty acid binding protein (FABP4; AFABP) is a 131-aa intracellular The conformations of an enzyme are directly related to function: during catal- lipid binding protein involved in the transport of fatty acids between cell mem- ysis, conformational changes are required to bring catalytic sidechains into po- branes and organelles. FABP4 participates in several pathways including lipol- sition for the reaction. Ligand binding often results in gross conformational ysis and lipogenesis, and strongly impacts lipid and energy metabolism related changes as well. While X-ray crystallography provides atomic resolution diseases such as diabetes. Disruption of FABP4 reduces inflammation and pro- enzyme structures, they are static conformations captured in the crystallo- tects against obesity-induced insulin resistance in animal models, however the graphic state. Deviation of crystal structures from actual solution state confor- exact mechanism of this protective effect is unknown. It is known that FABPs mation can arise from crystal packing as well as the non-physiological increase the half-life of unstable epoxide-containing leukotriene A4 (the pre- conditions required for crystal growth. Combining atomic resolution data cursor to LTB4 and LTC4), and this suggests a mechanism whereby FABP4 from crystallography with solution small angle X-ray scattering (SAXS) data stabilizes LTA4 against hydrolysis and leads to increased inflammatory allows us to observe small conformational changes at resolutions greater signaling. It is possible that there may be a subsequent stabilizing interaction than SAXS alone under conditions that are a more accurate representation of between FABP4 and LTB4. While the structure of FABP4 has been determined a protein’s native environment. using x-ray crystallography and binding to several of its hydrophobic ligands Phosphoenolpyruvate carboxykinase (PCK) is a key metabolic enzyme respon- have been characterized, its conformational transitions upon binding to leuko- sible for catalyzing the first committed step of gluconeogenesis. It is a bilobate trienes have yet to be investigated. In this study, we characterize the structural enzyme with the active site located in the cleft between the two domains. From dynamics of apo-FABP4, holo-FABP4 bound to oleic acid, and holo-FABP4 crystallographic studies, substrate binding leads to domain rotation and cleft bound with LTB4 by solution-state NMR techniques and ascertain whether closing, and a 10-residue cap closes over the cleft. SAXS experiments on FABP4 undergoes a disorder-to-order transition upon binding that stabilizes apo PCK suggests that the cap is in a closed state and the domains do not LTB4. The project is a working collaboration between St. Catherine University, stay in a fully open state. Certain active site mutants of PCK show drastically the University of Minnesota, and the Minnesota NMR Center, and provides a different behavior in solution, including a constitutively open mutant that model of conducting collaborative undergraduate research in partnership be- showed a different SAXS scattering profile. Addition of ATP to WT PCK tween a PUI, a major research institution, and an instrument center.

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139-Plat orophores were introduced at the identical positions in the HIV-1 Env proteins Histone H3 Tail Conformation Regulates Nucleosome Association by the used for structural studies and the native Env on the surface of virions, and the BPTF PHD Finger resulting smFRET values compared. Surprisingly, smFRET data reveal that 1 2 1 Emma A. Morrison , Samuel Bowerman , Kelli Sylvers , both the soluble gp140 SOSIP.664 and PGT151-HIV-1JR-FL Env structures Jeff Wereszczynski2, Catherine A. Musselman1. correspond to the State 2 gp120 conformation observed on the virus. The 1Biochemistry, University of Iowa Carver College of Medicine, Iowa City, all-important structure of State 1 of HIV-1 Env, which is the target of the ma- IA, USA, 2Illinois Institute of Technology College of Science, Chicago, IL, jority of broadly neutralizing antibodies, remains unknown. Determining the USA. structure of this additional conformation should allow the design of a second Post-translational modification (PTM) of histone proteins is one of the principal generation of immunogens that specifically present the State 1 conformation mechanisms of chromatin regulation. These chemical modifications are thought of HIV-1 Env. to act either directly by impacting chromatin structure or indirectly by recruit- ing cofactors to modified nucleosomes or regulating their activity once there. 141-Plat The specific recognition of histone PTMs by cognate effector domains in Hybrid Structure of the RagA/C-Ragulator mTORC1 Activation Complex chromatin-regulatory complexes is key to these processes. The mechanism of Ming-Yuan Su1, Kyle K. Morris1, Do Jin Kim1,2, Yangxue Fu1, binding of histone effector domains to PTMs has largely been studied with Rosalie Lawrence1, Goran Stjepanovic1,3, Roberto Zoncu1,4, James H. Hurley1,3. modified histone tail peptides, which does not take into account the context 1 of the nucleosome. We have used the BPTF PHD finger, a well- Department of Molecular and Cell Biology and California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, characterized effector for tri-methylated Lys4 on histone H3 (H3K4me3), as 2 a model system to probe the effect of the nucleosomal environment on effector USA, Denali Therapeutics, South San Francisco, CA 94080, USA, Berkeley, CA, USA, 3Molecular Biophysics and Integrated Bioimaging Division, domain binding. 4 In this study, we demonstrate that the conformation of the H3 tail within the Lawrence Berkeley National Laboratory, Berkeley, CA, USA, The Paul F. nucleosome core particle (NCP) abrogates binding by the BPTF PHD finger Glenn Center for Aging Research at the University of California, Berkeley, by approximately two orders of magnitude and probe the basis for this inhibi- Berkeley, CA, USA. tion. A combination of solution NMR-based investigations and molecular dy- The lysosomal membrane is the main locus for sensing and signaling cellular namics simulations demonstrates that the H3 tail robustly interacts with the nutrient levels, which are transduced to the master growth regulator, mTORC1 DNA component of the nucleosome core competitively with the PHD finger. kinase, via the heterodimeric Rag GTPases and the Ragulator (a.k.a. Lamtor) Our results support a model where the H3 tails predominantly exist in a dy- complex. To gain insight into the mTORC1 scaffolding and regulatory func- namic ensemble of conformations collapsed onto the nucleosomal DNA, in tions of the Ragulator-Rag assembly, we solved the crystal structure of the or- ˚ equilibrium with an ensemble of extended states. It naturally follows that chem- dered core of the five-subunit human Ragulator at 1.5 A resolution. Lamtor1 ical modification of the H3 tail that perturbs this linked equilibrium, such as wraps around the other four subunits to stabilize the assembly. The Lamtor2: lysine acetylation, in turn affects effector domain binding. Thus, histone tail Lamtor3 roadblock dimer stacks upon Lamtor4:Lamtor5 to create a platform accessibility, which is modulated by histone PTMs, is involved in dictating for Rag dimer binding. Hydrogen- deuterium exchange was used to map the specificity, providing key mechanistic insight into the active role of histone Rag binding site to the outer face of the Lamtor2:Lamtor3 dimer and to a region tails in chromatin signaling and supporting the existence of higher order of the N-terminal intrinsically disordered region of Lamtor1. Electron micro- PTM cross talk. scopy was used to reconstruct the assembly of the full-length RagAGT- P:RagCGDP dimer bound to Ragulator at 16 A˚ resolution, revealing that the 140-Plat G-domains of the Rags project away from the Ragulator core. The combined Single-Molecule FRET Reveals an Additional Conformational State of structural model shows how Ragulator functions as a platform for the presen- HIV-1 Envelope Glycoprotein Critical for Vaccine Design tation of active Rags for mTORC1 recruitment, and suggests an unconventional Maolin Lu1, Xiaochu Ma1, Castillo-Menendez Luis R.2,3, Utz Ermel1, mechanism for Rag GEF activity. Terry Daniel S.4, Jay Gorman5, Nick Reichard1, Kevin Wang1, Jonathan Grover1, Andres Finzi6,7, James B. Munro8, Peter D. Kwong5, 142-Plat Scott C. Blanchard4, Joseph Sodroski2,3, Walther Mothes1. Vinculin and its Fundamental Role in Actin Bundling Formation 1Dept Microbial Pathogenesis, Yale University, New Haven, CT, USA, Ernesto Alva Sevilla1, Andrey Krokhotin2, Nikolay V. Dokholyan2. 1 2Department of Microbiology and Immunobiology, Harvard Medical School, Physics and Astronomy, University of Texas at San Antonio, San Antonio, 2 Boston, MA, USA, 3Department of Cancer Immunology and Virology, Dana- TX, USA, Biophysics and Biochemistry, University of North Carolina at Farber Cancer Institute, Boston, MA, USA, 4Department of Physiology and Chapel Hill, Chapel Hill, NC, USA. Biophysics, Weill Cornell Medical College of Cornell University, New York, Vinculin is an abundant and essential cytoskeletal protein that localizes to focal NY, USA, 5Vaccine Research Center, National Institutes of Health, Bethesda, adhesions and adherent junctions. The interaction between vinculin and actin MD, USA, 6Department of Microbiology and Immunology, McGill plays a pivotal role in linking transmembrane receptors to the cytoskeleton, University, Montreal, QC, Canada, 7Department of Microbiology, which in turn, is important for controlling cellular force transmission, cell Infectiology and Immunology, Universite de Montreal, Montreal, QC, morphology and motility. Vinculin binds to F-actin and undergoes a conforma- Canada, 8Department of Molecular Biology and Microbiology, Tufts tional change that induces formation of a cryptic dimer necessary for actin fila- University School of Medicine, Boston, MA, USA. ment bundling, but nature of the dimer formed remains unknown. Here, we The HIV-1 envelope glycoprotein (Env) mediates virus entry into cells. Bind- employ computational approaches, including discrete molecular dynamic sim- ing to receptor CD4 and coreceptor induce conformational changes in HIV-1 ulations, to investigate actin-induced conformational changes in the vinculin Env that lead to fusion. The functional HIV-1 trimer mainly exists in a closed tail (Vt) domain that facilitate dimer formation and actin bundling. We find conformation (State 1), which is driven by CD4 binding through an interme- that actin engagement with Vt alters both the N-terminal helix (H1) and C-ter- diate conformation (State 2) to the open CD4-bound conformation (State 3). minus of Vt, and that conformational changes within the N- and C-terminus are These functional Env states can be visualized by single-molecule Fluores- necessary for the formation of stable interfaces with the actin surface. We argue cence Resonance Energy Transfer (smFRET). A breakthrough in the structural that this interface is important for Vt dimerization. We show that residue dele- characterization of the HIV-1 Env trimer has been the generation of recombi- tions with the C-terminus (DCT1, DCT2, and DCT5) affect the stability of this nant cleaved soluble gp140 SOSIP.664 trimers stabilized by a disulfide bond interface, consistent with previously published experimental findings that between the gp120 and gp41 subunits (SOS), an I559P change in gp41 (IP), C-terminal deletions within Vt reduce actin bundling activity. These observa- and a truncation at gp41 residue 664. Parallel cryo-electron microscopy tions are supported by additional mutagenesis data. Intriguingly, we find that studies have been performed with the mature HIV-1JR-FL Env in complex the presence of tryptophan at position 912 destabilizes H1 helix and postulate with the PGT151 neutralizing antibody. Both approaches resulted in similar that instability introduced by W912 plays an important role allowing H1 to un- structures. It is generally assumed that these structures represent the ground fold upon actin association. Experiments are in progress to test our actin- state of HIV-1 Env (State 1). Here we apply smFRET to determine the confor- induced vinculin dimer model and examine the role of this W912 in mational state of HIV-1 Env in these constructs and antibody complexes. Flu- vinculin-mediated actin bundling.

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Platform: Bacterial Electrophysiology: From 146-Plat Surface Sensing, Motility Appendages, and Extracellular Electron Trans- Single Cells to Biofilms port in P. Aeruginosa and S. Oneidensis Gerard Wong. 143-Plat Bioengineering, UCLA, Los Angeles, CA, USA. Voltage and Calcium Mediate E. Coli Mechanosensation Biofilms are integrated communities of cells that adhere to surfaces and Joel Kralj. are fundamental to the ecology and biology of bacteria. The accommodation BioFrontiers/MCDB, University of Colorado - Boulder, Boulder, CO, USA. of a free-swimming cell to a solid surface is more complex than simple Electrically excitable cells harness voltage coupled calcium influx to transmit modulation of cell adhesion. We investigate the interplay between motility intracellular signals, typically studied in neurons and cardiomyocytes. Despite appendages, molecular motors, metabolism, and extracellular electron intense study in higher organisms, investigations of voltage and calcium transport during bacterial surface detection and adaptation using tools signaling in bacteria have lagged due to their small size and a lack of sensitive from different fields that are not usually combined, including theoretical tools. Only recently were bacteria shown to modulate their membrane potential physics, community tracking with single cell resolution, genetics, and on the timescale of seconds, and little is known about the downstream effects microbiology. of this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in 147-Plat E. coli revealed calcium transients in single cells. A fusion sensor that simul- Membrane Tension Inhibits Wall Synthesis via Electrical Depolarization taneously reports both voltage and calcium indicated that calcium influx is to Balance Bacterial Cell Envelope Expansion induced by voltage depolarizations, similar to metazoan action potentials. Kerwyn Casey Huang, Enrique Rojas, Julie Theriot. Cytoplasmic calcium levels and calcium transients increased upon mechanical Stanford, Stanford, CA, USA. stimulation with a hydrogel, and single cells altered protein concentrations Feedback mechanisms are required to coordinate balanced synthesis of dependent on the mechanical environment. Blocking voltage and calcium subcellular components during cell growth. To probe these mechanisms, flux inhibited protein concentration differences, though the identity of the cal- we used osmotic shock to acutely perturb the growth of Gram-positive cium effectors remains unknown. Thus, voltage and calcium relay a bacterial bacteria. Upon hypoosmotic shock, cells swelled and then underwent a period sense of touch, and alter cellular lifestyle. These data open a host of new ques- of arrested growth, while hyperosmotic shock reduced growth rate for tens of tions about E. coli, including the identity of the underlying molecular players, minutes. These behaviors agreed quantitatively with a model in which mem- as well as other potential signals conveyed by voltage and calcium. These data brane tension induces growth arrest by inhibiting cell-wall synthesis and also provide evidence that dynamic electrophysiological flux exists as a turgor pressure drives mechanical expansion of the cell wall. Supporting signaling modality in the oldest kingdom of life, and therefore studying elec- this model, hypoosmotic shock halted the motion of Mbl, a reporter of cell- trophysiology beyond canonical electrically excitable cells could yield exciting wall synthesis, and reduction of membrane tension prior to hypoosmotic new findings. shock attenuated growth arrest. Hypoosmotic shock electrically depolarized cells, and depolarization halted Mbl and arrested growth independently of 144-Plat shock, indicating that membrane tension inhibits cell-wall synthesis via Electrical Signaling in Biofilms depolarization. In summary, membrane tension and cell-wall mechanical Gurol Suel. stress compete to ensure balanced synthesis of the Gram-positive cell UC San Diego, La Jolla, CA, USA. envelope. Bacteria can reside in densely packed communities known as biofilms. Our work has recently uncovered ion channel-mediated electrical cell-to-cell 148-Plat signaling that emerges within such biofilm communities. I will present our A Physiological Role for the KCH KD Channel in E. Coli most recent findings aimed at elucidating the mechanisms and functions of Steve Lockless, Sarah Beagle. electrical signaling in bacterial biofilms. Texas A&M University, College Station, TX, USA. In eukaryotic organisms, potassium (Kþ) channels have been well- 145-Plat characterized for their role in action potentials and long-range signaling, but Probing Phenazine Electron Transfer and Retention in Pseudomonas their function remains poorly defined in prokaryotes. E. coli possesses a single, þ Aeruginosa Biofilms uncharacterized K channel, kch, which was predicted by our bioinformatics- Scott H. Saunders1, Matthew D. Yates2, Edmund C.M. Tse3, based analysis to interact with many proteins involved in maintenance of the Jacqueline K. Barton3, Leonard M. Tender2, Dianne K. Newman4. proton motive force (PMF). Because bacterial flagellar motility is directly influ- 1Biology and Biological Engineering, California Institute of Technology, enced by the PMF, we examined how deletion of the channel (Dkch) affected þ Pasadena, CA, USA, 2Center for Bio/Molecular Science and Engineering, E. coli’s motility behavior. Surprisingly, in the absence of the K channel, the Naval Research Laboratory, Washington D.C., DC, USA, 3Chemistry and cells are motile and swim under conditions that are non-permissive for swim- Chemical Engineering, California Institute of Technology, Pasadena, CA, ming in a parental strain. However, genotypic rescue of the Dkch deletion did USA, 4Biology and Biological Engineering, Geological and Planetary not restore parental motility phenotypes, which led us to perform whole- Sciences, California Institute of Technology, Pasadena, CA, USA. genome sequencing to identify other mutations in the background. We are The opportunistic pathogen Pseudomonas aeruginosa forms metabolically currently using the identified mutations to further understand the physiological þ stratified biofilms. Redox-active phenazine metabolites shuttle metabolic function of the kch K channel. electrons from the oxygen depleted biofilm interior to the oxygen saturated exterior, facilitating anoxic survival. We are testing the hypothesis that 149-Plat phenazine retention and/or charge transfer through the biofilm matrix are Is there a Role for Mechanosensitive Channels in Formation and Mainte- mediated by extracellular DNA (eDNA), which is the most abundant polymer nance of Bacterial Biofilms? in the P. aeruginosa matrix and is known to provide structural integrity to Boris Martinac. the biofilm. Specifically, we are growing biofilms on electrode arrays and Molecular Cardiology and Biophysics Division, Victor Chang Cardiac performing in vitro studies of phenazine interactions with DNA. Biofilm Research Institute, Darlinghurst (Sydney) NSW, Australia. experiments show that conductivity through the matrix is mediated by Bacteria are among the most successful organisms to inhabit Earth. Their sequential electron transfer between phenazine molecules. Transferring world is filled with mechanical cues probably the most ancient of which electrode grown biofilms to fresh liquid medium demonstrates that some are osmotic forces. To cope with osmotic forces bacteria have developed phenazines are retained tightly in the biofilm matrix, while others rapidly mechanosensitive (MS) channels, which play essential role in osmoregula- diffuse away. The retention of different phenazines corresponds to their tion and provide one of the simplest paradigms for the study of mechanosen- affinity for DNA in vitro, consistent with the idea that some phenazines might sory transduction. In my talk, I will discuss the structure and function of bind the eDNA in the biofilm. We are testing whether phenazines bound to bacterial MS channels including the alternative mechano-sensory responses DNA can also mediate charge transfer. Collectively, these experiments are that they may mediate in complex microbial communities, such as bacterial helping us better understand how P. aeruginosa utilizes its self-produced biofilms. Mechanical forces modulate the formation of biofilms and the extracellular electron shuttles to survive biofilm oxidant limitation, and are shear stresses applied from fluid flow regulate these communities. Although opening up new perspectives on the role(s) that eDNA may play in the biofilm a recent transcriptomic analysis of E. coli suggests that many genes are matrix. differentially modulated by shear flow, the levels of MS channel genes

BPJ 8544_8555 Sunday, February 18, 2018 29a seemed not to be affected. Nevertheless, given that the bacteria in the 152-Plat biofilm emit an electrical signal (i.e. the release of Kþ)andthereare Multiscale Modeling and Simulation of Multivalent Cation Induced DNA five MscS-like homologues in E. coli of unknown physiological function Condensation these MS channels may play an important role in the biofilms. In fact, Tiedong Sun1, Alexander Mirzoev1, Nikolay Korolev1, the MscS-like MscK channel is regulated by external ionic environment, Alexander Lyubartsev2, Lars Nordenskio¨ld1. þ þ þ þ 1 2 requiring high concentrations of external K ,Rb,Cs or NH4 for its Nanyang Technological University, singapore, Singapore, Stockholm activation by membrane tension, which suggests its more specialized phys- University, Stockholm, Sweden. iological role compared to MscS or MscL channels, whose physiological DNA molecules are highly charged under physiological condition, hence repel- role in bacterial osmoregulation has been firmly established. Furthermore, ling each other. However, the effective interaction between DNA can become results linking bacterial MS channels to biofilm formation have been attractive in the presence of multivalent ions, which is crucial to storing DNA in reported showing that ablation of the quorum sensing system in Pseudo- the small space of nucleus. Cation-induced DNA condensation has been well monas aeruginosa, affects biofilm formation and downregulates MscK studied by in vitro experiments with condensing agent such as Cobalt(III) Hex- 3þ expression. These findings apparently link the MscK expression and ammine, [Co(NH3)6] . Despite the celebrated mechanism of cation-induced function to adhesive forces and metabolic cues within a biofilm and may polyelectrolyte condensation proposed by Manning and Oosawa, the dynamics also explain how mechanical forces determine biofilm development and of cation-induced DNA condensation is not clearly understood. maintenance. In this study, we utilized a coarse-grained DNA model, which is derived from all-atom simulation, to elucidate the structure of DNA condensate as well as the dynamics of condensation. Several microseconds long all-atom molecular dy- 150-Plat namics simulation trajectories were generated and used as the reference simu- Membrane Tension and the Charge State of Cells lation upon which the coarse-grained model was built. Inverse Monte Carlo Rob Phillips. method was adopted to determine all effective potentials for its capability Biology & Bioengineering, California Institute of Technology, Pasadena, to treat models with correlated degrees of freedom, such as our DNA CA, USA. model. In total, two models were derived respectively for two multivalent Many of the ways that cells talk and listen to the external world center on the ions, Cobalt(III) Hexammine and Spermidine. The resulting coarse-grained presence of proteins on the cell surface. Indeed, the cell membrane is an amaz- models have reproduced the structures of DNA condensates as compared to ingly diverse lipid environment, riddled in turn with a host of different proteins all-atom simulations. They also enable us to simulate cation-induced DNA that perform tasks ranging from sensing and measuring chemical signals to the condensation in a much larger space and time scale. Our large scale coarse transport of sugars needed for cell division to the detection of potentially lethal grain molecular dynamics simulations have suggested patterns and pathways osmotic pressures. This talk will focus on recent progress in the dissection of of DNA aggregation that are not accessible to traditional simulation methods. the mechanisms of mechanosensation in bacteria with special reference to At the same time, the accuracy of current simulations is expected to surpass the rich interplay between certain classes of ion channels and the surrounding other models from other coarse graining methods such as iterative boltzmann lipids and how that interplay sculpts the response of bacteria to osmotic insults. inversion (IBI). Using simple arguments from elasticity theory, I will describe the membrane deformation footprint surrounding ion channels and how this deformation foot- 153-Plat print contributes to the free energy of channel gating. In turn, I will show Structure and Dynamics of Nucleic Acid Molecules Studied by Pulsed EPR how these ideas can be parlayed into a higher-level coarse-grained model of Thomas F. Prisner1, Claudia M. Grytz1, Markus Graenz1, bacterial volume and an experimental strategy for better understanding Philipp E. Spindler1, Nicole Erlenbach1, Andriy Marko1, Pavol Cekan2, mechanosensation. Snorri Th Sigurdsson2. 1Institute of Physical Chemistry, Goethe University Frankfurt, Frankfurt am Main, Germany, 2Department of Chemistry, University of Iceland, Platform: DNA Structure and Dynamics Reykjavik, Iceland. Pulsed Electron-Electron Double Resonance spectroscopy (PELDOR or DEER) 151-Plat has been used to investigate structure and dynamics of nucleic acid (NA) mole- Effect of Pressure on the Conformational Landscape of a Large Loop DNA cules which are double-labeled by a paramagnetic cytidine analogue [1,2]. This Hairpin in the Presence of Salts and Osmolytes new type of spin-label, rigidly incorporated into double stranded NA motives, 1 1 2 1 Satyajit Patra , Vitor D. Schuabb , Rosario Oliva , Roland Winter . allows measuring the distance R and the mutual orientation between both two 1Physikalische Chemie I – Biophysikalische Chemie, Fakult€at fur€ Chemie 2 spin-labeled cytidine rings. Performing the experiment with several distinct und Chemische Biologie, TU Dortmund, Dortmund, Germany, Department probe frequencies and at different magnetic field strengths allows obtaining un- of Chemical Sciences, University of Naples ‘‘Federico II’’, Naples, Italy. ambiguous information on the structure and conformational flexibility from a The effect of pressure on the conformational landscape of a DNA hairpin small set of double-labeled NA molecules [3,4]. Extending the PELDOR mea- has been investigated in the absence and presence of salts and osmolytes surements to room temperature gives access to the time scale of the conforma- using both ensemble and single-molecule Fo¨rster resonance energy tional dynamics. Quantitative comparison with MD studies will be given [5] transfer (FRET) techniques.1 We use monovalent (Kþ), divalent (Mg2þ) 3þ and newly designed pulse sequences with superior performance discussed. and trivalent (Co )saltsandureaaswellasTMAOasosmolytes.Unlike [1] N. Barhate, P. Cekan, A. P. Massey, S. T. Sigurdsson, Angew. Chem., Int. the canonical DNA duplex structures of similar melting points, this Ed., 2007, 46, 2655-2658. [2] O. Schiemann, P. Cekan, D. Margraf, T. F. Pris- large loop DNA hairpin is rather sensitive to pressure.2 The transition 3 1 ner, S. T. Sigurdsson, Angew. Chem., Int. Ed., 2009, 48, 3292-3295 [3] A. volume upon unfolding is found to be 17 cm mol in neat buffer Marko, V. Denysenkov, D. Margraft, P. Cekan, O. Schiemann, S. T. Sigurds- solution. We found that the stabilizing effect of salt follows the order 3þ > 2þ > þ 2þ 3þ son, T. F. Prisner, J. Am. Chem. Soc. 2011, 133, 13375-13379. [4] C. M. Grytz, Co Mg K .Above1mMMg and 0.3 mM Co ,pressurehas A. Marko, P. Cekan, S. T. Sigurdsson and T. F. Prisner, Phys. Chem. Chem. a negligible effect on the conformation of the DNA hairpin, which is due Phys., 2016, 18, 2993-3002. [5] L. S. Stelzl, N. Erlenbach, M. Heinz, T. F. Pris- to the compensation of the negative charge density of the phosphate back- ner, G. Hummer, J. Am. Chem. Soc., 2017, 139, 11674-11677. bone, favoring the formation of loops by base pairing of complementary se- quences, and thus the closed conformation. The compatible osmolyte TMAO 154-Plat also stabilizes the closed conformation even at high pressures and tempera- Equilibrium Conformational Distributions of Bent DNA in Complex with tures, while urea destabilizes the closed conformation synergistically with IHF Mapped with Fluorescence Lifetime Measurements pressure and temperature. Intermediate states are populated by urea and Mitch Connolly, Viktoriya Zvoda, Anjum Ansari. high temperatures, indicating that the conformational landscape of the University of Illinois at Chicago, Chicago, IL, USA. DNA hairpin is in fact a rugged one. The results obtained are interpreted The dynamics and mechanism of how site-specific DNA-bending proteins in terms of preferential hydration and interaction effects of the cations and interrogate and recognize their target sites have remained elusive for most osmolytes. systems. Our group has previously applied laser temperature-jump spectros- References: copy with time-resolved FRET to investigate the kinetics of DNA bending 1. Patra, S.; Anders, C.; Erwin, N.; Winter, R. Angew. Chem. Int. Ed. 2017, 129, induced by Integration Host Factor (IHF), a nucleoid-associated architectural 5127-5131. protein that is known to bend a 35 base pair (bp) cognate DNA sequence 2. Dubins, D. N.; Lee, A.; Macgregor, R. B.; Chalikian, T. V. J. Am. Chem. Soc. (the H’ site) into a U-turn by sharply kinking the DNA at two sites. 2001, 123, 9254-9259. While the T-jump studies revealed the DNA-bending rates during preliminary

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‘‘interrogation’’ then ‘‘recognition’’ by IHF, what remained elusive was the 157-Plat equilibrium conformational distributions for the specific IHF-H’ complex. Chromatin Organization by an Interplay of Loop Extrusion and Compart- Indeed, for most DNA bending proteins, it is not known whether the DNA in mental Segregation the complex stays bent/kinked in a particular conformation, or whether the Johannes Nuebler1, Geoffrey Fudenberg2, Maxim Imakaev1, complex samples a range of bent conformations. To address this question Nezar Abdennur1, Leonid Mirny1. and to unveil the dynamic nature of the IHF-DNA complex, we used 1Massachusetts Institute of Technology, Cambridge, MA, USA, 2University picoseconds-resolved fluorescence lifetime spectroscopy to map the population of California, San Francisco, Gladstone Institutes, San Francisco, CA, USA. distribution as measured from the distribution of FRET between fluorescein and Eukaryotic chromatin, is far from being simply a randomly arranged polymer TAMRA attached at either end of the DNA duplex. Our results revealed a sur- in the cell nucleus. Rather, Hi-C and imaging reveal a high degree of spatial prisingly dynamic complex; while the majority of the population exhibited a organization on various length scales. In particular, active formation of FRET consistent with the end-to-end distance expected from the crystal struc- growing chromatin loops emerges as a general organizational principle ture, nearly 25-30% exhibited FRET indicative of unbent or partially bent throughout the cell cycle. We report in particular how loop extrusion com- DNA, under conditions where all DNA are expected to have a protein bound. petes with spatial segregation of active and inactive chromatin. This interplay We anticipate that the extent of this conformational flexibility in the complex explains a large number of experimental perturbations, namely removal of the could be modulated by variations in the cognate site sequence. Similar studies loop extruder cohesin, removal of the cohesin boundary element CTCF and with other cognate sites of IHF, and in particular with H1 that lacks an A-track removal of the cohesin unloader Wapl, and makes specific predictions about on one side of the IHF binding site (as in H’) are ongoing. joint changes in TADs and compartments for variations in the compartmental interaction. 155-Plat Dissecting the Mechanism of HP1 Mediated Chromatin Compaction via 158-Plat Single Molecule DNA Curtains Long-Range Structural Changes in the Meiotic Nucleus Revealed by Madeline M. Keenen, Adam G. Larson, Geeta J. Narlikar, Sy Redding. Changes in Stress Communication Along the Chromosome Biochemistry, UCSF, San Francisco, CA, USA. Trent Newman1, Bruno G. Beltran2, James McGehee1,3, Cori Cahoon1,4, Silenced genes are typically associated with densely compacted regions of the Daniel Elnatan1,5, Daniel Chu1,6, Sean Burgess1, Andrew Spakowitz2. genome. Initial compaction occurs through the wrapping of DNA around his- 1Molecular and Cellular Biology, UC Davis, Davis, CA, USA, 2Biophysics, tone octamer to form a nucleosome, but the structures of further compaction Stanford University, Stanford, CA, USA, 3California Institute of Technology, and its functional consequence on gene expression remains enigmatic. Hetero- Pasadena, CA, USA, 4Stowers Institute for Medical Research, Kansas City, chromatin protein 1 (HP1) is a major mediator of this process, and contains a MO, USA, 5Department of Biochemistry & Biophysics, University of globular chromo-domain (CD) that facilitates binding to H3K9me3, a chro- California, San Francisco, San Francisco, CA, USA, 6Department of moshadow domain (CSD) that mediates dimerization, and a flexible hinge Orthopaedic Surgery, University of California, San Francisco, San Francisco, that binds to DNA. The higher-order oligomerization of HP1 along a chromatin CA, USA. scaffold is thought to induce compaction. However, HP1 compacted structures Homologous recombination drives structural reorganization of the nucleus in and their functions have been difficult to study in vitro at physiologically early meiosis. In order to investigate the connection between homolog pairing, relevant length scales due to the technical limitations of bulk biochemical ap- meiotic progression, and the dynamics of the underlying chromatin, we tracked proaches. We utilize single molecule DNA curtains to visualize this compac- flourescently labeled homolog pairs in synchronized S. cerevisae. Various pre- tion, and find that the addition of HP1a leads to rapid compaction of 50kb viously unreported statistics of the anomalous inter-loci motion correlate with lambda DNA at a rate of 10kbs per second, faster than its own packaging motor. meiotic progression and can be quantitatively reproduced by a simple polymer Initially, a fluorescent DNA puncta appears at the end of the DNA strand, model of the sister chromatids. implying a cooperative mechanism of HP1a recruitment and compaction. The first of these is the distribution of waiting times for the homologous loci to Mutations in the DNA binding motif of the hinge region or in the CSD domain come into and out of contact with each other (loosely, inter-locus ‘‘looping’’ abrogate compaction, indicating that both the binding to DNA and HP1 oligo- and ‘‘unlooping’’ times). The full shape of the looping time distribution can merization are necessary. Further, of the three human paralogs only one is be quantitatively reproduced by a simple model of two polymers diffusing inde- capable of this rapid compaction, leading to a model in which the intrinsic pendently in a spherical confinement. This finding suggests a dominant role for compaction ability of each paralog could induce differential gene expression diffusion-limited, undirected search in homolog pairing in early meiosis. This in the cell. is in sharp contrast with the intuition that a heavy-tailed search process could never drive such a critical cell-cycle stage. 156-Plat We further show that the inter-locus velocity-velocity correlation (VVC) quan- Nuclear Architecture Controls the Timescales of Genomic Interactions titatively matches analytical results for the inter-locus VVC of our polymer Yaojun Zhang1, Nimish Khanna2, Olga Dudko3, Cornelis Murre2. model, allowing us to leverage our analytical theory to extract the time scale 1Princeton Center for Theoretical Science, Princeton University, Princeton, of stress communication between the labeled loci along the chromosome. We NJ, USA, 2Molecular Biology, University of California San Diego, San show that stresses can take tens of minutes to propagate between loci on paired Diego, CA, USA, 3Physics, University of California San Diego, San Diego, chromosomes, and that the increasing connectivity between the chromosomes CA, USA. as the cell progresses through meiosis can be quantified by the shortening of Many processes in biology, from antibody production to tissue differentiation, this communication time. involve a common fundamental step - establishing physical contact between Our study highlights the power of coarse-grained polymer models to analyze distant genomic segments. The segments must find each other quickly despite dynamic structural properties of the nucleus in vivo and the importance of their remoteness along the DNA sequence, the multi-level chromatin packing, analytical theory for uncovering intracellular connections that might be and the crowded environment in which they move. A key outstanding question obscured by lag times of many minutes. is then: What are the consequences of the chromatin architecture for the time- scales of genomic interactions? To address this question quantitatively, we analyzed 3D genomic trajectories from a novel multi-color imaging approach Platform: Protein-Small Molecule Interactions in live immune cells. The analysis indicates that anomalous diffusion in a viscoelastic environment is a dominant mechanism of chromatin motion. Meta- 159-Plat bolic activities in addition to thermal noise contribute to the jiggling of gene A Central Role for Biophysics in Cancer Drug Discovery - Development of segments. We used Molecular Dynamics simulations and insights from statis- Candidate Small Molecule Inhibitors in Mutant KRas tical and polymer physics to reveal some of the principles by which the nuclear Andrea Gohlke, Justin Bower, Peter N. Brown, Ken S. Cameron, architecture controls the genomic timescales. Specifically, we built a hierarchy Martin Drysdale, Gillian Goodwin, Christopher Gray, Jen Konczal, of polymer physics models reflecting a spectrum of chromatin configurations, Duncan McArthur, Heather McKinnon, Mokdad Mezna, Angelo Pugliese, such as loops and loop domains. We established a quantitative relationship be- Alexander W. Schuettelkopf. tween the encounter time of two segments and their spatial distance within a Drug Discovery Programme, CRUK Beatson Institute, Glasgow, United given configurations. We then developed a larger-scale model of the locus as Kingdom. a stable yet dynamic three-dimensional network phase-separated from the nu- The Ras GTPase family is comprised of three proto-oncogenes (HRas, KRas clear environment. Together, these models provide quantitative, physical inter- and NRas) cycling between inactive GDP and active GTP bound states. Active pretation of the experimentally observed features of genomic motion and KRas localises to the plasma membrane and signals through a functionally generate testable predictions regarding the structure-dynamics relationship at diverse set of down-stream effector proteins (including RAF1) to influence different levels of the genome organization. cellular differentiation, growth, survival and apoptosis. Mutant KRas, which

BPJ 8544_8555 Sunday, February 18, 2018 31a is ostensibly stabilised in the active GTP form, is well validated and linked to ITC, and iTC200 (manufactured by Malvern Instruments) and Affinity ITC over 20% of human cancers, making it a highly desirable target in oncology (by TA Instruments). The protein-ligand binding reaction was repeated under drug discovery. We have employed a combination of biophysical techniques identical conditions to obtain the statistical standard deviation of the measure- as central drivers for drug discovery, interacting with medicinal chemistry to ments performed at 25 C at various protein concentration. The enthalpy and establish structure-activity relationships (SAR), increasing affinities and Gibbs energy mean values plus minus the standard deviation of acetazolamide improving the binding kinetics of candidate small molecules targeting mutant binding to CA II at 10 mM and 25 C were equal to DH= 51.1 5 3.1 kJ/mol KRas. Fragment based screens using surface plasmon resonance (SPR) and nu- and G= 45.2 5 1.3 kJ/mol as determined by averaging the results of all four clear magnetic resonance (NMR) yielded a number of different chemical start- instruments and applying the NITPIC-SEDPHAT software analysis that usu- ing points. These initial hits, which exhibited high millimolar affinities, have ally reduced the standard deviation of the enthalpy. evolved and grown to deliver a set of molecules that exhibit submicromolar af- finity with additional help of crystallography, isothermal titration calorimetry 163-Plat (ITC) and computational modelling. The improved binding is also translating Binding Pockets Under Mechanical Stress to functional effects in vitro in additional biochemical assays. This study dem- Matteo Tiberti1, Bob-Dan Lechner2, Arianna Fornili1. onstrates the impact of Biophysics in a critical area of cancer drug discovery 1School of Biol and Chem Sciences, Queen Mary Univ of London, London, and how its interplay with medicinal chemistry has developed promising com- United Kingdom, 2School of Physics, University of Exeter, Exeter, United pounds that aim to interrupt KRas signalling and thus decrease KRas dependent Kingdom. oncogenesis. The binding of small molecules to proteins usually occurs at specific clefts or cavities on the protein surface known as binding pockets. In the past years it has 160-Plat become increasingly clear that binding pockets are dynamical entities, whose Identification of the Flexible Regions Differentiating Ligand-Binding Af- shape and size can change due to the intrinsic motions of the protein. In partic- finity for Mdm2 and MdmX ular, some pockets open transiently, so that they can be detected only if the dy- Zheng Su, Xiyao Cheng, Lingyun Qin, Rong Rong, Yongqi Huang. namics of the protein is taken into account. Biopharmaceutical Science, Hubei University of Technology, Wuhan, China. The aim of the present work is to understand how the surface and in particular The interactions of p53 protein with the overexpressed mouse double minute 2 the binding pockets of a protein can be modified by an external force (Mdm2) and mouse double minute X (MdmX) impair the cancer suppression of mimicking a mechanical stress. Indeed, proteins can often be subjected to me- p53 in approximately half of cancers. Although Mdm2 and MdmX are highly chanical forces while performing their function, for example if they are homologous, currently only Mdm2 inhibitors are available but exhibit weak af- involved in muscle contraction or cell adhesion. However, most of the single finity for MdmX. The structural mechanism underlying how the two proteins molecule studies of protein mechanics performed so far have focused on me- distinguish a ligand remains elusive. Here we found that the distinct difference chanical unfolding, requiring the application of forces much larger than the between their ligand-binding pocket flexibilities differentiated ligand-binding ones observed under physiological conditions. In this work we investigated affinity. Through rigidifying the ligand-binding pocket on MdmX using disul- instead the effect of low forces on the surface plasticity of proteins while fide staples to tether flexible regions, we were able to identify the flexible re- they are still in their folded state. gions that control ligand-binding affinity. Thus, we utilized a reverse- Remarkably, our Steered Molecular Dynamics simulations indicate that me- thinking strategy to transform nutlin-3a into potent MdmX inhibitors. The strat- chanical stress can lead to the formation of new pockets in the folded protein. egy should be applicable for lead optimization in drug discovery. These mechanically-induced pockets are energetically stabilised by the pres- 161-Plat ence of external forces and can bind probe molecules in druggability tests. Photoswitchable Drugs and Insulin Release: Molecular Events in EPAC2A Our findings have a high potential impact on drug design, since they suggest Protein the existence of a new class of drug targets that has not been exploited so far Wieslaw A. Nowak1, Lukasz Peplowski1, Jakub Rydzewski1, and that is expected to be particularly relevant for the treatment of heart and Tomoo Miyahara2, Haruki Nakamura3, Hiroshi Nakatsuji2. muscle diseases. 1Biophysics and Medical Physics, Institute of Physics, N. Copernicus This research has been supported by the British Heart Foundation. University, Torun, Poland, 2Quantum Chemistry Research Institute, Kyoto, Japan, 3Institute for Protein Research, Osaka, Japan. 164-Plat An increasing wave of new type 2 diabetes (T2D) cases affected millions of Neutron Diffraction Studies of a Non-Canonical Catalytic Triad of a Less people worldwide and calls for new and more efficient ways of blood glucose Promiscuous Aminoglycoside Acetyltransferase level control. Insulin is secreted from pancreatic beta cells a process facilitated Fnu Prashasti. by sulfonylurea drugs. Precise dynamical control of this process is not possible Genome Science and Technology, University of Tennessee, Knoxville, TN, yet. Photopharmacology [1] brings a promising strategy for an improved treat- USA. ment of T2D. We used a range of quantum-chemical methods (HF, DFT, SAC- Bacteria can acquire resistance against antibiotics by covalently modifying CI) to study the molecular and electronic structure of a prospective them. This is achieved by plasmid-encoded enzymes called as aminoglycoside azobenzene-based drug JB253 [2]. We present UV-VIS and circular dichroism modifying enzymes (AGMEs). More than 50 AGMEs are known, having var- spectra of this compound embedded in a protein environment. We developed iable levels of substrate promiscuity. However, no correlation has been confir- CHARMM force field parameters for the drug and probed, via systematic dock- matively observed between the sequence or structure of an AGME and its ing [3] and modeling in atomic detail, how its interactions with EPAC2A substrate profile. We aim to understand the molecular principles underlying enzyme depend on the cis-trans conformations of JB253. Classical MD simu- this ligand selectivity by deciphering the thermodynamic, structural and dy- lations on a nanoseconds timescale reveal that the optically activated conforma- namic properties of enzyme-ligand complexes.This work describes kinetic, tional change triggers a signal transduction. However, current data and quality thermodynamic and structural properties of the aminoglycoside N3 acetyltrans- of protein structures are too limited to elucidate the full mechanism of the op- ferase VIa (AAC-VIa). AAC-VIa’s substrate profile is limited to only five ami- tical control of the insulin release. noglycosides, making it one of the least promiscuous AGMEs, despite having This work is supported by National Science Centre, Poland grants no. 2016/23/ significant sequence similarity to other highly promiscuous acetyltransferases. B/ST4/01770(WN) and 2015/19/N/ST3/02171(JR). X-ray crystallography studies suggested a novel catalytic mechanism involving a non-classical catalytic triad. The catalytic process was also hypothesized to 162-Plat involve protonated form of the active site histidine, which was demonstrated Repeatability of Enthalpies and Gibbs Energies of a Protein - Ligand Bind- by neutron diffraction studies. Thermodynamic studies determined the binding ing Reaction Measured by ITC of ligands to be enthalpically driven and entropically unfavorable. Unlike other Vaida Paketuryte, Vaida Linkuviene, Daumantas Matulis. AGMEs, the formation of binary and ternary complexes was accompanied by a Institute of Biotechnology, Vilnius, Lithuania. net deprotonation of the enzyme, ligand or both. Another significant difference During the rational drug design, it is important to accurately determine the was observed in the structure of AAC-VIa and other AGMEs in solution. protein-ligand binding constant (the affinity or the Gibbs energy change upon Analytical ultracentrifugation (AUC) studies showed that AAC-VIa exists in binding), the enthalpy, and the entropy change upon binding. These three ther- a monomer-dimer equilibrium, with more dimeric form appearing with modynamic parameters of the association reaction are often determined by the increasing concentrations of the enzyme. Binding of ligands drive the enzyme isothermal titration calorimetry (ITC). Here, the repeatability of the measure- to the monomeric form. Also, dimer formation, was observed to be achieved ment of the Gibbs energy and enthalpy of acetazolamide interaction with the through polar interactions. Crystal structures of different complexes showed recombinant human carbonic anhydrase II (CA II) was evaluated by four that structures of apo-and ligand-bound forms are similar which suggests isothermal titration calorimetry (ITC) instrument models: PEAQ-ITC, VP- that, unlike other AGMEs, more rigid structure of AAC-VIa may limit the

BPJ 8544_8555 32a Sunday, February 18, 2018 active site to accommodate only few selected aminoglycosides, hence low sub- modified KRAS4b provided by the Frederick National Laboratory, we investi- strate promiscuity. gated the role of specific lipids in the recruitment of KRas4b to a Nanodisc membrane surface of defined composition. Application of a single frequency 165-Plat fluorescence anisotropy decay experiment to this system revealed that KRas4b Measure Small Molecule-Membrane Protein Binding Kinetics with Nano- has a significant binding preference for Nanodisc bilayers containing PIP2. We Oscillators conducted molecular dynamics simulations to look for an origin of this speci- Guangzhong Ma. ficity. In the case of membranes containing PIP2 the protein formed long-lived Biodesign Institute, Tempe, AZ, USA. salt bridges with PIP2 head groups but not the monovalent DMPS, explaining Most FDA-approved drugs are small molecules and many membrane proteins the experimentally observed lipid specificity. Additionally, we report that PIP2 are preferred drug targets. However, measuring binding kinetics between small forms key contacts with Helix-4 on the catalytic domain of KRas4b that orient molecules and membrane proteins has been extremely challenging, because the protein in a manner expected to facilitate association with upstream and these proteins have to be embedded in lipid bilayers to maintain their proper downstream signaling partners. conformation to be functional. We developed a method to quantify small molecule-membrane protein binding kinetics using self-assembled nano-oscil- 168-Plat lators, each consisting of a nanoparticle tethered to a surface by flexible poly- How Topology Correlates to Dynamics and Function for Membrane Pe- mer linker. The nanoparticle is either a virion displaying membrane proteins on ripheral Protein Complex the viral envelope or a bead coated with membrane protein-embedded nano- Zhenlu Li1, Matthias Buck2. discs. By tracking the oscillation of the nanoparticles under an applied electric 1Case Western Reserve University, Cleveland, OH, USA, 2Department of field, we managed to measure the ka and kd values between small molecules and Physiology, School of Medicine, Case Western Reserve University, membrane proteins (e.g., G-protein coupled receptors and ion channels) in a Cleveland, OH, USA. real-time, label-free fashion. This method provides a unique platform for In this study, we reveal that the topology of membrane peripheral protein measuring affinities, drug screening, biomarker discovery, and analysis of complexes as well as the geometry restraint due to membrane proximity cellular processes that involve molecule-membrane protein interactions. largely affects protein dynamics and function. As monomeric proteins, both 166-Plat of K-Ras4B [1] and C-Raf CRD interact with anionic lipid molecules through Porphyrin-Induced Multimerization of Solution-State Proteins a certain interface. However, in a complex of K-Ras4B: C-Raf RBD-CRD, the Oleksandr Kokhan, Daniel Marzolf, Coleman Swaim. geometric restraint, provided by the tight binding between the two proteins, Chemsitry and Biochemistry, James Madison University, Harrisonburg, VA, does not allow the two favorable membrane binding interfaces to interact USA. with the membrane simultaneously. This leads to a dynamic protein complex Interactions between charged porphyrins and complimentary or similarly at the membrane sampling two dominant orientations with rather K-Ras4B he- charged proteins provide important models systems for studies of electron lix 3, 4 or C-Raf CRD membrane binding region in contact with the mem- transfer processes, artificial photosynthesis, and control of protein-protein in- brane [2]. In another example, two Rho-GTPases, Rnd1 and Rac1, were teractions. Typically, the experimental results are analyzed and discussed found to positively and negatively regulate the GAP activity of plexin-B1, assuming that the proteins exist in a monodisperse state. Here, we explored respectively. Plexin-B1: Rnd1 complex reserves enough space (between interaction of four solution-state proteins (horse heart cytochrome c, hen plexin-B1 and the membrane) for Rap1b association with plexin-B1 GAP egg-white lysozyme, 3-heme c-type cytochrome (cyt) PpcA from Geobacter domain. In contrast, membrane anchored Rac1 brings the Plexin-B1 RBD sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several and accordingly the Plexin-B1 GAP domain to the membrane. The membrane cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Com- proximity of the GAP domain leaves little space between Plexin-B1 and mem- bined small- and wide-angle X-ray scattering experiments revealed formation brane for Rap1b association, hence blocking the GAP activity. Our study re- of multimers with a wide range of complex sizes. Thermodynamic interaction veals the detailed steric organization/dynamics of two peripheral membrane parameters and complex binding stoichiometries were established with complexes through molecular modeling and molecular simulation on the ms isothermal calorimetry. Locations of porphyrin binding sites were determined scale. The observations suggest a new paradigm in that the geometric features with heteronuclear single quantum coherence (HSQC) and total correlation of membrane peripheral protein complexes have a role in functional spectroscopy (TOCSY) NMRs for PpcA,.while covalent labeling shielding ex- regulations. periments followed by LC-MS analysis of tryptic digests were used to map [1] Li, Z. and Buck, M. Structure 2017, 25: 679-689 ligand binding sites on cyt c, cyt c4 and lysozyme surfaces. All-atom molecular [2] Li, Z., Prakash, P. and Buck, M. BioRxiv 2017, Doi: https://doi.org/10. dynamics simulations revealed quick complex formation with binding sites 1101/181347 well matching the areas identified in our experimental work. The obtained re- sults demonstrate that multimerization of solution-state proteins by large water- 169-Plat soluble ligands appears to be a wide-spread phenomenon controlled by a deli- Interaction Between Myristoylated Human Arf1 and ASAP1 at the Mem- cate interplay of electrostatic and hydrophobic forces. Molecular level mapping brane Surface Yifei Li1, Olivier Soubias1, Jess Li1, Paul A. Randazzo2, R. Andrew Byrd1. of the binding sites allows us to build a theory explaining the size of the formed 1 2 complexes and provides opportunities for targeted design and assembly of NCI, NIH, Frederick, MD, USA, NCI, NIH, Bethesda, MD, USA. multi-subunit protein complexes. Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an understanding of how they interact at the membrane surface has remained elusive. Here, we focused on ADP ribo- Platform: Protein-Lipid Interactions I sylation factor (Arf) GTPase Arf1, which orchestrate a variety of regulatory functions in vesicular formation and trafficking, and their deactivation by 167-Plat the Arf GTPase-activating protein (ArfGAP) ASAP1. The interaction of the Mehanisms in Cancer Signaling: The Role of the Membrane in the PH domain of ASAP1 with the lipid surface was studied by 15N and 13C Recruitment of the Oncogene KRas4b methyl NMR, using phosphatidylinositol 4, 5- bisphosphate (PIP2) - contain- Stephen G. Sligar, Michael C. Gregory, Mark A. McLean. ing nanodiscs as mimics of the membrane surface. Analysis of the chemical Biochemistry, University of Illinois, Urbana, IL, USA. shift perturbations within PH showed that the two loops between b sheets Ras proteins are a key element of signal transduction where extracellular 1, 2 and 3, 4 are involved in the membrane surface binding interface. This growth factors control nuclear transcription events involved in cell division, suggests an oriented interaction with the membrane, in which charged lipids proliferation, and apoptosis. Ras activity is controlled, and signaling mediated bind to two binding sites of the PH domain as proposed recently. We followed by, critical protein-protein interactions. GTPase activating proteins (GAPs), the formation of the myristoylated Arf1/ASAP1 complex with a PIP2- such as p120, bind to activated Ras, dramatically increasing the rate of GTP hy- containing bilayer, using NMR and fluorescence. Methyl chemical shifts of drolysis thus returning the system to the inactive GDP bound state. Guanine ex- Arf1 reveal that the nanodisc surface supports the binding of Arf1 triggered change factors (GEFs), such as Son of Sevenless (SOS), bind and effect the by GTP and several other GTP analogs. Rates of GTP hydrolysis by a full exchange of GDP for GTP, thus turning ‘‘on’’ KRas4b. Most importantly, these length ASAP1 functional domain confirm that a nanodisc lipid surface sup- multi-protein complexes all operate on a membrane surface, which is a critical ports the formation of an active complex. Interestingly, a short deletion at partner in signaling. Despite this critical role of the membrane, there is incom- the N term of ASAP1 functional domain strongly reduces its efficiency. Taken plete knowledge as to the role of the bilayer composition in anchoring the pro- together, this suggests that the high efficiency of ASAP1 requires a well- tein to the membrane and the importance of specific lipid type in dictating the defined orientation on the membrane and critical contacts between Arf1 and final orientation of KRas4b on the surface. Using a fully post-translationally the N-term of ASAP1.

BPJ 8544_8555 Sunday, February 18, 2018 33a

170-Plat dramatically, and even promote partial penetration of full-length Bax. This pro- Membrane Bound Structure of the HIV-1 Accessory Protein Nef cess was characterized by a combination of solid state NMR spectroscopy, dif- Rebecca Eells1, Kindra Whitlatch2, Bradley Treece1, Frank Heinrich3,1, ferential scanning calorimetry and fluorescence spectroscopy. More recently, John Jeff Alvarado2, Thomas E. Smithgall2, Mathias Lo¨sche1,3. we found that Bax under these conditions is already able to induce MOM 1Carnegie Mellon University, Pittsburgh, PA, USA, 2University of pore formation if the right OxPl species is present. To provide further insight Pittsburgh, Pittsburgh, PA, USA, 3National Institute of Standards and into this process and its dependence on the lipid matrix we study this process Technology, Gaithersburg, MD, USA. currently by a combination of NMR, neutron reflectometry and fluorescence Human immunodeficiency virus-1 Nef is a myristoylated accessory protein based approaches. essential for viral replication, immune evasion of infected cells, and AIDS pro- gression. Nef functions in a membrane-bound state through interactions with 173-Plat numerous host cell proteins involved in signal transduction and endocytic traf- Importance of Membrane Curvature Near Hole Edges in Plasma Mem- ficking. These include members of the Src and Tec kinase families, which are brane Repair constitutively activated by interaction with Nef. Nef dimerizes at the plasma Theresa Louise Boye1, Weria Pezeshkian2, Adam Cohen Simonsen2, membrane in cell-based model systems, and dimerization has an important orga- Jesper Nylandsted1. 1Research Center, Danish Cancer Society, Copenhagen, Denmark, nizational role in Nef:kinase complex formation and activation. To characterize 2 the biophysical aspects of this key Nef function, we aim to reconstitute com- Department of Physics, Chemistry and Pharmacy, University of Southern plexes of Nef with Tec kinases at a membrane interface utilizing model mem- Denmark, Odense M, Denmark. branes. Here we report an initial structure of myristoylated, recombinant Nef Maintaining integrity of the plasma membrane is essential for cell life. Thus, bound to fluid lipid bilayers determined using neutron scattering on solid- efficient cell membrane repair mechanisms are crucial for handling membrane supported sparsely-tethered lipid bilayer membranes (stBLMs). As expected, disruptions resulting from external perturbations of eukaryotic cells. Cancer myristoylation is essential for high affinity membrane interactions with anionic cells in particular, experience enhanced membrane stress when navigating stBLMs. The folded Nef core is displaced from the bilayer in a position that is through the dense extracellular matrix, which increases the frequency of mem- brane injuries. Yet the underlying molecular details of plasma membrane repair presumably amenable to engagement with membrane-bound kinases while N- 2þ terminal lipidation and a cluster of basic residues anchor the protein on the mem- are not well understood. Plasma membrane injury followed by Ca influx, ac- brane. The distance of the Nef core from the membrane depends on surface con- tivates the recruitment of Annexins to membrane wound edges. In cells, we find centration of the protein and appears to differ slightly for wild-type Nef and a that Annexin A4 binding promotes repair of lesions generated by local laser dimerization-deficient Nef mutant (D123N). MD simulations constrained by treatment or other types of controlled damage. In planar model membranes, the experimental neutron scattering results are currently exploring the confor- we show that curvature stress induced by annexin-binding leads to roll-up of mational flexibility of the membrane-bound protein. These studies will releval the membrane as initiated from free membrane edges. The observation of rolling the molecular conformation of Nef in its natural membrane environment, and identify plasma membrane curvature near hole-edges as a potential key event in address the impact of dimerization on Nef structure at the lipid bilayer. the plasma membrane repair (PMR) process. In the geometry of a membrane hole we propose that curvature leads to the formation of a characteristic neck 171-Plat structure around the hole as the first step in the repair process. Here we examine Molecular Interactions of the Matrix Domain of HIV-1 Gag Protein at the the phenomenon of annexin-induced membrane curvature near edges. This in- Membrane Interface cludes results obtained from model membranes, cells studies and theoretical Viviana Monje-Galvan. modeling of curvature near a hole. Together, the results provide an enhanced Department of Chemistry, The University of Chicago, Chicago, IL, USA. mechanistic insight into the role of annexins for initiating membrane repair. Binding and aggregation of the HIV-1 Gag protein on the plasma membrane (PM) enable budding and release of immature virons, which propagate the viral 174-Plat infection once they reach maturity. The matrix (MA) domain of the Gag protein Not Just Ionic Mimicry: Biophysics of Toxic Metal Ion Interactions with is responsible for Gag-membrane interactions through the myristate group Peripheral Membrane Targets (Myr), a fatty acid covalently attached to the N-terminus of the protein, and Tatyana I. Igumenova1, Taylor R. Cole1, Sachin Katti1, Krystal A. Morales1, a highly basic region (HBR) of residues located close to the Myr. The binding Samuel G. Erickson1, Min Woo Sung1, Sarah B. Nyenhuis2, Alexander B. Taylor3, P. John Hart3, Andreas Holzenburg4, David S. Cafiso2. mechanism and energetics of interaction between MA and the inner leaflet of 1 the PM are still uncertain. NMR studies suggest MA trimerization facilitates Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA, 2Department of Chemistry and Biophysics Program, University of Myr exposure from its sequestered conformation inside the hydrophobic pocket 3 of the MA domain, leading to Myr anchoring. Interactions between the HBR of Virginia, Charlottesville, VA, USA, Department of Biochemistry and MA and acidic lipids could also play key roles to enable Myr exposure, suggest- Structural Biology and the X-ray Crystallography Core Laboratory, ing the need of lipid domains for MA, therefore Gag, binding. We examined University of Texas Health Science Center at San Antonio, San Antonio, TX, USA, 4The University of Texas Rio Grande Valley, Brownsville, TX, USA. MA-membrane interactions with model membranes using enhanced sampling 2þ simulation techniques to gain basic understanding of its binding mechanism. Ca is a major regulator of signal transduction processes that occur at the We looked at the effect of lipid composition, specifically the presence of membrane surface. Toxic heavy metal ions interfere with these processes by targeting Ca2þ-sensing proteins in the cell. The conserved-homology 2 (C2) PIP2, on MA binding events as well as the role of Myr in this process. Metady- namics were used to understand the conformational changes that occur during domains, a class of independently folded peripheral membrane modules that > Myr exposure and MA trimerization that stabilize protein binding. are found in 100 proteins, are one of those targets. To understand the molec- ular mechanisms of heavy metal ion toxicity, we sought to determine how the 172-Plat interactions with Pb2þ, a potent neurotoxin, and Cd2þ, a known carcinogen, in- Oxidatively Stressed Mitochondrial Membranes: Insight into their Orga- fluence the structure, dynamics, and membrane interactions of three paradig- nization and Function during Apoptosis matic C2 domains: one from Protein Kinase Ca, and the other two from Artur P.G. Dingeldein1, Tobias Sparrman1,Jo¨rgen A˚ den1, Hanna Wacklin2, Synaptotagmin 1. To address these questions, we used an array of biophysical Radek Sachl 3, Sa´rka Pokorna´3, Martin Hof3, Gerhard Gro¨bner1. techniques, such as solution NMR spectroscopy, X-ray crystallography, SAXS, 1Chemistry, Biophysical Chemistry, Umea˚, Sweden, 2ESS, Lund, Sweden, 3J. EPR, and fluorescence spectroscopy. Our results revealed that the common Heyrovsky´ Institute of Physical Chemistry, Prague, Czech Republic. feature shared by the C2 domains is the presence of a high-affinity Pb2þ site Programmed cell death (apoptosis) is an essential mechanism in life. Key reg- whose population prevents Ca2þ from further binding, despite the existing co- ulators of the intrinsic mitochondrial apoptotic pathway are pro- and anti- ordination vacancies. We found that despite sharing the same binding site on apoptotic members of the Bcl-2 family who meet at the mitochondrion’s surface the C2 domains, Pb2þ and Cd2þ have distinct functional effects: while Pb2þ - as defined by its outer membrane system - where they arbitrate a life or death supports the membrane-binding function of C2 domains, Cd2þ does not – un- decision. Our main objective was to address this molecular regulation mecha- less extremely high local concentrations of lipids are present. The effect of nism occurring at this mitochondrial outer membrane (MOM). For this purpose Cd2þ and Pb2þ extends beyond structural ionic mimicry: through opportunistic we use the anti-apoptotic Bcl-2 protein itself which is an integral membrane pro- binding to the disordered linker region that brackets the C2 domain, Pb2þ and tein, and its counterpart, the pro-apoptotic Bax protein. Elevated oxidative stress Cd2þ promote the self-association of the regulatory region with the formation levels which initiate apoptosis, severely alter the mitochondrial membranes due of high-molecular weight oligomers that we characterized using NMR, SAXS, to the generation of oxidized lipids (OxPls). We could show that damaged mito- and EM. Our work illustrates the diversity of responses of signaling proteins to chondrial outer membranes - generated upon oxidative stress - increase the af- toxic metal ions and suggests a mechanism by which low concentrations of finity and therefore the translocation of Bax towards these membranes Pb2þ can interfere with the Ca2þ-dependent function of proteins in the cell.

BPJ 8544_8555 34a Sunday, February 18, 2018

Symposium: Membrane Bending: Mechanisms theoretical model, in vitro, and in vivo experiments, that reveals how a protein scaffold — a rigid sleeve made up of these proteins — can cut tubular mem- and Consequences branes connected to a quasi-flat membrane reservoir. We found that the scaf- fold — which assembles from protein subunits in solution onto the tube and 175-Symp near the junction with the reservoir— bound to the underlying tube creates a Stochastic Mechanisms in Membrane Traffic frictional barrier for lipid diffusion; tube elongation thus builds up local mem- Jeanne Stachowiak. brane tension until the membrane undergoes scission through lysis. As I will University of Texas at Austin, Austin, TX, USA. discuss, the lysis step was modeled as a stochastic pore nucleation event at Membrane traffic, an essential cellular process that plays a role in many human the scaffold/bare tube boundary that leads to scission. We call this mechanism diseases, requires key biophysical steps including formation of membrane friction-driven scission (FDS). In cells, motors pull tubes, in particular during buds, loading of these buds with specific molecular cargo, separation from endocytosis. Through reconstitution, we showed that motors can not only pull the parent membrane, and fusion with the target membrane. The prevailing out and extend protein-scaffolded tubes, but also cut them by FDS. view has been that structured protein motifs such as wedge-like amphipathic helices, crescent-shaped BAR domains, curved coats and constricting dynamin 178-Symp rings drive these processes. However, many proteins that contain these struc- Molecular Mechanisms of Membrane Remodeling tural motifs also contain large intrinsically disordered protein (IDP) domains Ralf Langen. of 300-1500 amino acids, including many clathrin and COPII coat components. Inst Gen Med, University of Southern California, Los Angeles, CA, USA. While these IDP domains have been regarded primarily as flexible biochemical Membrane remodeling plays an active role in many membrane-related pro- scaffolds, we have recently discovered that IDPs are highly efficient physical cesses, including membrane fusion and fission. Recent evidence implicates a drivers of membrane budding. Further, our work demonstrates that IDP do- number of proteins in this remodeling processes. The central goal of our studies mains serve as strong drivers of membrane fission. How can molecules without is to identify the molecular mechanisms by which proteins can remodel cellular a defined structure drive membrane budding and fission? Our results support membranes and how this ability can be regulated. Toward this end, we have the idea that disordered domains generate entropic pressure at membrane sur- used a combination of biophysical methods including electron microscopy faces, which is critical to overcoming key biophysical barriers to membrane and electron paramagnetic resonance to determine the structural features of traffic. IDPs are particularly efficient generators of entropic pressure owing membrane curving proteins bound to membranes of defined curvatures. Of to their very large hydrodynamic radii, potential for electrostatic repulsion particular interest have been BAR-domain containing proteins, dynamin-like owing to high net charge, and the substantial entropic cost of extending proteins, as well as a number of proteins involved in amyloid diseases. The them. More broadly our findings suggest that any protein, regardless of struc- seemingly simplest membrane remodeling proteins were the amyloidogenic ture, can contribute to membrane remodeling by increasing entropic pressure, proteins alpha-synuclein and IAPP, where the membrane curvature induction and paradoxically, that proteins that lack a defined secondary structure, IDPs, coincided with the formation of amphipathic alpha-helices that are likely to may be among the most potent drivers of membrane traffic. Our ongoing act as wedges in the presence of membranes. While some of the BAR proteins work focuses on understanding how entropic pressure influences membrane (amphiphysin and endophilin) also use amphipathic helices for generation of traffic, and designing biophysical tools for manipulating receptor recycling membrane curvature, we find different structures are taken up depending on and signaling. what type of membrane curvature is induced. In particular, we find that tubular structures are more strongly stabilized by scaffolding than vesicular structures. 176-Symp Finally, we have investigated how the aforementioned membrane interactions Flipping Helices: Membrane Insertion of Amphiphilic Helices and Extru- are controlled by post-translational modifications. sion of Transmembrane Segments Torsten Walther, Lena Steger, Erik Strandberg, Ariadna Grau Campistany, Symposium: Channel Mechanisms: Sensing and Parvesh Wadhwani, Benjamin Zimpfer, Jochen Burck,€ Dirk Windisch, Katharina Becker, Stephan Grage, Johannes Reichert, Sergiy Afonin, Gating Anne S. Ulrich. 179-Symp IOC & IBG-2, Karlsruhe Institute of Technology, Karlsruhe, Germany. Molecular Rearrangements Underlying Function of Large Conductance It is commonly perceived that (a) amphiphilic peptides bind to the membrane Calcium- and Voltage-Regulated Potassium Channels surface, while (b) hydrophobic transmembrane protein segments traverse the Teresa Giraldez1,2. lipid bilayer. Here, we will demonstrate several cases and conditions where 1Basic Medical Sciences, Universidad de La Laguna, Tenerife, Spain, these scenarios can be reversed due to the flipping of membrane-bound a-heli- 2Institute of Biomedical Technologies, Tenerife, Spain. ces or b-hairpins. þ þ Large conductance Ca2 - and voltage-gated K channels (BK, hslo or KCa1.1) With regard to (a), some straightforward examples include the insertion of anti- are high conductance potassium channels that are synergistically regulated by microbial peptides into lipid bilayers to form oligomeric pores, or the folding þ þ voltage and intracellular Ca2 . Numerous studies have identified the Ca2 and membrane insertion of b-barrel proteins [1]. Having systematically studied binding sites within an intracellular structure known as the gating ring. Howev- the physicochemical parameters that need to be fulfilled in order to reach and er, many questions remain about the complex structural rearrangements stabilize such inserted state, we found that the spontaneous lipid curvature þ involved in coupling Ca2 binding to channel gating. Information about the plays a critical role, besides hydrophobic mismatch [2,3]. Other features that pore region, voltage sensing domain or isolated intracellular domains has can stabilize amphiphilic structures in a transmembrane alignment are polar in- been obtained separately using electrophysiology, biochemistry and crystallog- teractions between helices, such as charge zippers or H-bond zippers [4,5]. raphy. Using a combination of imaging and electrophysiology, which we corre- Concerning (b), we will present some examples and evidence where an uncon- late with protein structure analysis, we have investigated the structural ventional flipping of largely hydrophobic ‘‘transmembrane’’ segments onto the þ dynamics underlying the molecular coupling of Ca2 , voltage and activation membrane surface seems to be intimately related to protein function [unpub- of BK channels in the membrane environment, its regulation by accessory sub- lished]. Again, hydrophobic mismatch and lipid shape play a major role, as units and channel effectors. well as the distribution of polar residues. References: Giraldez et al. JGP 2005, 126:429; Miranda et al. PNAS [1] Strandberg et al. (2015) Biochim. Biophys. Acta, 1848: 1944. [2] Grau- 2013, 110:5217; Miranda et al. PNAS 2016, 113(49), 14055. Giraldez and Campistany et al. (2016) J. Phys. Chem. Lett. 7, 1116. [3] Grau-Campistany Rothberg, JGP 2017, 149:431. Kshatri et al 2017, BBA doi:10.1016/ et al. (2015) Sci. Rep., 5: 9388. [4] Walther et al. (2014) Curr. Opinin. Struct. j.bbamem.2017.09.023. Biol., 27: 63. [5] Walther et al. (2013) Cell, 152: 316. Funding:European Research Council ERC-CoG-2014-648936 (NANOPDICS). 177-Symp 180-Symp Friction-Driven Scission of Membrane Tubes Structure and Mechanisms of Selectivity Gating, Inhibition and Activation Andrew Callan-Jones. in an Ion Channel Laboratoire Matie`re et Syste`mes Complexes, Universite Paris-Diderot, Paris, Robert Stroud, Alexander F. Kintzer. France. Biochemistry & Biophysics, University of California, San Francisco, San Membrane scission is essential for intracellular trafficking. Whereas curved Francisco, CA, USA. BAR domain proteins such as endophilin have been reported to be involved The X-ray crystal structure of a voltage gated intracellular channel reveals the in dynamin-independent scission of tubular membrane necks, the cutting mech- mechanism of ion permeation, calcium activation and inhibition, location of anism remained unknown. In this talk, I will present recent work, combining a phosphoregulatory sites and the first resting state voltage-sensing domain in

BPJ 8544_8555 Sunday, February 18, 2018 35a an ion channel. Our structure suggests a mechanism for channel opening, most contribute to this electric field, with the positioned general base (residue whereby Ca2þ concentrations, voltage, and phosphoregulation are integrated 40) contributing only about 20%.[1] through conformational changes in the voltage sensing domains, selectivity fil- Next, I will present our strategy to exploit these electric field calculations to ter, gate, N-terminal domains and C-terminal domains to govern the conduction improve the catalytic activity of the computationally designed Kemp Eliminase of ions. Acknowledgements: We are grateful to J. M. Holton and G. Meigs at KE15. I will explain how we identified mutations that enhance electric fields to ALS beamline 8.3.1 Work supported by NIH grant GM24485 to R.M.S. Beam- contribute to free energy stabilization of the transition state. Starting from the 1 1 line 8.3.1 at the Advanced Light Source is operated by the University of Cali- design that has a kcat/KM of 27M s , our most improved quadruple mutant 1 1 fornia Office of the President, Multicampus Research Programs and Initiatives exhibits a kcat/KM of 403 M s .[2] Most importantly, almost all of the grant MR-15-328599. enzyme improvement comes through a 43-fold improvement in kcat, indicative of a direct impact on the chemical step. 181-Symp [1] ‘‘The role of electric fields in Ketosteroid Isomerase’’, in preparation [2] Insights into Gating of GIRK (KIR3) Channels through G Protein- ‘‘Computational Optimization of Electric Fields for Improving Catalysis of a Independent Pathways Designed Kemp Eliminase’’, submitted Paul A. Slesinger. Dept. Neuroscience, Icahn School of Medicine at Mount Sinai, New York, 184-Plat NY, USA. Designed Enzymes: Creating a more Efficient Nitric Oxide Dioxygenase Activation of G protein-gated inwardly rectifying potassium (GIRK) channels Mia C. Brown, Kelly Greenland, Lei Zhang, Ronald L. Koder. alters the excitability of cardiac and brain cells. GIRK channels were first City College of New York, New York, NY, USA. We have designed an artificial nitric oxide dioxygenase (NOD) which, when described as K channels that are activated directly by G protein Gbg subunits. þ GIRK channels are also modulated by small molecules but much less is known paired with ferredoxin NADPH oxidoreductase (FNR) as an electron donor, about the underlying gating mechanisms. One drawback to previous studies has transforms nitric oxide into nitrate ions at rates comparable to that of natural been the inability to control intrinsic and extrinsic factors. Here, we describe NOD enzymes. Nitric oxide has been implicated in several neurodegenerative recent studies investigating the molecular interactions of alcohol, cholesterol disorders, and unwanted NOD activity has been identified as a major problem in artificial blood substitutes. We have characterized the binding affinities and and the membrane phospholipid PIP2 that underlie GIRK channel activation in the absence of G proteins. the kinetics of each step along the reaction pathway and competing off- pathways. While capable of acting as a NOD, HFHF has several deficiencies: 182-Symp poor selectivity in gaseous binding and a slow rate of electron transfer to the Can KD be Conducted through a Narrow Pore? Investigating the role of porphyrin cofactor from FNR. As we found electron transfer to the porphyrin Conformational Change in Gating Kir Channels cofactor to be rate-limiting, we created an improved NOD by covalently linking Jacqueline M. Gulbis1, David M. Miller1, Katrina Black1, Adam P. Hill2, the artificial hemoglobin directly to an electron donor. Building upon the orig- Derek Laver3. inal design, we combined a natural and a designed protein to create a chimera in 1Structural Biology Division, Walter and Eliza Hall Institute of Medical which the diflavin domain of cytochrome P-450 BM3 is connected to a variant Research, Victoria, Australia, 2Computational Cardiology Laboratory, Victor of our designed enzyme that contains an additional porphyrin binding site in- Chang Cardiac Research Institute, Darlinghurst, NSW, Australia, 3School of tended to relay electrons from the diflavin domain to the site of NOD activity. Biomedical Sciences and Pharmacy, The University of Newcastle, This single chain protein contains one FMN, one FAD, and two porphyrin co- Newcastle, NSW, Australia. factors. The chimera reacts with NADPH, taking in its two electrons at the FAD Potassium channels are gates to passive Kþ diffusion across cell membranes, cofactor, breaking them into single electrons at the FMN cofactor, and then regulating conduction in response to cellular signals. Their exquisite selectivity transferring them into the artificial porphyrin domain. This construct increases for Kþ over other cations is achieved by direct interaction between Kþ and the the electron transfer rate to the porphyrin by more than an order of magnitude. ion selectivity filter, wherein each Kþ is coordinated by backbone carbonyls and, at the innermost of the four binding sites, threonine hydroxyls. Outside 185-Plat the selectivity filter, these carbonyl ligands are replaced by six to eight water A Bayesian Integrative Structure Model of the Yeast Centrosome þ Shruthi Viswanath1, Massimiliano Bonomi2, Seung Joong Kim1, molecules, such that K ions diffuse fully hydrated between selectivity filter 3 4 5 5 and cytosol. In keeping with this, the perception of gating is of reversible steric Vadim A. Klenchin , Keenan Taylor , King C. Yabut , Neil T. Umbreit , Janet Meehl6, Michele H. Jones6, Javier Velazquez-Muriel1, Mark Winey6, occlusion of the ion conduction pathway, with the pore alternating between Ivan Rayment3, Trisha N. Davis5, Andrej Sali1, Eric D. Muller5. wide ‘open’ and narrow ‘closed’ conformations at a constriction point known 1Bioengineering and Therapeutic Sciences, UCSF, San Francisco, CA, USA, as the ‘helix bundle crossing’. Within the constraints of this gating model, a 2 3 requirement for activation and conduction is that the pore expands (relative University of Cambridge, Cambridge, United Kingdom, University of Wisconsin, Madison, WI, USA, 4University of Cambridge, Madison, WI, to resting channels) to at least the breadth of a hydrated ion (approximately 8 5 6 A˚ ). To investigate this, we have embarked upon a coordinated structure- USA, University of Washington, Seattle, WA, USA, University of function approach utilising the inward rectifier KirBac3.1 as the subject. The Colorado, Boulder, CO, USA. Microtubule organizing centers (MTOCs) form, anchor and stabilize the polar- talk will describe the approach, its challenges and some unexpected results. ized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and during mitosis assembles a bipolar Platform: Protein Structure, Prediction, and spindle to capture and segregate sister chromatids. Yet, despite their impor- tance in cell biology, the physical structure of MTOCs is poorly understood. Design Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in 183-Plat vivo FRET, small-angle X-ray scattering (SAXS), X-ray crystallography, elec- Electric Field Optimization in Enzymes tron microscopy and two-hybrid analysis. The model is validated by several Valerie Vaissier. methods that include a genetic analysis of the conserved PACT domain that re- University of California, Berkeley, Berkeley, CA, USA. cruits Spc110, a protein related to pericentrin, to the SPB. The model suggests The catalytic efficiency of enzymes is difficult to rationalize due to the inherent that calmodulin can act as a protein cross-linker and Spc29 is an extended, flex- complexity of the complexes they form with their substrate. Kinetic studies, ible protein. The model led to the identification of a single, essential heptad in with the determination of the Michaelis Menten parameters (KM and kcat)in the coiled coil of Spc110 and a minimal PACT domain. It also led to a proposed particular, greatly contribute to the understanding of enzymatic catalysis. How- pathway for the integration of Spc110 into the SPB. ever, a detailed molecular picture is missing, preventing us from designing cat- alysts that can rival natural enzymes. In this talk, I will present atomistic 186-Plat simulations of enzyme-substrate complexes, from which we calculate electric Coiled Coil Proteins as Scaffold for Broad Range, Ultrafast Biosensors fields projected onto the bonds that break and form during the reaction, a Ameed Hashmi, Florence R. Lucey, Mourad Sadqi, Victor Mun˜oz. descriptor that correlates with kcat. University of California, Merced, CA, USA. I will start by looking at the natural enzyme Ketosteroid Isomerase (KSI) and its Proteins that toggle between two states (unfolded/folded) upon specific binding inhibitor 19-norstestosterone, for which electric fields have been estimated to a target molecule, behave as conformational switches. Devices based on experimentally. I will show how our calculated electric fields are (i) in quanti- these proteins can be used for developing real-time sensors. Our work takes tative agreement with experimental data and (ii) correlates with measured cat- this approach forward to design high-performance conformational rheostat sen- alytic efficiency, validating our method. Further, I will identify the residues that sors. The rationale is to develop sensors with expanded dynamic range and

BPJ 8544_8555 36a Sunday, February 18, 2018 faster response time by coupling, binding to the folding process of fast folding features of interest is cotranslational protein folding. Traditional ab initio pro- protein modules. As proof of concept we investigate the pH sensing capabilities tein structure prediction takes place by performing small perturbations on a of anti-parallel coiled-coil proteins. Our results reveal that such a pH sensor ex- fully-elongated sequence. We implemented a sequential approach in SAINT2 hibits a linear response over 4 orders of magnitude in analyte concentration, akin to cotranslational folding, in which the perturbations are performed on a compared to the 2 orders of magnitude for switches. growing peptide. We performed an unbiased comparison between SAINT2 in its sequential and non-sequential modes, and show that sequential protein struc- 187-Plat ture prediction is faster and more efficient than the traditional non-sequential Molecular Design and X-Ray Crystal Structure of Engineered Phospho- approach. We provide a mathematical rationale as to why this is the case. lamban Transmembrane Variant Finally, we contextualise our results in light of the most recent Critical Assess- Marco Mravic, J. Thomaston, William F. DeGrado. ment of protein Structure Prediction (CASP) experiment, where our postdiction University of California, San Francisco, San Francisco, CA, USA. results suggest that SAINT2 can produce models of comparable quality to the Phospholamban is an oligomeric transmembrane (TM) protein thoroughly stud- state-of-the-art. Our research paves the way for exploring our full understand- ied as a contractility regulator in cardiomyopathies and as a model system for ing of protein folding in the context of protein structure prediction. spectroscopy and protein folding. While phospholamban forms a narrow apolar channel, it debated given conflicting biochemical studies and NMR-based 190-Plat models whether this protein simply regulates the SERCA2 Ca2þ pump or also Predicting Protein Contact Maps Directly from Primary Sequence without conducts ions. Additionally, the ‘‘Leu-Ile zipper’’ motif at its core poses a puz- the Need for Homologs zling paradigm in TM protein folding: how does hydrophobic side chain packing Thrasyvoulos Karydis, Joseph M. Jacobson. drive association of alpha-helices in the already hydrophobic lipid bilayer. Media Lab, MIT, Cambridge, MA, USA. Meanwhile, phospholamban’s X-ray crystal structure has not been reported, Predicting the three-dimensional structure of protein directly from its amino but would provide invaluable answers. We re-designed this protein, guided by acid sequence remains one of the most challenging problems in computational simulation, and solved the variant’s structure. First, we conducted 300 ns all- biology. A relaxed version of the problem is when some contacts between pro- atom molecular dynamics simulations in lipid bilayers to assess the structural ri- tein residues are known and used as constraints in the energy minimization pro- gidity and pore size of the phospholamban TM domain from a reported NMR- cess of protein folding. In recent work, protein contacts prediction methods based model. We observed the channel to widen, but remain dehydrated. The showed notable progress, in particular by the use of novel deep learning algo- N-terminal Leu-Ile zipper motif remained very rigid (<1.5 A˚ RMSD), while rithms. In all of the current frameworks, though, a significant number of its C-terminal polar region was dynamic and splayed open forming a water- sequence homologs are needed as an input to the neural networks, for the con- filled pore. Our engineered variant PL5 (50% similarity) has an extended Leu- tact prediction algorithm to provide enough long-range constraints to the fold Ile zipper to replace the C-terminal polar region. In simulation, the now apolar the protein correctly. The latter renders it impossible to use these methods to C-terminus was rigidified and maintained the narrow dehydrated pore as previ- fold proteins where there are very few or low-variance homologs found in pro- ously observed. We showed PL5 is an alpha-helical coiled-coil pentamer in so- tein sequence databases. We overcome this limitation and introduce a novel lution by analytical ultracentrifugation and CD spectroscopy in micelles. We deep learning method to reconstruct the protein contact map from an amino- ˚ then solved the X-ray crystal of PL5 in C8E4 micelles (3.17 A), which is in strik- acid sequence, without the need for homology. To achieve that, we begin by ing agreement to the design model: 1.16 A˚ RMSD. Our data shows PL5 is a reli- decomposing the entire collection of known protein sequences and structures able model for phospholamban and the Leu-Ile zipper motif, and suggests into a set of hierarchical sequence and structural motifs, respectively. We sub- phospholamban forms a dehydrated channel intermediate in size between the sequently use the extracted motif representation to embed the sequences and two reported NMR models unlikely to conduct ions. structures into latent spaces and train a meta-model to associate a sequence embedding with a structural embedding. Finally, we deconvolve the structural 188-Plat embedding into a two-dimensional contact map and proceed with contact- Mainmast: De Novo Main-Chain Model Tracing for EM Maps using Tree- assisted protein folding. In this work, we build on top of our sequence embed- Graph Optimization Method ding framework, CoMET, developing the structural embedding pipeline and Genki Terashi1, Daisuke Kihara2. 1 train the end-to-end framework to characterize the fidelity of the predicted con- Biological Sciences, Purdue University, West Lafayette, IN, USA, tact maps for protein folding. 2Biological&Computer Sciences, Purdue University, West Lafayette, IN, USA. An increasing number of protein structures are determined by cryo-electron mi- Platform: Cardiac Muscle Mechanics, Structure, croscopy (cryo-EM) at near atomic resolution. However, tracing the main- chains and building full-atom models from EM maps of 4-5 A˚ is still not triv- and Regulation I ial and a demanding task. Here, we introduce a novel de novo structure modeling method MAINMAST (MAINchin Model trAcing from Spanning 191-Plat Tree) that builds an entire three-dimensional model of a protein from a near- Allosteric Modulation of Cardiac Myosin Dynamics by Omecamtiv atomic resolution EM map. The method directly traces the main-chain and Mecarbil identifies Ca positions as tree-graph structures in the EM map. The method Shaima Hashem, Matteo Tiberti, Arianna Fornili. has substantial advantages over the existing methods: i) MAINMAST directly School of Biological and Chemical Sciences, Queen Mary, University of constructs protein structure models from an EM density map without requiring London, London, United Kingdom. reference structures; ii) The procedure is fully automated and no manual setting New strategies for the pharmacological treatment of heart muscle diseases rely is required; iii) a pool of models are produced, from which a confidence score is on molecules that can directly bind to sarcomeric proteins and either inhibit or computed that indicates accuracy of structure regions. We tested MAINMAST enhance their activity. Compared to traditional drugs, direct sarcomeric modu- on 40 simulated density maps at 5A˚ resolution and 30 experimentally deter- lators have reduced side effects and a higher potential to be tailored to specific mined maps at 4-5 A˚ resolution and showed that MAINMAST performed disease phenotypes. significantly better than existing software. Omecamtiv Mecarbil (OM) is a sarcomeric modulator of cardiac myosin that is currently being tested in clinical trials for the treatment of heart failure. While 189-Plat the overall effect of OM is an increased contractility of the cardiac muscle, its Exploring Folding Features in Protein Structure Prediction molecular mechanism of action is still elusive. Here, we present an in silico Saulo H.P. de Oliveira, Charlotte M. Deane. study of the cardiac motor domain bound to OM, where the effects of the Department of Statistics, University of Oxford, Oxford, United Kingdom. drug on the dynamical properties of the protein are investigated for the first While the boundaries of our understanding of how proteins fold have expanded, time with atomistic resolution using Molecular Dynamics simulations. computational approaches for structure prediction have struggled to keep up. We found that OM binding has a double effect on myosin dynamics, since a) it Experimental evidence has shown us that all proteins, at some level, fold co- induces an increased coupling of the converter and lever arm subdomains to the translationally. Stable intermediates occurring during translation have been rest of the protein, resulting in a strong reduction in the amplitude of their mo- experimentally characterised and translational kinetics has been shown to tions and b) it rewires the network of dynamic correlations within the motor play a key role in accurate folding. Nonetheless, structure prediction software domain, producing preferential communication pathways between the OM has traditionally disregarded our knowledge of the mechanistics of protein binding site and functional regions in the U50K subdomain. Based on these folding. To address this issue, we have implemented SAINT2, a protein struc- findings, we suggest that the development of improved drugs could be achieved ture prediction protocol. We used SAINT2 to assess whether the incorporation by changing the balance between the two types of OM-induced effects on of folding features leads to improvements in structure prediction. One of our myosin dynamics.

BPJ 8544_8555 Sunday, February 18, 2018 37a

This research is supported by the British Heart Foundation and the UK High- 194-Plat End Computing Consortium for Biomolecular Simulation, HECBioSim. High-Throughput Screening for Actin-Binding Compounds that Affect Actomyosin Structure and Function using Time-Resolved FRET 192-Plat Piyali Guhathakurta1, Ewa Prochniewicz1, Kurt C. Peterson2, Phosphoinositide-Mediated Myosin-1 Membrane Targeting during Endo- Benjamin D. Grant2, Gregory D. Gillispie2, David D. Thomas1. cytosis 1Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 1 2 1 Girish Rajendraprasad , Tim Scholz , Matthias Preller , Minneapolis, MN, USA, 2Fluorescence Innovations Inc, Minneapolis, MN, 1 Georgios Tsiavaliaris . USA. 1Biophysical Chemistry, Hannover Medical School, Hannover, Germany, 2 We have used a novel fluorescence resonance energy transfer (FRET) based Molecular and Cell Physiology, Hannover Medical School, Hannover, assay to detect small-molecule modulators that alter actin-myosin structure Germany. and function. Interaction between actin and myosin plays a crucial role in mus- Cells couple the dynamic organization of the cytoskeleton with changes in mem- cle functions. Several mutations, both in actin or in myosin, disrupt the normal brane shape to direct cell polarity and facilitate endocytosis and intracellular traf- skeletal and cardiac muscle functions and cause life-threatening syndromes. ficking. Class-1 myosins play key roles in these processes as they act as motorized Despite extensive efforts, treatment options remain limited. We utilized a cross-linkers between the cytoskeleton and membrane systems, generating the FRET biosensor to identify modulators that bind to the actomyosin interface forces required for remodeling the membrane. Myosin-1 lipid associations are pri- and alter structural dynamics of actomyosin. We have attached a fluorescent marily electrostatic and mediated via the PH-domain found in the tails of the mo- donor (fluorescein-5-maleimide) to actin at C374, and a non-fluorescent tors. However, little is known about the specificity of direct phospholipid acceptor (Dabcyl) at the N-terminus of a peptide (DNT) containing the 12 N- associations and the mechanisms regulating myosin-1 activities at the terminal amino acids of myosin’s long essential light chain isoform. The membrane-cytoskeleton interface. One less well understood issue is how DNT binding site on actin overlaps with that of myosin, as indicated by (a) a myosin-1s specifically target the membranes, how they switch and differentiate similar distance observed in the actin-DNT complex as in the actin-myosin between various membrane systems e.g. cortical membrane, membrane protru- complex and (b) a significant decrease in actin-DNT FRET upon binding sions, endosomal vesicles, and whether different isoforms have distinct or over- myosin. A high-throughput FRET screen of a small-molecule library (NCC) lapping roles in the process of endocytosis they have been implicated in. showed that actin-DNT FRET is significantly affected by several compounds. Following the hypothesis that the modular architecture of the class-1 myosin A significant number of these ‘‘Hit’’ compounds inhibit actin-activated myosin tail combines all features for targeting the motors to specific subcellular locations, ATPase and affect the microsecond dynamics of actin as detected by transient we performed a comparative study with three long-tailed class-1 myosins in vitro phosphorescence anisotropy. We conclude that the actin-DNT system allows and within cells. By combining fluorescence-based protein-lipid and protein- detection of the effects of pharmacologically active compounds on actin struc- cytoskeleton interaction assays including TIRF-microscopy-based single mole- tural states and consequently on functional properties of actomyosin. This assay cule experiments with recombinant protein and proteins fragments, we achieved sets the groundwork for a large-scale campaign of small-molecule screening, to to specify and quantify the distinct binding abilities of myosin-1s towards phos- develop needed therapies to treat actomyosin-based muscle disorders. phoinositides and filamentous actin. By confocal live cell imaging we show how myosin-1s cooperate during different stages of endocytosis, from the formation of 195-Plat an endocytic cup till subsequent endosome maturation. Based on these results and Controlling Cardiac Contractility at the Single Molecule Level confirmed by cellular inhibition experiments including theoretical considerations Chao Liu, Dan L. Song, Masataka Kawana, Kathleen M. Ruppel, of the binding kinetics and structural modeling, we present a model that describes James A. Spudich. a phosphoinosited-based targeting mechanism of myosin-1s in response to Biochemistry, Stanford University, Stanford, CA, USA. changes in PIP concentrations important for fine-tuning the subcellular localiza- Previous studies have found that cardiomyopathy-causing mutations and small tion of the motors during endocytosis in a temporally controlled manner. molecule drugs can alter contractility of the heart muscle. However, the molec- 193-Plat ular mechanism underlying this modulation is unclear. A key parameter of car- The Molecular Defects in Ca2D Regulation due to Mutations that Cause diac contractility at the molecular level is the detachment rate of cardiac Hypertrophic Cardiomyopathy can be Reversed by Small Molecules that myosin, the motor that produces force during muscle contraction. This rate de- Bind to Troponin termines the time that myosin is bound to actin in a force-producing state, and it Steven B. Marston1, Andrew E. Messer1, Juan Eiros-Zamora2, Ian Gould2, depends on the load (force) against which myosin works. Measurement of the Maria Papadaki3, Afnan Choudry1, Alice Sheehan1. load-dependent detachment rate of cardiac myosin under physiological ATP 1NHLI, Imperial College London, London, United Kingdom, 2Chemistry, conditions (2 mM) had been challenging due to its fast kinetics until the devel- Imperial College London, London, United Kingdom, 3Physiology, Loyola opment of Harmonic Force Spectroscopy (HFS) in the Spudich lab for optical University Medical School, Chicago, IL, USA. trapping, which presented a simple and efficient solution without the need for Mutations in contractile proteins can cause familial hypertrophic cardiomyop- fast feedback. Here, we measured the detachment rate of single molecules of athy (HCM) or familial dilated cardiomyopathy (DCM). HCM has been linked human b-cardiac myosin under load using HFS. We found that the rate could to a higher myofilament Ca2þ sensitivity. In addition we have identified a mo- be modulated to various extents by both small molecule compounds and lecular level dysfunction common to both HCM and DCM-causing mutations. cardiomyopathy-causing mutations, and effects of mutations could be reversed This is an uncoupling of the relationship between troponin I (TnI) phosphory- by introducing appropriate compounds. Furthermore, we discuss implications lation and modulation of myofilament Ca2þ-sensitivity, essential for normal re- for cardiac myosin force production under the various perturbations. Our re- sponses to adrenaline. Adrenergic response is blunted in vivo which sults suggest that cardiac contractility can be controlled by tuning the load- predisposes to heart failure under stress. We have identified compounds that dependent kinetics of single myosin molecules. can specifically reverse these abnormalities in vitro and therefore have potential for treatment. Based on our lead compound, Epigallocatechin-3-Gallate 196-Plat (EGCG), we examined 40 compounds: variants of EGCG lacking the pyrogal- Non-Linear Model for Mechanical Entrainment of Cardiomyocytes lol ring, variants of EGCG lacking the galloyl ring, silybin, its variants and ste- Ohad Cohen, Samuel A. Safran. reoisomers and unrelated Hsp90 inhibitors and Ca2þ-desensitisers. We found Chemical and Biological Phyiscs, Weizmann Institute, Rehovot, Israel. 23 compounds that reversed the uncoupling; many of these can re-couple inde- Recent experiment by the group of Tzlil (Nitsan et al., Nat. Phys. 2016) have pendent of Ca2þ-desensitization. 3 compounds desensitized but did not recou- shown that nearby cardiac cells seeded ( 100 micrometers apart) on an elastic ple, one compound has the reverse effect (P-TnI had higher Ca2þ-sensitivity gel, synchronize their beating phase and frequency even without direct contact. than unP TnI). We mapped EGCG, Silybin A and Silybin B binding to whole By introducing an inert probe that induced periodic elastic deformations in the troponin by molecular dynamics simulations and found that they are usually substrate, the experiments showed that one can pace beating cardiac cells that located between the N-terminal phosphorylatable peptide of TnI and the N-ter- are relatively far from the probe. The time required to pace the cell was on the minal Ca2þ regulatory domain of TnC and differentially alter troponin dy- order of 15 min, and the cell maintained the new beating frequency for as namics. We have proposed a 4-state model to account for coupling, long as 1 hr after the probe was removed. These long time scales are in com- uncoupling and recoupling. We have established a biological assay platform plete contrast to the very short time scales ( 1 sec) that characterize relaxation for screening EGCG and related analogues in intact cardiomyocytes to study after electrical stimulation is removed. their effects on contractile regulation in vivo, using an E99K ACTC We construct a simple, analytical model based on the works of Julicher and heterozygous-mutant HCM mouse model. Preliminary findings suggest that Duke (Duke et al., PNAS, 2000), and treat the deterministic dynamics of the response to dobutamine is blunted and that recoupling molecules appear beating. The model predicts spontaneous, entrained beating (with the probe fre- to restore the adrenergic response. quency) and ‘‘bursting’’ (short periods of entrainment to the probe separated by

BPJ 8544_8555 38a Sunday, February 18, 2018 quiescence) of paced cells, and how these depend on the probe amplitude and crease force development, but only within a small concentration range. These frequency, in agreement with experiment (Nitsan et al., Nat. Phys. 2016). We findings are consistent with our hypothesis that pre-activation can increase further consider the interesting effects of small noise on the non-linear oscil- force development. Highly compliant titin allows cells to function at higher dia- lator model of the beating cell, and show how it affects the coherence of stolic Ca2þ, but this is expected to result in limited contractile reserve. beating. Finally, we predict the dependence of time required for a cell to tran- sition from spontaneous to entrained beating once the probe is applied as well as its dependence on the probe amplitude. We account for the origin of the Platform: Voltage-gated Na and Ca Channels much longer time scale (minutes) required to entrain spontaneously beating cells by considering biological adaptation (which delays the response of the 199-Plat cell to the external signal). Fenestration Mutants of a Voltage-Gated Sodium Channel that Modify Channel Blocker Ingress 197-Plat Giulia Montini, Altin Sula, Andrew J. Miles, B.A. Wallace. Substrate Stiffness and Work Affects Myocyte Hypertrophy and CapZ Dy- Institute of Structural and Molecular Biology, Birkbeck College, University namics via PKC-Epsilon and PIP2 Signaling Pathways of London, London, United Kingdom. Christopher Solı´s1, Michael Mkrtschjan2, Brenda Russell1. The high-resolution crystal structure of the voltage-gated sodium channel pore 1Department of Physiology and Biophysics, University of Illinois at Chicago, of NavMs from M. marinus (Bagneris et al, 2014) was previously solved in the Chicago, IL, USA, 2Department of Bioengineering, University of Illinois at presence and absence of channel blocker drugs. In this study, mutants were de- Chicago, Chicago, IL, USA. signed, expressed and characterised in order to block the transmembrane fenes- Cardiac muscle remodeling is a physiological response to mechanical and trations that are proposed to enable ingress of drugs into the hydrophobilic neurohumoral signaling. Physiological hypertrophy is an adaptation to binding cavity, and which subsequently block sodium ion translocation through increasing cardiac output demand, like exercise. However, pathological hyper- the pore. Guided by the HOLE analysis programme (Smart et al, 1996) we iden- trophy is caused by decompensated mechano-signaling The hypothesis tested is tified critical residues in helix S6 adjacent to the fenestration. We were then that the physiologic or pathologic mechanical environment controls PKC 3 and able to produce a number of mutants with side chains of different sizes, aimed PIP2 signaling pathways leading to muscle differential remodeling. Here, at narrowing or blocking the fenestration, in order to prevent drug entry into the chronic loading by substrate stiffness provides the pathological stimulus while internal pore cavity. acute additional drug treatment increases workload physiologically. Neonatal Using circular dichroism (CD) spectroscopy we identified whether the mutants rat ventricular myocytes (NRVMs) were cultured under varying substrate stiff- had any differential effect on channel stability in the presence/absence of the ness that resemble physiological conditions (10 kPa) or cardiac fibrosis (100 drugs, and then we used X-ray crystallography to determine the high resolution kPa and glass). Localization of PIP2 and PKC 3 was observed and the redistri- structures of the fenestration mutants. bution compared with additional physiologic workload mimicked by 15-min- Supported by grants from the BBSRC. ute, 1-hour, or 24-hour treatment with isoproterenol. Actin dynamics, 200-Plat assessed by fluorescence recovery after photobleaching (FRAP) using actin- Characterization of Photoswitchable Sodium Channel Inhibitors by GFP, were altered by changes in workload. Chronic stiffness and isoproterenol Planar Patch Clamp treatment increased kFRAP significantly compared to untreated cells on 10 kPa Nils Winter1, Andrea Bruggemann€ 2, Claudia Haarmann2, Michael George2, < 3 (p 0.05). Immunostaining confirmed that PKC and PIP2 localize with a-ac- Niels Fertig2, Martin Sumser1, Dirk Trauner3. tinin at the Z-disk. PKC 3 immunostains showed total (tPKC )3 and phosphory- 1Dep. Chemistry, LMU Munich, Munich, Germany, 2Nanion Technologies, lated PKC 3 (pPKC )3 in the Z-lines; however, only pPKC 3 demonstrated a time- Munich, Germany, 3Dept. of Chemistry, NYU, New York, NY, USA. dependent response on 10 kPa substrates. PIP2 colocalization in the Z-lines Photopharmacology is an emerging technique for applications in biophysics changed with increasing substrate stiffness and PIP2 localization was only and medicine without the need of genetic manipulations. The energy of light diminished by neomycin on 10 kPa substrates. FRET-based colocalization as- is used to change the shape and pharmacological properties of molecules, re- says indicated that PIP2 directly interacts with CapZ. PIP2 interactions with sulting in different biological activities. By introducing a photoswitch, the CapZ were predicted using a ligand docking software. PIP2 binds CapZ in molecule has two different states between which it can be switched with light. two regions: the actin binding phase and a cleft conformed by a hydrophobic Since both states have a different structure, the activity of the molecule on the pocket in the a-subunit and the b-tentacle. In conclusion, PKCe and PIP2 are target can be changed with a light pulse. involved in Z-disc regulatory pathways that dictate pathological and physiolog- Here we show the high-throughput characterization of different classes of so- ical response to mechanical and neurohumoral signaling. Funding HL 62426. dium channel inhibitors and their photoregulation. For the characterization a planar patch clamp system (SyncroPatch 384PE) with 96 built in diodes was 198-Plat used to illuminate the 384 patched cells. In this configuration several concen- Pre-Activation of Cardiomyocytes Determines Contractile Force and trations of compounds and ion channels could be monitored at the same time. Speed of Contraction; Role of Titin and Calcium The light was applied to all cells simultaneously. We describe a new class of Diederik W. Kuster, Michiel Helmes, Aref Najafi, Maike Schuldt, photoswitchable sodium channel inhibitors. These molecules may serve as use- Jolanda van der Velden. ful tools in neurobiology and could have therapeutic value as well. Physiology, VU University Medical Center, Amsterdam, Netherlands. Cardiomyocytes have, through splicing, an exquisite control over the length of 201-Plat titin, enabling it to regulate passive stiffness. As titin sets the preload of the car- Structural Dynamics of Slow-Inactivation in a Voltage-Gated Sodium þ diomyocyte, we hypothesized that together with diastolic Ca2 it pre-activates Channel the cardiomyocyte during diastole and that this is a major determinant of force Soumili Chatterjee1, Rajan Vyas1, Sreevatsa Chalamalasetti1, production in the subsequent systolic phase. Through this mechanism titin Indra D. Sahu2, Jerome Clatot3, Gary A. Lorigan2, Isabelle Deschenes1, would play an important role in active force development and length- Sudha Chakrapani1. dependent activation. Mutations in the splicing factor RNA binding motif pro- 1Physiology and biophysics, Case western Reserve University, Cleveland, tein (RBM20) results in the expression of large, highly compliant titin isoforms. OH, USA, 2Chemistry and Biochemistry, Miami University, Oxford, OH, We measured single cardiomyocyte work-loops that mimic the cardiac cycle in USA, 3Heart and Vascular Research Center, Casewestern Reserve University, wildtype (WT) and heterozygous (HET) RBM20 deficient rats, with long Cleveland, OH, USA. compliant titin. In addition we studied membrane-permeabilized cardiomyo- Slow-inactivation in voltage-gated sodium channels (NaV) directly regulates cytes with different amounts of diastolic calcium. At low pacing frequencies, excitability of neurons, cardiac myocytes, and skeletal muscles. Although myocytes isolated from HET left ventricles were unable to produce normal NaV slow-inactivation appears to be conserved across the phylogeny, from bac- levels of work (55% of WT), but this difference disappeared when diastolic cal- teria to humans, the structural basis for this mechanism remains unclear. Using cium increased at high pacing frequencies. HET myocytes operated at higher X-ray crystallography, in combination with spin-labeling/EPR spectroscopic SL to achieve the same level of work (2.10 mm vs. 1.94 mm at 6 Hz). To delin- measurements in membrane reconstituted prokaryotic NaV homologues, we þ eate the effects of diastolic Ca2 and titin pre-activation on force generation, characterize the conformational dynamics of the selectivity filter region in the measurements were performed in detergent-permeabilized cardiomyocytes iso- conductive and slow-inactivated states to determine the molecular events under- lated from rat hearts. In these cells cardiac twitches were simulated by tran- lying NaV gating. Our findings reveal profound conformational flexibility of the þ siently (1 s) exposing the cell to a Ca2 -concentration of 2 mM. Increasing pore in the slow-inactivated state. We find that the P1 and P2 pore-helices un- pre-activation by pre-stretching the myocyte increased the kinetics of force dergo opposing movements with respect to the pore-axis, resulting in changes þ development and total force development. Increasing diastolic Ca2 did in- in volume of both the central, as well as the intersubunit cavities. These regions

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form pathways for lipophilic drugs that modulate slow-inactivation; hence, our Among voltage-gated L-type calcium channels (CaV1.1-1.4), the skeletal mus- findings provide novel insights into the molecular basis for state-dependent ef- cle isoform (CaV1.1) exhibits the most unique voltage- and time-dependent fects of these drugs on channel function. properties. Until recently, studies of CaV1.1 biophysical properties have been hampered by 202-Plat negligible expression level in heterologous systems. Structural Modeling of Local Anesthetic and Antiarrhythmic Drug Bind- Thanks to the discovery of STAC-mediated trafficking of CaV1.1 channels ing to the Human Cardiac Voltage Gated Sodium Channel (Polster 2015) we are now able to optically track the activation of human 1,2 1,2 2,3 Phuong T. Nguyen , Kevin R. DeMarco , Igor Vorobyov , CaV1.1 voltage sensing domains (VSDs) using the voltage-clamp fluorometry Colleen E. Clancy2,3, Vladimir Yarov-Yarovoy2. technique. We expressed the pore-forming subunit a with STAC3 and b 1 2 1s 1a Biophysics Graduate Group, UC Davis, Davis, CA, USA, Department of in Xenopus oocytes and labeled VSD I with thiol-reactive fluorophores at a Physiology and Membrane Biology, UC Davis, Davis, CA, USA, strategically-introduced Cysteine (L159C). We resolved voltage-dependent 3 Department of Pharmacology, UC Davis, Davis, CA, USA. fluorescence changes from VSD I, which report the movement of this The human voltage-gated sodium (Nav) channel, hNav1.5, is predominantly voltage sensor. The voltage dependence of these structural transitions (FV: 0 expressed in cardiac myocytes and is responsible for the rapid upstroke of Vhalf= 28.752.2 mV; z= 2.150.1 e ; N=4) closely follows the pore 0 the cardiac action potential. hNav1.5 channel plays a central role in congenital voltage-dependence (GV: Vhalf= 33.651.0 mV; z= 1.850.1 e ). Impor- and acquired cardiac arrhythmias and has been a key target for drug develop- tantly, the L159C mutation did not alter the channels biophysical properties 0 . ment. Mutagenesis studies have previously identified key residues in Nav chan- (WT GV: Vhalf= 31.851.5 mV; z= 2.150.04 e ; N=5) We also found that nels S6 segments from central pore that form a receptor site for binding of local depolarized holding potentials (þ40 mV) shifted VSD I voltage dependence anesthetic and antiarrhythmic drugs. However, the structural details of how to more hyperpolarized potentials by 80mV, recapitulating properties these drugs affect Nav channel function are not well understood. In this study, observed in the gating current. Finally, no voltage dependent fluorescence we used Rosetta computational modeling software to build a homology model changes were detected from WT channels when incubated with thiol reactive of human Nav1.5 in open-inactivated and closed states based on the cryo-EM fluorophores. structures of electric eel Nav1.4 (PDB ID: 5XSY) and American cockroach VSDs II, III and IV are currently under investigation. NavPaS (PDB ID: 5X0M), respectively. We applied the RosettaLigand molec- Based on our findings in its ‘‘closest relative’’ (CaV1.2), which revealed a stag- ular docking program to study hNav1.5 channel interactions with local anes- gering heterogeneity in the time- and voltage-dependent properties of the four thetic and antiarrhythmic drugs, including lidocaine, etidocaine, QX-314, VSDs, we expect that the four skeletal VSDs will exhibit repeat-specific fea- ranolazine, flecainide, and GS967. Our lowest energy models have shown tures, likely underlying CaV1.1 distinct properties. that both local anesthetic and antiarrhythmic drugs bind to hNav1.5 via a com- mon receptor site formed by S6 segments from domains III and IV in the central 205-Plat pore. Our results may further advance structural understanding for molecular Isoproterenol Promotes Augmentation of L-Type Cav1.2 Channel Clus- mechanisms of local anesthetic and antiarrhythmic drug interaction with tering and Cooperative Gating in Ventricular Myocytes hNav1.5 and provide useful insights towards the rational design of novel mod- Danica W. Ito, Karen I. Hannigan, Luis F. Santana, Rose E. Dixon. ulators of ion channel activity for the treatment of cardiac arrhythmias. Physiology and Membrane Biology, University of California Davis, Davis, CA, USA. 203-Plat L-type CaV1.2 channels are essential components of cardiac excitation- Insights into Sodium Channel Gating Enabled by Transplantation of an contraction coupling. We have previously reported that CaV1.2 channels Aryl Sulfonamide Drug Binding Site in Combination with Genetically- form clusters arranged along the z-lines on the t-tubule membranes of ventric- Encoded Cross Linking ular myocytes. The C-termini of neighboring channels within these clusters Daniel T. Infield1, Samuel J. Goodchild2, Jason D. Galpin1, can undergo dynamic, physical and allosteric interactions in a Ca2þ-calmod- Christopher A. Ahern1. ulin dependent manner, enabling cooperative gating of the channels, 1 2þ Physiology and Biophysics, University of Iowa, Iowa City, IA, USA, enhancing channel Po, and resulting in amplification of Ca influx through 2Current Address - Xenon Pharmaceuticals, Burnaby, BC, Canada. the channels. During the ‘fight or flight response’, activation of the b-adren- Upon opening, voltage gated sodium channels rapidly terminate conductance ergic receptor-cAMP-PKA pathway leads to increased mean open time and 2þ through a process called fast inactivation. Defects in inactivation can underlie Po of CaV1.2 channels and thus enhanced Ca influx. Since CaV1.2 channel multiple human diseases including pain disorders, epilepsies, and cardiac ar- cooperativity is a Ca2þ dependent process, we hypothesized that the b-AR rhythmias. To better understand this process, we have used nonsense suppression agonist isoproterenol (ISO) could affect CaV1.2 channel clustering and coop- to encode the photoactivatable crosslinker p-benzoly-l-phenylalanine (Bpa) in erativity. To test this hypothesis, we used Ground State Depletion (GSD) and F1486 of the IFMT inactivation motif of the DIII/DIV linker in hNav 1.5 in Zeiss AiryScan detector super-resolution microscopies, Bimolecular Fluores- HEK cells. These channels displayed fast inactivation that was essentially normal cence Complementation (BiFC), electrophysiology and Ca2þ imaging ap- ( 4 percent late current). If UV light was applied under depolarizing voltage proaches. Mean Cav1.2 channel cluster size in myocytes treated with ISO clamp after channels had inactivated, channels quickly developed an inactivation was 4566 5 146 nm2, equating to a 26% (p = 0.0029) increase over those gate ‘‘locked closed’’ phenotype; if applied under hyperpolarizing conditions in untreated control cells (3618 5 201 nm2). While the mean area of channel (140mV), inactivation was largely abolished. To dissect the structural conse- clusters increased, the number of clusters per mm2 decreased, suggesting that quence in the DIV voltage-sensing domain of locking the IFMT motif in the chan- clusters fused together to form larger super-clusters in response to ISO. We nel’s resting state, we built a chimeric channel wherein the putative binding site observed this effect dynamically in tsA-201 cells expressing CaV1.2 channels from Nav 1.7 for the aryl sulfonamide GX 674 was engineered into the cardiac using an AiryScan detector. In BiFC experiments, 100 nM ISO increased sodium channel DIV voltage-sensing domain (VSD). Previously published crys- protein-protein interactions between CaV1.2 channels. Recording of CaV1.2 tallographic data suggests that this drug binds the DIV voltage sensor with high single channel activity using both electrophysiology and Ca2þ sparklet ap- affinity only when S4 is in the ‘‘up’’ position; therefore, affinity can thought of as proaches revealed enhanced cooperative openings of CaV1.2 channels in a reporter of the conformation of the DIV VSD. To this end, crosslinking of the response to 100 nM ISO. Thus, dynamic augmentation of CaV1.2 channel inactivation motif into its resting state position by UV caused a more than 1000- clustering in response to b-AR signaling may represent a novel EC- fold loss of affinity (low nano- to micromolar) between the drug and channel. coupling regulatory mechanism. These results suggest that Nav inactivation involves the exchange of conforma- tional poses of the inactivation motif, and furthermore, that the binding of the 206-Plat motif to its resting state site is incompatible with the ascension of DIV S4, a Disturbances of Transretinal Signaling After Ablation of CaV2.3 / R-Type movement itself proposed to be intimately related to fast inactivation. Calcium Channels Toni Schneider1, Jan Niklas Luke€ 1, Isha Akhtar1, Felix Neumaier1, 204-Plat Gerrit Alexander Schubert2, Hans Clusmann2,Jurgen€ Hescheler1, Optically-Tracked Structural Rearrangements of the Voltage Sensing Do- Matthias Luke€ 1, Walid Albanna1,2. 1 mains in the Human CaV1.1 Channel University of Cologne, Institute for Neurophysiology, D-50931 Ko¨ln, Nicoletta Savalli1, Fenfen Wu2, Marbella Quinonez2, Stephen C. Cannon2, Germany, 2RWTH Aachen, Department of Neurosurgery, RWTH Aachen, Riccardo Olcese1,2. Aachen, Germany. 1 Anesthesiology & Perioperative Medicine, University of California, Los Four different Cav2.3 deficient mouse models have been produced worldwide Angeles, Los Angeles, CA, USA, 2Physiology, University of California, Los (1). In the present model exon 2, encoding the transmembrane segment IS1 Angeles, Los Angeles, CA, USA. and the loop to IS2, was deleted (2). Two methods are used for the analysis

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of retinal signaling in control and Cav2.3-deficient mice: in anesthetized mice, contraction. Recent studies have shown that failure of skeletal muscle EC neurovascular coupling is tested by using flicker light as a trigger to increase coupling occurs in mice and fish null for the protein STAC3 (SH3 and metabolic energy demands and subsequent changes in retinal blood flow. In cysteine-rich domain 3) and has led to the conclusion that STAC3 is critical the superfused murine retina, transretinal signaling and the effect of drugs on in trafficking and maintaining components of the EC coupling machinery in a a- and b-wave of the electroretinogram is analysed to evaluate the potency of functional state. We present isothermal titration calorimetry data showing a the retina model for noninvasive analysis of neurological processes. This report direct interaction between STAC3 and CaV1.1 a1s. This association is shows the ‘proof-of-principle’ for retinal vessel analysis (RVA) by a prototype conserved among the members of the STAC family of adaptor proteins, as camera for the murine eye. This technique was successfully intro-duced for pa- STAC1 and STAC2 show similar low-micromolar affinity binding. The tients with subarachnoidal hemorrhage (SAH) to estimate their risk for succes- STAC3 Native American Myopathy (NAM) mutation significantly reduces sive vasospasm after SAH (3). Since Cav2.3 is known to be upregulated after the affinity of association. Our binding studies corroborate with our structural SAH during subsequent vasospasms, its regulatory behavior was analyzed in findings. We present the first high-resolution (1.2 - 2.6A˚ ) crystallographic in- control and Cav2.3-deficient mice to deduce the role of Cav2.3 during signaling sights into the three STAC protein isoforms in apo-form and a complex be- for disturbances in neurovascular coupling. tween STAC2 and our CaV1.1 a1s binding determinant. EC coupling assays Ex vivo ERG exhibit a difference in the half-maximal peak width of the b- and two-electrode voltage clamp electrophysiology was used to assess the wave, which is markedly reduced in Cav2.3 KO when compared to control. functional effect of STAC3 on CaV1.2 a1c. Furthermore, the effect of hemoglobin degradation products on transretinal signaling is genotype dependent. In conclusion, the murine retina proves to 209-Plat be a powerful and reliable model system for testing neural network communi- De Novo Reconstitution of Skeletal Muscle Voltage-Induced Calcium cations under physiological and pathophysiological conditions. Release 1 2 1 1. Weiergr€aber, M., Kamp, M. A., et al., Schneider, T. (2006) Neurosci. Bio- Stefano Perni , Manuela Lavorato , Kurt G. Beam . 1Physiology and Biophysics, University of Colorado, Aurora, CO, USA, behav. Rev. 30, 1122-1144 2 2. Pereverzev, A., et al., Schneider, T. (2002) Mol. Endocrinol. 16, 884-8953. Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Albanna, W., et al., Schubert, G. A. (2016) PLoS One 11, e0158781 PA, USA. Depolarization of vertebrate skeletal muscle causes intracellular calcium release, an event depending on specialized junctions between the plasma Platform: Excitation-Contraction Coupling membrane (PM) and sarcoplasmic reticulum (SR). It is known that depolar- ization is ‘‘sensed’’ by a voltage-gated calcium channel located in the PM 207-Plat and containing CaV1.1 as its principal subunit, that calcium is released 2þ Stac Proteins Associate with the Domain of the CaV1.1 II-III Loop Critical from the SR via RyR1, and that this does not require external Ca entry. for EC Coupling Thus, it is thought that activation of RyR1 is driven by depolarization- 1 2 3 Alexander Polster , Benjamin R. Nelson , Symeon Papadopoulos , induced conformational changes in CaV1.1 which are mechanically trans- Eric N. Olson2, Kurt G. Beam1. mitted to RyR1, either directly or via intervening proteins. Based on muscle 1Physiology and Biophysics, University of Colorado Denver, Aurora, CO, gene knockouts, at least two additional proteins are required for this confor- USA, 2Department of Molecular Biology, UT Southwestern Medical Center, mational coupling, but knockouts cannot reveal whether yet-to-be identified Dallas, TX, USA, 3Vegetative Physiology, University Hospital of Cologne, proteins, or sets of proteins with overlapping functions, are also critical. Cologne, Germany. Thus, we used reconstitution in tsA201 cells to identify a minimal set of pro- In skeletal muscle, residues 720-764/5 within the CaV1.1 II-III loop represent teins required for conformational coupling. We found that expression of junc- a ‘‘critical domain’’ that plays an essential role in transmitting the excitation- tophilin2 was effective at promoting ER-PM junctions, and that CaV1.1 and 2þ contraction (EC) coupling Ca release signal to the type 1 ryanodine receptor RyR1 both targeted to these junctions in the presence of the CaV1.1 auxiliary (RyR1) in the sarcoplasmic reticulum (SR). However, the identities of pro- subunit b1a. Moreover, tsA201 cells expressing these four proteins, and the þ teins that are directly contacted by the loop and its critical domain, or how adapter protein Stac3, produced depolarization-triggered Ca2 transients þ the II-III loop regulates RyR1 gating, remain unknown. Recent work has which were independent of extracellular Ca2 entry and which increased in shown that EC coupling in skeletal muscle of fish and mice depends on the amplitude as a saturating function of voltage, as expected for conformational presence of Stac3, an adaptor protein highly expressed only in skeletal mus- coupling. Additionally, freeze-fracture electron microscopy indicated that cle. Here, we utilized co-localization as an indicator of interaction, and found CaV1.1 and RyR1 were physically linked in these cells, thus establishing that Stac3, as well as Stac1 and Stac2 (predominantly neuronal Stac isoforms), that the five expressed proteins are sufficient for conformational coupling. interact with the CaV1.1 II-III loop, promote the functional expression of The ability to reconstitute conformational coupling with a minimal set of pro- CaV1.1 in tsA201 cells, and support EC coupling in Stac3-null myotubes, teins may provide a system for obtaining high resolutions structures of with Stac3 being most effective. Co-expression in tsA201 cells revealed CaV1.1 and RyR1 as part of a functioning complex, which will be necessary that Stac3 interacted only with II-III loop constructs containing the majority for understanding the molecular mechanism of depolarization-evoked cal- of the CaV1.1 critical domain residues. We also co-expressed Stac3 in cium release in skeletal muscle. dysgenic (CaV1.1-null) myotubes together with CaV1 constructs which had chimeric II-III loops and had previously been tested for functionality. The 210-Plat Every Action Potential Activates Store-Operated Ca2D Entry in Skeletal ability of Stac3 to interact with these CaV1 constructs paralleled their content of critical domain residues and ability to mediate skeletal type EC coupling. Muscle Based on co-expression in tsA201 cells, the interaction of Stac3 with the II-III Xaver Koenig, Bradley S. Launikonis. loop critical domain did not require the presence of a PKC C1 domain in School of Biomedical Sciences, University of Queensland, Brisbane, Australia. Stac3, but did require the presence of both SH3 domains. 2þ Taken together, our results indicate that the activation of RyR1 Ca2þ release by Store-operated Ca entry (SOCE) is rapidly activated during pharmacological activation of ryanodine receptor (RyR) Ca2þ release in skeletal muscle. Fast CaV1.1 depends upon Stac3 being bound to critical domain residues in the II-III loop. SOCE is supported by the invagination of the plasma membrane, the tubular (t-) system that forms junctions with the sarcoplasmic reticulum (SR) terminal 208-Plat cisternae where STIM1 and RyRs reside. However, the physiological activation Structural Insights into the STAC Adaptor Protein and Voltage-Gated of SOCE in muscle is not defined, thus clouding its physiological role. To Calcium Channel Interaction address this, we developed a novel fluorescence technique to directly measure þ Siobhan Wong King Yuen1, Marta Campiglio2, Ching-Chieh Tung1, SOCE during physiological Ca2 release. We trapped rhod-5N inside the t-sys- Bernhard Flucher2, Filip Van Petegem1. tem of mechanically skinned fibres from rat extensor digitorum longus muscles þ 1Biochemistry and Molecular Biology, University of British Columbia, and bathed them in a K -based cytoplasmic solution that allowed the sealed t- Vancouver, BC, Canada, 2Physiology and Medical Physics, Innsbruck system to repolarize. The skinned fibre was excited by field pulses across flank- Medical University, Innsbruck, Austria. ing platinum electrodes to generate propagating action potentials (AP). The Skeletal muscle Excitation-Contraction (EC) coupling relies on the proper cytoplasmic solution contained fluo-4 to allow simultaneously tracking of cyto- 2þ 2þ 2þ trafficking of CaV1.1 to regions of the plasma membrane to facilitate its phys- plasmic Ca and t-system Ca during AP-evoked Ca release with confocal ical interaction with the ryanodine receptor (RyR1). CaV1.1 acts as a voltage microscopy. Our results show that SOCE is activated phasically following þ sensor for RyR1, allowing the plasma membrane depolarization to be trans- every AP that evokes Ca2 release. The pattern and duration of SOCE was þ duced into a rapid torrent of cytosolic calcium that culminates in muscle shaped by AP frequency and the activity of the SR Ca2 pump. The in-phase

BPJ 8544_8555 Sunday, February 18, 2018 41a activation of SOCE with each AP and RyR opening makes SOCE a counter- 213-Plat flux to fibre Ca2þ loss and a potential signal that transduces muscle usage pat- Fibroblast-Mediated Atrial Mechanical Dysfunction in HFpEF and Hy- terns for gene expression. pertensive Heart Disease David Bode1,2, Rafael Doerr1,2, Diana Lindner3,4, Michael Schwarzl3,4, 211-Plat Dirk Westermann3,4, Uwe Primessnig1,2, Burkert Pieske1,2, Role of the CaCC Channel ANO1 in Electromechanical Coupling of Mu- Frank R. Heinzel1,2, Felix Hohendanner1,2. rine Pulmonary Artery Smooth Muscle 1Internal Medicine and Cardiology, Charite University Medicine, Campus Katie Mayne1, Michael D. Young1, Nathan Grainger2, Julius C. Baeck1, Virchow-Klinikum, Berlin, Germany, 2German Center for Cardiovascular Kenton M. Sanders2, Sean M. Ward2, Iain A. Greenwood3, Simon A. Bulley4, Research (DZHK), Partner Site Berlin, Berlin, Germany, 3Klinik fur€ Jonathan H. Jaggar4, Normand Leblanc1. Allgemeine und Interventionelle Kardiologie, Universit€ares Herzzentrum 1Department of Pharmacology, University of Nevada, Reno, Reno, NV, USA, 4 2 Hamburg, Hamburg, Germany, German Center for Cardiovascular Research Department of Physiology and Cell Biology, University of Nevada, Reno, (DZHK), Partner Site Hamburg, Hamburg, Germany. Reno, NV, USA, 3Department of Vascular Pharmacology, St. George’s 4 Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about University of London, London, United Kingdom, Department of 50% of HF patients, often related to metabolic syndrome. Atrial remodeling is Physiology, University of Tennessee, Memphis, TN, USA. 2þ - common in HFpEF, associated with atrial mechanical stretch leading to In VSMCs, Ca -activated Cl channels (CaCCs) are encoded by the gene changes in secretory activity (e.g. adversely altered TNF-alpha and IL-10) TMEM16A/Anoctamin 1 (ANO1). The mechanism by which ANO1 influence and independently increases mortality. We hypothesize that atrial cardiomyo- the excitability of VSMCs remains to be elucidated due to questionable phar- cyte (CM) function and its reaction to extracellular-matrix-triggers is pivotal macology and lack of a reliable genetic knockdown mouse model of ANO1. for the manifestation and progression of atrial remodeling and mechanical The aim of this study was to re-evaluate the role of ANO1 in electromechanical dysfunction in HF. coupling of pulmonary artery (PA) smooth muscle using newer generation Atrial mechanical function in-vivo was assessed using echocardiography. ANO1 blockers and a novel smooth muscle-specific inducible ANO1 knockout Excitation-contraction-coupling (ECC) was examined using Ca-imaging (Ca- mouse model (SMC-iANO1-KO). Wire myography was used to determine the transients; CaT) in atrial CM of ZFS-1 rats without (Ln; hypertension) and vascular reactivity to 5-HT of PA from wild-type and SMC-iANO1-KO mice. with metabolic syndrome (Ob; diabetes, hypertension, obesity). CaT were re- Calcium imaging experiments were also carried out using SMC-iGCaMP3 2þ corded after treatment with conditioned medium of unstressed or stressed mice, which genetically express the Ca biosensor GCaMP3 in smooth muscle (Flexercell system) fibroblasts isolated from Ln and Ob. CM were also exposed cells. 5-HT elicited a dose-dependent contraction (0.01-30 mM) that was simi- to TNF-alpha and IL-10 and ECC was studied. larly inhibited ( 50-70%) by the ANO1 blocker CaCCInh-A01 (10 mM), the Ob show impaired atrial function in-vivo associated with an increased dia- CaV1.2 blocker nifedipine (1 mM) or the SERCA2 pump inhibitor cyclopia- stolic Ca-content, prolonged CaT time-to-peak and Ca-removal after treat- zonic acid (CPA; 10 mM). Genetic ablation of ANO1 produced a reduction ment with activated fibroblast-medium. While Ca-spark frequency was in 5-HT-induced tone ( 60% at 1 mM 5-HT) that was similar to that produced 2þ unaltered, conditioned medium significantly increased amplitude and altered by CaCCinhA01, nifedipine or CPA. Ca imaging experiments in the intact PA time to peak, duration and width in Ob. This indicates changes of ECC due of SMC-iGCaMP3 mice revealed that 5-HT evoked spatially and temporally 2þ 2þ to mechanical stress in metabolic syndrome mediated by fibroblasts. In Ln localized Ca transients. These Ca oscillations were potently inhibited by however atrial in-vivo function was preserved and a shortening of Ca- CaCCInh-A01 or nifedipine, and were abolished by CPA. In conclusion, 5- 2þ 2þ removal could be observed after exposure to stressed fibroblast secretome, HT elicited highly localized Ca oscillations that were promoted by Ca en- supporting the notion of an in-vivo compensatory phenotype. Ob showed no try through CaV1.2, most likely involving transient depolarizations evoked by 2þ change in ECC when exposed to TNF-alpha. IL-10 however, had beneficial ANO1 activated by a balance between oscillatory SR Ca release through IP3 2þ effects on ECC as it significantly accelerated Ca removal by 29% in receptors and Ca entry through CaV1.2. We propose that the stable agonist- Ob CM. induced PA contraction results from the integration of stochastic and localized 2þ During metabolic syndrome, impaired in-vivo atrial mechanical function might Ca events supported by a microenvironment comprising ANO1, CaV1.2 and be related to adversely affected CM ECC due to fibroblast secretome. In hyper- IP3 receptors. tensive atrial remodeling, stressed fibroblasts enhance CM Ca-removal and in- vivo atrial function is preserved. 212-Plat Expression of Orai1 Restores Normal Sarcoplasmic Calcium Release in 214-Plat Cmpt Mice Surface Mechanosensors and the Fundamental Conundrum of Homeomet- Mo´nika T. Sztretye,Peter Szentesi, La´szlo´ Csernoch, Beatrix Dienes. ric Regulation Physiology, University of Debrecen, Debrecen, Hungary. Rafael Shimkunas, Zhong Jian, Zana Coulibaly, Ye Chen-Izu, In our mouse model, a naturally occurring 12-bp deletion in the myostatin gene Leighton T. Izu. is considered responsible for the compact phenotype (MstnCmpt-dl1Abc, Cmpt) Pharmacology, University of California, Davis, Davis, CA, USA. labeled by a tremendous increase in body weight along with signs of muscle More than 100 years ago von Anrep observed the eponymous Anrep effect weakness, easier fatigability and decreased store operated calcium entry whereby the heart initially dilates in response to an increase in outflow resis- (SOCE, Sztretye et al. 2017). While the voltage dependence of SR calcium tance but over several minutes the end-diastolic volume decreases suggesting release was not statistically different between WT and Cmpt fibers an increase in contractility. About 50 years later Sarnoff et al. made the (26.0351.35 mV for WT vs. 28.8650.77 mV for Cmpt), the amount of important discovery that in the face of increased outflow resistance (after- releasable calcium was significantly reduced in the latter, indicating smaller load) the heart increased the force of contraction even at the same fiber SR content. To assess the immediate role of SOCE in replenishing the SR, length (preload), which is in clear contradistinction from Starling’s Law of the evolution of intracellular calcium concentration during a train of long- the Heart. Subsequent research supports the idea that the Anrep effect is lasting depolarizations to a maximally activating voltage were monitored. intrinsic to the heart but the cellular mechanisms remain unclear. The conun- Cmpt mice exhibited a faster decline in calcium release suggesting a compro- drum is thus: Given the same starting length (precluding the Frank-Starling mised ability to refill the SR. We found SOCE having a role in maintaining and mechanism), how can a myocyte ‘‘know’’ it is contracting against two þ refilling SR Ca2 stores not only in repetitive tetanic stimulation, but on an im- different resistances? We propose that on the myocyte surface, mechanosen- mediate basis. When reconstructing the Cmpt fibers with venus-Orai1, we sors oriented orthogonal to the longitudinal axis enable the myocyte to detect found a slight shift to more positive potentials in the voltage dependence of stress (afterload) independently of myocyte strain (preload). During SR calcium release (23.5750.85 mV) When Cmpt cells were loaded with 50 contraction the myocyte expands transversely and the extent that surface me- nM TMRE fiber segments with damaged mitochondria were identified covering chanosensors are compressed depends on the stiffness of its environment, on average. 27.751.7% of the fiber area (n=8). Our results favor the idea that which in the heart is a function of wall stress. Our model makes the surpris- SOCE is immediately activated upon voltage-dependent SR calcium release. ing prediction that within a certain range, the Ca2þ transient will increase By doing so it plays an important role in regulating SR calcium content both with increasing mechanical stiffness thus providing the cellular basis for on the long run and also during a contraction-relaxation cycle. A new finding what Sarnoff called homeometric autoregulation and the Anrep effect. This is that the Cmpt phenotype in mice is associated with abnormal mitochondrial prediction was confirmed in experiments using our Cell-in-Gel system where function.Funded by: OTKA PD-108476, Bolyai Fellowship to MSz, GINOP- myocytes embedded in viscoelastic hydrogel are subjected to controlled me- 2.3.2-15-2016-00044. chanical loads.

BPJ 8544_8555 42a Sunday, February 18, 2018

Platform: Computational Methods and 218-Plat SEEKR: Simulation Enabled Estimation of Kinetic Rates, A Multiscale Bioinformatics Approach for the Calculation of Protein-Ligand Association and Dissocia- tion Kinetics 215-Plat Benjamin R. Jagger1, Lane W. Votapka2, Rommie E. Amaro1. A Dimension Reduction Method for cryo-EM Image Analysis 1Department of Chemistry and Biochemistry, University of California San I-Ping Tu. Diego, La Jolla, CA, USA, 2Department of Chemistry, Point Loma Nazarene Institute of Statistical Science, Academia Sinica, Taipei, Taiwan. University, San Diego, CA, USA. In the field of structural biology, X-ray crystallography and NMR spectroscopy We present recent developments to our SEEKR software, a hybrid molecular dy- have been two major high-resolution techniques. The former requires crystalli- namics, Brownian dynamics, and milestoning approach for the calculation of as- zation of macromolecules while the latter is a solution method but would meet sociation and dissociation rates as well as binding free energies for protein-ligand limit when the macromolecule is large. Very recently, breakthrough in the cam- systems. The advantage of this approach, particularly the use of milestoning the- era together with microscopy automation, advancement of algorithm and GPU- ory, is that it is highly parallel; resulting in a significant speedup and increased accelerated computations has transformed cryo-EM to become a standard tech- sampling as compared to conventional long-timescale molecular dynamics sim- nique to solve structures of macromolecules at near atomic resolution. In contrast ulations. This extensive sampling, in conjunction with milestoning theory pro- to X-ray crystallography, single particle cryo-EM does not need crystalline sam- vides a statistically robust framework for the calculation of kinetic parameters. ple because it focuses the transmitted electrons to directly provide electron den- We also present recent successes including calculation of association and disso- sity information of a macromolecule. As such, atomic structures of several ciation rates for the well-studied trypsin-benzamidine system that are in good difficult macromolecules not amenable to X-ray crystallography have now agreement with experimental rates. Furthermore, we discuss the potential of been rapidly revealed by single particle cryo-EM and single particle cryo-EM this approach for the quantitative ranking of ligands by their kinetic parameters. has been chosen as the method of 2015 by Nature Method. Because biological molecules are extremely vulnerable, very low doses of electrons are used by 219-Plat cryo-EM, leading to very noisy images. Here, we propose a dimension reduction Accurate Prediction of Protein-Ligand Binding by Combined Molecular method 2SDR to a set of single-particle cryo-electron-microscopy (cryo-EM) Dynamics-Based Docking and QM/MM Methods images and demonstrated that 2SDR can realize effective de-noising. Iris Antes, Antoine Marion, Chen Zheng, Okke Melse. Biosciences, Technical University of Munich, Freising, Germany. 216-Plat Protein-ligand interactions are crucial for many important biological processes Identifying Metastable States of Protein Folding with Deep Clustering and thus the selective inhibition of disease relevant proteins is one of the major Techniques strategies for drug design in pharmaceutical industry. In this context computer- Debsindhu Bhowmik, Arvind Ramanathan. based methods are routinely used to screen large compound libraries for the Biomedical Sciences, Engineering & Computing, Oak Ridge National identification of potential drug candidates. Thus many sampling and scoring Laboratory, Oak ridge, TN, USA. methods have been developed for this purpose over the last two decades, which The global features of the folding free energy landscape of proteins and other perform very well for ‘‘classical’’ drug design applications, namely predicting bio-macromolecules can be characterized by metastable states, which consist of the binding of non-covalently bound small molecule-based ligands to specific conformations sharing similar structural and energetic features. However, char- target proteins. Nevertheless, these approaches experience considerable limita- acterizing metastable states is challenging, even for simple systems mainly tions for the prediction of covalently bound inhibitors, ligands binding to metal because (1) the intrinsic dimensionality of folding processes is difficult to deter- containing binding sites, and very flexible ligands like peptides or macrocycles. mine a priori, (2) the features that constitute biophysically relevant reaction co- In addition, the calculation of quantitatively accurate ligand binding affinities is ordinates are difficult to identify, and (3) defining boundaries between still a challenge in the field. We are developing new and optimizing existing conformational states is sensitive to parameters of different algorithms (e.g., ki- approaches for these areas of applications. Thereby our focus is on the improve- netic clustering methods versus geometric metrics). To overcome these chal- ment of classical molecular docking approaches by the use of biophysical sam- lenges, we propose an unsupervised deep learning based approach that pling methods and QM/MM approaches. We will present several case studies, automatically clusters long time-scale protein folding simulations into meta- which show how the accuracy of protein-ligand binding predictions can be stable states. Our approach can robustly identify low dimensional manifolds improved considerably by the new, combined approaches for e.g. covalently on which conformations from the simulations can be organized such that bound inhibitors or metal-containing binding sites. each state possesses similar structural and energetic similarities. Further, we References: also show that our approach can correct for boundary errors by separating bar- [1] C. Hartmann, I. Antes, T. Lengauer, Proteins 74(3), 712 (2009). riers between interstate and intrastate fluctuations. We demonstrate our [2] I. Antes, I., Proteins 78(5), 1084 (2010). approach on two model protein folding systems, namely Fs peptide (14 micro- [3] M. Marcinowski, M., M. Rosam, M., C. Seitz, J. Elferich, J. Behnke, seconds aggregate sampling) [i] and the mixed b-b-a(BBA) fold (325 micro- C. Bello, M.J. Feige, C.F. Becker, I. Antes, I., J. Buchner, J. Mol. Biol. seconds sampling) [ii] and show that the rates of folding determined from 425(3), 466 (2013). our approach are in good agreement with experimental observations. Taken [4] M. Zachmann, G. Mathias, I. Antes, ChemPhysChem 16(8), 1739 (2015). together, we show that deep learning approaches can be leveraged to analyze [5] M. Schneider, M. Rosam, M. Glaser, A. Patronov, H. Shah, K.C. Back, long time-scale simulations. M.A. Daake, J. Buchner, I. Antes, Proteins 84(10), 1390 (2016). [i] Beauchamp et al., J Chemical Theory Computat, 2011; [ii] Lindorff-Larsen et al., Proteins: Struct. Func. Bioinform , 2010. 220-Plat A Novel Finite Volume Method for Diffusion Equation Coupled with Cell 217-Plat Surface Reaction Effectively Detect Metastable States of Proteins by Non-Equilibrium Myles Kim. Simulations Mathematics, Florida Polytechnic University, Lakeland, FL, USA. Xin Zhou. Computational models for multicellular biological systems, both in vitro or School of Physical Sciences, Univ Chin Acad Sci, Beijing, China. in vivo environments, require solving systems of differential equations to incor- Non-equilibrium driving can efficiently accelerate molecular dynamics simula- porate molecular transport and their reactions such as release, uptake, or decay. tion to visit complex conformational spaces of proteins thus improve the effi- Examples can be found from drugs, growth nutrients, and signaling factors. The ciency of simulations. However, suitable driving should be designed to avoid systems of differential equations frequently fall into the category of the compelling systems too far away from equilibrium, i.e., still mainly exploring diffusion-reaction system due to the nature of the spatial and temporal change. the relevant conformations of proteins. Here we proposed a general method, Due to the complexity of equations and complexity of the modeled systems, named as adaptive non-equilibrium simulations (ANES), to automatically analytical solution for the systems of the differential equation is not possible. adjust the magnitude of external driving by estimating the response of system Therefore, numerical calculation schemes are required and have been used on the fly, then reaches both high efficiency and accuracy in simulation of pro- for multicellular biological systems such as bacterial population dynamics or teins, by approximately keeping the local equilibrium and efficiently acceler- cancer cell dynamics. Finite volume methods in conjunction with agent- ates global motions of systems at the same time. We illustrated the capability based models have been popular choices to simulate such reaction-diffusion of the ANES in detecting metastable conformations of HP35 protein, and systems. In such implementations, the reaction occurs within each control vol- show that the 0.2 us ANES simulation approximately captures the important ume and control volumes interact one another following the law of diffusion. states and folding/unfolding pathways found in the recent 398 us equilibrium The characteristic of the reaction can be determined by the agents in the control MD simulation on Anton. volume. In case of cancer cell growth dynamics, it is observed that cell

BPJ 8544_8555 Sunday, February 18, 2018 43a

behavior can be different within two or three cell size distance because of the protein and cytochrome c. Unlike the canonical complex III (e.g., bc1 or b6f), its chemical gradient in that distance. Therefore, in the modeling of such systems, biochemical properties are poorly characterized and its structure is unknown. a spatial resolution is required to be comparable to the cell size. Such spatial Previously, we were able to isolate functional ACIII from Flavobaterium john- resolution poses an extra challenge in the development and execution of the soniae along with the aa3 cytochrome oxidase as a supercomplex using styrene computational model due to the agents sitting over multiple control volumes. maleic acid (SMA) copolymer. Here with the same preparation, we have carried In this presentation, a new computational method for cell surface-based reac- out a structural study on the ACIII/ aa3 supercomplex using single particle elec- tion for the finite volume method will be introduced and compared to other tron cryomicroscopy. From 160k particles, the density map of the supercomplex methods in terms of accuracy and computation speed. is determined to a resolution of 3.4 A˚ . We then have built, refined and validated the first ab inito atomic structural model of the ACIII using a wide variety of 221-Plat computational tools including Coot, Phenix and molecular dynamics flexible Studying Stem Cell Organization using ‘‘Label-Free’’ Methods and a fitting (MDFF). The final model of ACIII consists of 6 subunits and more than Novel Generative Adversarial Model 2,300 residues, covering 90% of the protein sequence. The structural integrity Gregory Johnson, Rory Donovan-Maiye, Chek Ounkomol, and stability of the ACIII model is further confirmed with a 700-ns atomistic mo- Mary M. Maleckar. lecular dynamics simulation. Furthermore, we have built 10 lipid molecules and Modeling, Allen Institute for Cell Science, Seattle, WA, USA. portions of the aa3 oxidase based on the well-resolved density map and lipid An integrated representation of the cell and subcellular structures would enhance biochemistry. Overall, not only does this cyro-EM structure offer insights into understanding of cellular organization and how it produces characteristic pheno- how the ACIII functions, but also it exhibits many lipid-protein and protein- types and changes as cells progress through the cell cycle, differentiate and other- protein interactions within the supercomplex that are functionally important. wise change state, offering key insight into cells’ function. Fluorescence microscopy allows the imaging of labeled cellular components; however, current 224-Plat methodology limits the number of fluorescent tags that can be imaged simulta- Modulation of the Mitochondrial Potassium Channel Activity by Infrared neously without gross cellular perturbations from tagging and imaging. To address Light this, we developed two deep learning tools: (a) a ‘‘label-free’’ method to predict Adam Szewczyk1, Piotr Bednarczyk2. fluorescently labeled structures patterns solely from 3D transmitted light micro- 1Nencki Institute of Experimental Biology, Warsaw, Poland, 2Warsaw scopy images, and (b) a conditional 3D model of cell organization (the ‘‘Integrated University of Life Sciences, Warsaw, Poland. Cell’’) to predict the integrated location of cellular organelles from high replicate Mitochondrial potassium channels are regulated by a variety of ligands. More- fluorescent microscopy images. In combination, the result is 3D integrated repre- over, gaseous signaling molecules such as NO or CO were recognized as new sentations of cellular organization. In initial evaluation of model performance, we and important modulators of mitochondrial function. Recently, also infrared see excellent correspondence for several well-stereotyped intracellular structures. light being absorbed by molecules and/or protein complexes was shown to Ongoing work includes expanding predictions to several additional intracellular modulate function of mitochondria. For example, it was suggested that mito- structures, increasing image resolution, and enhancing model interpretability. chondria interact with near-infrared light (wavelengths between 700 and 1400 nm) absorbed by complexes of the electron transport chain (ETC). In 222-Plat the near-infrared region, the 820 nm absorption band belongs mainly to the In Silico Identification of Rescue Sites by Double Force Scanning relatively oxidized CuA and the 760 nm absorption band to the relatively Matteo Tiberti1, Alessandro Pandini2, Franca Fraternali3, Arianna Fornili1. reduced CuB chromophore of cytochrome c oxidase. Absorption of photons 1 School of Biological and Chemical Sciences, Queen Mary University of (at 760 and 820 nm) by cytochrome c oxidase is hypothesized to enhance 2 London, London, United Kingdom, Department of Computer Science, ETC function and increase synthesis of ATP. Mitochondrial potassium chan- 3 Brunel University London, Uxbridge, United Kingdom, Randall Division of nels have been proposed to regulate the mitochondrial membrane potential, Cell and Molecular Biophysics, King’s College London, London, United respiration, matrix volume and Ca2þ ion homeostasis. It has been suggested Kingdom. that mitochondrial potassium channels participate in ischemic preconditioning Motivation. Deleterious amino acid changes in proteins can be compensated and neurodegenerative disorders. In our research, mitochondrial large- by second-site rescue mutations. These compensatory mechanisms can be conductance calcium-regulated potassium channel (mitoBK channel) of the as- mimicked by small molecules. Therefore, the location of rescue mutations trocytoma (glioblastoma) U-87 MG cell line were investigated using a patch- can be used to identify protein regions that can be targeted by drugs to reacti- clamp with illumination system. We have found that mitoBK channel is modu- vate a damaged mutant. lated by illumination by infrared light. Activation of the mitoBKCa channel Methods. Here we present the first general computational method to detect (depending on ETC redox state) was observed after illumination with using rescue sites. By mimicking the effect of mutations through the application of specific wavelengths of light: 760 nm and 820 nm. Our findings indicate the forces, the Double Force Scanning (DFS) method identifies the second-site res- functional coupling of the respiratory chain via cytochrome c oxidase (complex idues that make the protein structure most resilient to the effect of pathogenic IV) to mitoBK channel and its regulation by infrared light. This work was sup- mutations. The method is based on an elastic network model. All the possible ported by Polish National Science Center, grant no. 2015/17/B/ NZ1/02496. residue pairs in the protein are scanned and a rescue effect is detected when the simultaneous application of forces at the two sites affects the protein structure 225-Plat less than a force at a single site. Structural Dynamics of Light-Harvesting Complex II in Native Thylakoid Results. We tested DFS predictions against two datasets containing experimen- Membranes Detected by Solid-State NMR tally validated and putative evolutionary-related rescue sites. A remarkably Anjali Pandit. good agreement was found between predictions and experimental data. Indeed, Leiden Institute of Chemistry, Leiden University, Leiden, Netherlands. almost half of the rescue sites in p53 was correctly predicted by DFS, with 65% Photosynthetic pigment-protein complexes contain multiple chromophores to of remaining sites in contact with DFS predictions. Similar results were found perform delicate photophysical and photochemical tasks, which strongly rely for other proteins in the evolutionary dataset. Finally, we showed how the pre- on the conformational dynamics of their protein matrix. Striking examples diction of rescue sites can be used to identify potential pockets for the binding are the light-harvesting antenna complexes of plants and algae that can switch of reactivating drugs. The source code for DFS is available at https://fornililab.- between light-harvesting and excitation-quenched, photoprotective states via github.io/dfs. hitherto unresolved pigment and protein conformational changes. Obtaining atomic-level information of their pigment and protein dynamics in native- like environments is an essential step forward for solving their molecular Platform: Energy Transducing Complexes and switches that are controlled by subtle changes in inter-pigment interactions. Mitochondria in Cell Life and Death We herein report on the simultaneous solid-state NMR spectroscopic detection of protein, pigment and lipid moieties in a native, uniformly-13C,15N-enriched, 223-Plat Light-Harvesting Complex II of Chlamydomonas reinhardtii (Cr. LHCII) in cryo-EM Structure of Alternative Complex III/ AA3 Cytochrome Oxidase native thylakoid lipid membranes. Results reveal that membrane-embedded Supercomplex from Flavobacterium Johnsoniae Cr. LHCII has ordered loops and N-terminal regions, in agreement with the Chang Sun1, Padmaja Venkatakrishnan1, Samir Benlekbir1, Yuhang Wang1, plant-LHCII crystal structures, and overall dynamics similar to that of proteins John Rubinstein2, Robert B. Gennis1, Emad Takjhorshid1. in native thylakoids. NMR signals of the protein backbone including its large 1University of Illinois, Urbana, IL, USA, 2The Hospital for Sick Children coiled loops, chlorophyll rings, and xanthophylls are observed in dipolar- Research Institution, Toronto, ON, Canada. based spectra, while J-coupling based spectra reveal the presence of several en- Alternative complex III (ACIII) is an integral membrane protein that catalyzes dogeneous, protein-associated glycerolipid molecules. Interestingly, J-based the oxido-reduction between quinol and electron acceptors such as blue copper experiments also reveal new protein signals that we attribute to residues at

BPJ 8544_8555 44a Sunday, February 18, 2018 the N-terminal end, which is not resolved in LHCII crystal structures and con- relevance of P-Y to MCU channel function as well as mitochondrial functions. tains phosphorylation sites that are important for light-harvesting regulation. In this project, we determined specific Pyk2 phosphorylation site(s) in MCU 226-Plat and tested whether P-Y at these site(s) modulates MCU channel function. Structure of Photosystem I - Interplay between Robastness and Only three tyrosine residues were identified as potential phosphorylation candi- Complexity date sites for PTKs using phosphorylation prediction programs, and they are Nathan Nelson, Ido Caspy, Daniel Klaiman. conserved across all eukaryotic species. In-vitro kinase assays showed that pu- Biochemistry, Tel Aviv University, Tel Aviv, Israel. rified active Pyk2 phosphorylated purified full-length MCU. Next, P-Y levels at Plant Photosystem I (PSI) is one of the most intricate membrane complexes in Na- candidate sites were biochemically detected after Gq protein–coupled receptor ture. It is comprised of two complexes, a reaction center and light-harvesting (GqPCR) stimulation using cell lines stably expressing wild-type (WT)- or de- LHCI. An atomic-level structural model of higher plant PSI at 2.2 A˚ resolution phosphomimetic mutants of MCUs (MCU-YFs). This in-situ assay revealed has been constructed based on new crystal form. The structure includes 16 sub- that only two tyrosine sites increased P-Y levels in response to GqPCR stimu- lation. Finally, we assessed mtCa2þ uptake profiles in cells stably expressing units and more than 200 prosthetic groups, the majority of which are light harvest- þ WT- and MCU-YFs in response to cytosolic Ca2 elevation via live cell imag- ing pigments. The model reveals detailed interactions, providing mechanisms for þ ing using mitochondria-targeted Ca2 -sensitive biosensors. Although the over- excitation energy transfer and its modulation in one of Nature’s most efficient 2þ photochemical machine. Recently we solved the structure of trimeric PSI from expression of WT- and two of the MCU-YFs significantly accelerated mtCa ˚ uptake compared to non-transfected cells, overexpression of one of the MCU- Synechocystis at 2.5 A resolution. Several differences between the mesophilic 2þ 2þ and thermophilic PSI were revealed and the position of lipids between the mono- YFs failed to increase mtCa uptake in response to cytosolic Ca elevation. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2- mers was determined. Similarly the structure of monomeric PSI was determined. 2þ An operon encoding PSI was identified in cyanobacterial marine viruses. We dependent P-Y of MCU increases mtCa uptake via the MCU complex. generated a PSI that mimics the salient features of the viral complex containing 229-Plat PsaJ-F fusion subunit. The mutant is promiscuous for its electron donors and can UCP3: New Insights in Tissue Distribution and (Transport) Function accept electrons from respiratory cytochromes. We solved the structure of the Elena E. Pohl, Gabriel Macher, Karolina Hilse. PsaJ-F fusion mutant as well as a monomeric PSI at 2.8 A˚ resolution, with subunit Univ Vet Medicine, Vienna, Austria. composition similar to the viral PSI. The novel structures provided for the first UCP3 - a member of the mitochondrial uncoupling protein family - was discov- time a detailed description of the reaction center and antenna system from mes- ered in 1997. However, its physiological and transport functions are still far ophilic cyanobacteria, including red chlorophylls and cofactors of the electron from being understood. Previously we have described that UCP3 is present transport chain. Our finding extends the understanding of PSI structure, function in high amounts in brown adipose tissue, followed by heart and skeletal mus- and evolution and suggests a unique function for the viral PSI. cles1. Using two different models - embryonic stem cell differentiation and 227-Plat heart development - we have now demonstrated that UCP3 is a specific marker High-Resolution Sub-Ensemble Optical Spectroscopy Study of Protein for adult cardiomyocytes, which rely on fatty acid beta-oxidation. By investi- gation of the UCP3 recombinant protein reconstituted in planar bilayer mem- Dynamics and Energy Transfer in Pigment-Protein Complexes 2 Valter Zazubovits. branes , we revealed that its inhibition mechanism differs from that of UCP1 Physics, Concordia University, Montreal, QC, Canada. and we identified an inorganic phosphate, present in mitochondria at mM con- Pigment-protein complexes found in the organisms involved in photosynthesis centrations, as a new inhibitor of UCPs, capable to essentially decrease their offer a unique opportunity to explore native protein environments using optical activity under physiological conditions. spectroscopy methods. Single Complex Spectroscopy has recently been a tech- References: nique of choice for studying spectral dynamics in these pigment-protein systems. 1. Hilse, K. E. et al. The expression of UCP3 directly correlates to UCP1 abun- However, Non-Photochemical Spectral Hole Burning (NPHB) is capable of dance in brown adipose tissue. Biochim Biophys Acta 1857, 72-78 providing additional or competing information. (NPHB occurs due to small 2. Beck, V. et al. A new automated technique for the reconstitution of hydro- conformational changes in the protein that are triggered by optical excitation phobic proteins into planar bilayer membranes. Studies of human recombinant of the chromophore.) In particular, it can be shown that most of the spectral shifts uncoupling protein 1. Biochim.Biophys.Acta 1757, 474-479 (2006). observed in single complex experiments are in fact light-induced (and not occur- 230-Plat ring anyway while the observer is merely monitoring them) and constitute NPHB Mitochondrial Defects in Primary Osteocytes Derived from an ALS Mouse on a single-molecule level. Recently we undertook a detailed NPHB study of Model spectral dynamics in CP43 antenna complex of Photosystem II and Cytochrome Huan Wang, Jianxun Yi, Xinyang Xu, Xuejun Li, Yajuan Xiao, b6f. We also developed a unified approach to modeling NPHB and spectral hole Jingsong Zhou. recovery under various conditions. This approach relies on the presence of Kansas City University of Medicine and Bioscience, Kansas City, MO, USA. ‘‘spectral memory’’ - ‘‘burnt’’ systems returning to the original wavelengths While motor neuron death is a pathological hallmark of amyotrophic lateral scle- upon spectral hole recovery. All complexes we have explored so far exhibit bar- rosis (ALS), defects in other cell types may also contribute to ALS disease pro- riers in the same range, distinct from that of simple organic glasses. Qualita- gression. ALS patients experience progressive muscle wasting that may not only tively, however, barrier distribution shapes show great variability, with some exacerbate neuronal degeneration, but likely has a significant impact on bone local protein environments apparently being ‘‘glassier’’ than others. function. In our previous study, we have discovered severe bone loss in an I will also discuss the possible nature of the entities responsible for NPHB in ALS mouse model (G93A). The reduced bone density is closely related to the pigment-protein complexes, possible cooperative effects, how understanding development of muscle atrophy. In the current study, we examined mitochondria protein dynamics helps understand issues more directly related to photosyn- in osteocytes derived from the same ALS model at different disease stages. We thesis such as excitation energy transfer, etc. In particular, I will address how found that abnormal mitochondrial network and dynamics in primary osteocytes relatively slow excitation energy transfer, in Fo¨rster regime, affects various only occur in ALS mice after the ALS disease onset, indicating that mitochondria high-resolution spectra of pigment-protein systems. in osteocytes respond to muscle atrophy during ALS progression. Remarkably, 228-Plat the ALS mutation also directly causes mitochondrial dysfunction independent of Role of Tyrosine Phosphorylation of Mitochondrial Calcium Uniporter in muscle atrophy, because we observed abnormal mitochondrial morphology and Regulating Mitochondrial Calcium Homeostasis motility in cultured osteocytes (MLO-Y4) with overexpression of mitochondrial Jessica L. Cao1, Stephanie Adaniya1, Amy K. Landi1, Dong Qin Yang1, targeted SOD1G93A, when compared with osteocytes overexpressing the wild Bong Sook Jhun1, Shey-Shing Sheu2, Jin O-Uchi1. type SOD1. In addition, the overexpression of SOD1G93A enhanced the expres- 1Department of Medicine, Brown University, Providence, RI, USA, sion level of Dynamin-related protein 1 (Drp1), a key protein promoting mito- 2Department of Medicine, Thomas Jefferson University, Philadelphia, PA, chondrial fission activity, and reduced the expression level of optic atrophy USA. protein 1 (OPA1), a key protein related to mitochondrial fusion. A specific mito- Mitochondrial Ca2þ (mtCa2þ) uptake via the mtCa2þ uniporter (MCU) com- chondrial fission inhibitor (Mdivi-1) partially reversed the effect of SOD1G93A plex is a critical factor in determining cell survival or death. Basal tyrosine on mitochondrial network and dynamics, indicating that SOD1G93A likely pro- phosphorylation (P-Y) of the pore forming subunit, MCU has been reported motes mitochondrial fission, but suppresses the fusion activity. Our data provide via mass spectroscopy, and our lab reported that the activation of a Ca2þ- the first evidence that mitochondria show abnormality in osteocytes derived and reactive oxygen species (ROS)-sensitive protein tyrosine kinase (PTK), from an ALS mouse model. The accumulation of mutant SOD1G93A protein in- proline-rich tyrosine kinase 2 (Pyk2), increases P-Y of MCU and mtCa2þ up- side mitochondria directly causes dysfunction in mitochondrial dynamics in take. However, further research is required to determine 1) the identity of Pyk2- cultured osteocytes, which could be a potential mechanism underlying the specific phosphorylation sites within the MCU structure and 2) the functional bone loss during ALS progression.

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Posters 234-Pos Board B4 Mechanistic Picture of Allosteric Information Flow of HIV-1 Restriction Factor, SAMHD1 via MD Studies Posters: Protein Structure and Conformation: Kajwal K. Patra. Physics, IIT Guwahati, Aaaam, India, Guwahati, India. Computational Methods SAMHD1 is a human cellular enzyme that blocks HIV-1 infection in myeloid cells and non-cycling CD4þT cells. The enzyme is an allosterically regulated 231-Pos Board B1 triphosphohydrolase that modulates the level of cellular dNTP. Retroviral re- Ticking Mechanism of a Biological Clock striction is attributed to the lowering of the pool of dNTPs in the cell to a point Andy LiWang. where reverse transcription is impaired. A mechanistic understanding of the Chemistry & Chemical Biology, University of California at Merced, Merced, allosteric activation of the enzyme is still elusive. The catalytically active CA, USA. form of the protein is an allosterically triggered tetramerof the HD domain, Circadian clocks arose in organisms as an adaptation to the rotation of the earth. which is considered to be the necessary and sufficient structural unit for These clocks produce involuntary anticipation of sunrise and sunset by gener- dNTPase activity as well as restriction of HIV-1. The tetrameric form of the ating a succession of biochemical phases. In this talk, the mechanism of a protein complex is assembled by the GTP-dNTP combination. In study we model system, that of cyanobacteria, will be described. Briefly, it depends on have used both classical MD techniques along with the correlation network phosphorylation, long-range allostery, dynamics, and protein metamorphosis. analysis to study the dynamics and allosteric information flow across the active Because a simple mixture of clock proteins and ATP generate a persistent complex in the protein system. We have revealed the evidence of reciprocal macroscopic rhythm, the mechanism of the clock can be studied in real time allosteric ‘‘Handshakes’’ between adjacent monomers through correlation as it ticks. network analysis. We have also uncovered the allosteric links in SAMHD1 232-Pos Board B2 to demonstrate the flow of information across the assembled SAMHD1 Zooming in on Solvation Free Energy Surfaces in Atomistic Simulations tetramer. Matthias Heyden. School of Molecular Sciences, Arizona State University, Tempe, AZ, 235-Pos Board B5 USA. Investigating the Structure of the XPF-ERCC1 Functional Endonuclease Changes in free energy are generally the sum of multiple contributions that using a Computational Approach Francesco Gentile1, Jack A. Tuszynski2, Khaled H. Barakat3. include changes in the internal energy of flexible molecules, their conforma- 1 tional entropy and solvation free energy. Often these individual contributions Department of Physics, University of Alberta, Edmonton, AB, Canada, 2Department of Physics and Department of Oncology, University of Alberta, are large in magnitude and of opposite sign, leading to a significant compensa- 3 tion of favorable and unfavorable terms. To obtain a detailed understanding of Edmonton, AB, Canada, Faculty of Pharmacy and Pharmaceutical Sciences, thermodynamic driving forces that are responsible for conformational fluctua- University of Alberta, Edmonton, AB, Canada. Introduction: The XPF-ERCC1 endonuclease is responsible for the exci- tions of proteins and enzymes, aggregation, self-assembly, and molecular 0 recognition, microscopic insights into the origins of the distinct free energy sion of the damaged DNA strand at the 5 extremity in nucleotide excision contributions are required. We present here applications of a novel spatially repair (NER) and inter-strand cross-link (ICL) repair pathways. The activ- resolved analysis (3D-2PT) of local solvation enthalpy and entropy contribu- ity of this enzyme requires the dimerization of XPF and ERCC1 to obtain tions, which define the solvation free energy surface of individual biomolecules a functional endonuclease, and the DNA is cleaved within the catalytic site and solvent-mediated interactions between them. Our analysis is based on of the endonuclease, located on the XPF nuclease domain. Although atomistic molecular dynamics simulations, which we employ to quantify vari- different models have been proposed, currently there is not a consensus ations in the solvation free energy of proteins along conformational transitions. structural model for the heterodimeric form of the human endonuclease Further, we demonstrate how the results of our analysis can be employed to and for the DNA positioning on the surface of the enzyme. In this improve the description of solvent-mediated interactions in meso-scale simula- work, we propose a computational model for the functional XPF-ERCC1 tions with implicit solvent models. Finally, we correlate local solvent thermo- endonuclease and its binding to DNA. Methods: To build the minimal dynamics to chemical and topological properties of hydrated biomolecular functional structures of XPF (residues 667 to 916) and ERCC1 (residues surfaces and local retardations of dynamical processes in the hydration shell. 96 to 297), we used a combination of multiple sequence alignments and Such correlations elucidate the relevant microscopic factors that determine homology modelling. Representative conformations of the two proteins favorable and unfavorable solvation free energies and allow for the formulation were generated using long molecular dynamics simulations and clustering of computationally efficient predictions. analysis. Protein-protein and protein-DNA docking simulations, molecular dynamics simulations and free energy calculations were employed to 233-Pos Board B3 generate a model for the functional heterodimer and for the DNA binding Quantitative Understanding of Distances from Cross Linking Mass to it. Conclusions: We report the first computational study aiming to Spectrometry investigate the structural basis of the XPF-ERCC1 dimerization and Isaac Fillela-Merce1,2, Guillaume Bouvier1, Michael Nilges1,3. DNA binding at an atomistic level. Our model can help to interpret the 1Institut Pasteur, Paris, France, 2University Pompeu Fabra, Barcelona, Spain, deleterious effect of mutations occurring in the two proteins, and can be 3CNRS UMR 3528, Paris, France. also used for rational design new DNA repair inhibitors for combination Crosslinking mass spectrometry is increasingly used for structural charac- cancer therapy. terization of multisubunit protein complexes. We recently introduced an automated modeling strategy dedicated to large protein assemblies [Ferber 236-Pos Board B6 M et al., Nature Methods 2016] that uses a form of spatial restraints Dynamical Analysis Methods for Protein Folding Simulations that realistically reflects the distribution of experimentally observed cross- Ayori Mitsutake, Hiroshi Takano. linked distances; automatically deals with ambiguous and/or conflicting Physics, Keio University, Yokohama, Japan. crosslinks and identifies alternative conformations within a Bayesian As longer and larger MD simulations are performed, it is more important to framework; and allows subunit structures to be flexible during conforma- develop analysis methods to investigate dynamics or kinetics of proteins. tional sampling. Relaxation mode analysis (RMA) was developed to investigate ‘‘dynamic’’ The strategy relies on imposing distance restraints between the linked resi- properties of polymer, homo-polymer, systems. In RMA, slow relaxation dues with euclidian distances, a severe approximation since it neglects the modes are extracted from molecular dynamics simulations. Recently, RMA fact that the chemical linkers cannot overlap with the protein matrix. has been applied to proteins, hetero-polymer systems to investigate dynamic Methods have been proposed to calculate the shortest path between two res- properties of structural fluctuation. Here, we present results of RMA for idues through free space [Kahraman A et al, Bioinformatics 2011]. Here we simulations of folding protein simulations near transition temperature. present a novel and efficient method to perform this task and use it to get a RMA extracts effective order parameters from the simulations to identify better understanding of the reasons for the observation or non-observation of local-energy-minimum states and transition between them. The free-energy a particular crosslink. We also develop a model for the crosslinked side surfaces obtained from RMA provide a clear understanding of the transitions chains that reflects their flexibility. Finally we present a straightforward not only between local minimum-energy states but also between the folded way to integrate the shortest path distances as restraints in our modeling and unfolded states, even though the simulation involved large conforma- strategy. tional changes.

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237-Pos Board B7 In order to evaluate our hypothesis, we performed 6 molecular dynamics sim- PDB2CD: A Web-Based Application for the Generation of Circular ulations of each SCR7 isoform for a total of 35 ms per isoform. Though initial Dichroism Spectra from Protein Atomic Coordinates structures retrieved from the Protein Data Bank suggested conformations of Elliot D. Drew, Lazaros Mavridis, Robert W. Janes. isoforms Y402 (2jgx) and H402 (2uwn) are similar, Markov chain representa- School of Biological and Chemical Sciences, Queen Mary, University of tions of our simulations suggest that the conformational dynamics and equilib- London, London, United Kingdom. rium distribution of conformations for each isoform differ. For example in the Circular Dichroism (CD) spectroscopy is widely used to determine the second- most probable state for the tyrosine isoform, Y402 interacts solely with residues ary structure content of proteins. However, more structural features than sec- between turn 2 and 3; however, in the most probable state for the histidine iso- ondary structure alone contribute to the shape and magnitude of the spectra form, H402 interacts primarily with residues between the N-terminus and turn obtained by CD. The juxtapositioning of these secondary structure elements 1. Given the structural differences involving position 402 and proximal resi- in its tertiary structure can also contribute to the CD spectrum of a protein dues known to bind GAGs, our simulations suggest a structural basis for for example. decreased recognition of self in the risk isoform. PDB2CD is an empirical-based approach to the prediction of CD spectra from protein atomic coordinates and here we present extensions to the original pack- 240-Pos Board B10 age1. The method combines three levels of structural features within the confor- An Ambiguous View of Protein Architecture 1  2 2 mation of a protein; its percentage secondary structure content, the topological Guillaume Postic , Charlotte Perin , Yassine Ghouzam , Jean-Christope Gelly2. arrangement of discrete secondary structure elements, and the overall structural 1 2 similarity of the query protein to the 71 proteins in the SP175 dataset, the ‘gold University Pierre et Marie Curie, Paris, France, University Paris Diderot, standard’ set obtained from the Protein Circular Dichroism Data Bank Paris, France. (PCDDB). Qualifying something as ambiguous means that several interpretations are The approach employed in PDB2CD was able to generate a significant number possible to describe it. We have proposed that such view might be used to of the CD spectra associated with the 71 proteins in this dataset with excellent analyze protein architecture into structural domains (Postic et al., Science accuracy using a leave-one-out cross-validation process. The method also Adv, 2017).The idea of dividing a protein structure into subunits, called do- generated spectra in good agreement with those of a test set of 14 proteins mains, was introduced more than four decades ago by Donald B. Wetlaufer, from the PCDDB. PDB2CD is a user-friendly web-based package providing who defined protein domains as structurally compact and separate regions of a novel approach to enable researchers to produce CD spectra from protein the macromolecule. However, since this geometrical definition, many manual atomic coordinates. Our additions include, for example, an extended wave- and automated assignments of structural domains have been proposed, based length range for the predicted CD spectrum and the ability to download the im- on criteria such as folding independence, function, thermodynamic stability, age files. or motions. As a result, the same protein may be viewed differently depending Available at: http://pdb2cd.cryst.bbk.ac.uk on the criteria used for domain partitioning. Therefore, it may be more appro- Support: BBSRC UK Grant (J019194) priate to consider that protein structures may be ambiguous and have different, 1 Mavridis, L. and Janes, R.W. (2017) Bioinformatics 33: 56-63. but equally valid, domain delineations.Unfortunately, the a priori, based on our knowledge, can hamper our faculty to recognize more than one way to split an object—a protein, in this respect— thus leading to miss interesting pros- 238-Pos Board B8 pects.We have recently proposed a new automated method able to produce Structure-Function Relationships in Protein Complexes alternative domain delineations of protein structures (Postic et al., Science Petras Kundrotas, Saveliy Belkin, Ilya Vakser. Adv, 2017). Our conceptually new method is based on the hierarchical merging Computational Biology, University of Kansas, Lawrence, KS, USA. of Protein Units, which describe the protein structure organization at an inter- Structural and functional characterization of protein-protein interactions is mediate level, between secondary structures and domains.We have extensively important for understanding molecular mechanisms in a living cell. benchmarked our method using several reference datasets of domain assign- Structure-function relationships are usually studied for individual proteins. ments made by either human experts or algorithms. We have shown the signif- We present a quantitative analysis of structure-function relationships for pro- icance of our approach with examples covering a wide range of problems teins and protein-protein complexes, based on 4950 protein-protein structures related to protein structure, folding, function, and evolution. from the DOCKGROUND resource (http://dockground.compbio.ku.edu). Structural similarity of individual proteins, protein complexes, and protein- 241-Pos Board B11 protein interfaces was quantified by TM-score. Functional similarity was rep- Generalization of the Elastic Network Model for the Study of Large resented by GO-score, which quantifies similarity between two sets of Gene Conformational Changes in Proteins Ontology (GO) terms (we used only the molecular function domain of the Adolfo Poma, Panagiotis Theodorakis. GO annotation). The GO-scores were calculated for the individual proteins Institute of Physics, Polish Academy of Sciences, Warsaw, Poland. and the protein complexes separately, based on the complete sets of GO terms, The Elastic Network (EN) is a prime model that describes the long-time dy- and on the GO terms related to the function of the complex only. The results namics of biomolecules. However, the use of harmonic potentials renders showed only a weak correlation of structural and functional similarity in protein this model insufficient for studying large conformational changes. Here, we complexes. Proteins and their interfaces were further clustered based propose a model based on the EN, a harmonic approximation described by on the structural and functional similarity, and the functional/structural vari- Lennard-Jones interactions for far contacts, and Go-type native contacts ob- ability of the clusters was analyzed. The function of structurally similar inter- tained from the standard overlap criterion with the latter describing hydrogen faces was determined to be more variable than that of the structurally similar bonds, ionic bridges and hydrophobic/hydrophilic interactions. Our results full proteins. based on Normal Mode Analysis show excellent agreement with the EN model. Moreover, we apply large forces along the N- and C-termini in order to study a 239-Pos Board B9 large conformational change (i.e. protein stretching), our pulling simulations Molecular Basis for the Link between Macular Degeneration and a Single reproduce the experimental data on the maximum force of the unfolding of a Nucleotide Polymorphism protein domain. We anticipate that our work will provide new venues for the Reed E.S. Harrison, Dimitrios Morikis. EN in a broader range of problems in biology, including folding of proteins Bioengineering, University of California, Riverside, Riverside, CA, USA. and protein-docking prediction. A single nucleotide polymorphism, tyrosine 402 to histidine (Y402H), within the gene encoding complement Factor H (FH) is known to predispose individuals to 242-Pos Board B12 acquiring age-related macular degeneration later in life. This polymorphism has Secondary Structure Elements - Annotations and Schematic 2D Visualiza- been shown to significantly affect binding of the FH splice variant named FHL-1 tions Stable for Individual Protein Families to glycosaminoglycans (GAGs), extracellular matrix components, and products Radka Svobodova Varekova1,2, Adam Midlik1,2, Ivana Hutarova of oxidative stress. Structurally, 20 short consensus repeats (SCR) comprise FH, Varekova2, Jan Hutar2, Veronika Navratilova3, Jaroslav Koca1,2, while FHL-1 contains only the first 7 SCR. FHL-1 is thought to be an important Karel Berka3. regulator of complement in the macula where it can diffuse more freely through 1CEITEC - Central European Institute of Technology, Masaryk University, Bruch’s membrane than can FH. SCR7 from FHL-1 and FH contains position Brno, Czech Republic, 2National Centre for Biomolecular Research, Faculty 402 and exhibits a positive charge that promotes association with negatively of Science, Masaryk University, Brno, Czech Republic, 3Department of charged GAGs. Since the Y402H mutation results in decreased binding affinity Physical Chemistry, Palacky University, Olomouc, Czech Republic. of FHL-1 for GAGs, we hypothesized that the mutation results in side-chain re- Composition and organization of secondary structure elements (SSEs), such as arrangements of amino acids involved in binding GAGs. alpha-helices and beta-sheets, are characteristic for protein families and they

BPJ 8556_8558 Sunday, February 18, 2018 47a participate in formation of protein fold. They are however often influenced by ible fitting (MDFF) with de novo structure prediction algorithms in an the sequence variability and ligand binding. For this reason, identification of interactive way allowing for incorporation of user expertise into model build- similarities and differences between SSEs can help us in the analysis of individ- ing. This approach is in particular beneficial as presently it is highly chal- ual proteins within a protein family. lenging to employ crystallographic modeling software to obtain models for To utilize the SSEs for research of individual protein families, we need to have cryo-EM densities with a resolution of 4A˚ . the SSEs easily and automatically intercomparable within one protein family. Employing this approach we obtained in collaboration with the Baumeister Specifically, the corresponding SSEs should have the same name (annotation) department (MPI for Biochemistry) the first atomic structure of the human and there should be a transparent schema of their localization in the protein 26S proteasome with bound nucleotides (Schweitzer et al. PNAS 2016) based structures. on a 3.9 A˚ resolution cryo-EM density. In a follow up study, we derived four Unfortunately, SSE annotations are still performed mainly manually and uni- more structural models of the yeast proteasome in different conformational versal automatic approach to assign SSE names is not available yet. Moreover, states (Wehmer et al. PNAS 2017). These models provide the first atomic in- current methods focused on 2D visualization of SSEs (e.g., PROMOTIF, Pro- sights as to how ATP hydrolysis in the engine of the proteasome unwinds pro- origami, HERA) do not consider information about real distances of SSEs. teins and steers them towards the degradation chamber. Therefore, even when two proteins from the same family differ only slightly, their SSE 2D diagrams can be totally different. 245-Pos Board B15 For this reason, we developed a tool set which can perform SSE annotation and Structural Analysis of Human Glycoprotein Butyrylcholinesterase using 2D visualization in such a way that structural information is kept. Applicability Atomistic Molecular Dynamics: The Importance of Glycosylation Site of this approach is shown in a case study focused on cytochromes P450. This ASN241 Austen Bernardi1, Karl Kirschner2, Roland Faller1. protein family of drug-metabolizing enzymes has currently available more 1 2 than 750 structures from about 30 organisms and each cytochrome P450 con- Chem Eng, UC Davis, Davis, CA, USA, Bonn-Rhein-Sieg University of tains more than 20 SSEs for which there is a stable annotation used through the Applied Sciences, Sankt Augustin, Germany. community. Our approach can be further extended to other protein structural Human butyrylcholinesterase (BChE) is a glycoprotein capable of bio- families, which will allow family-wide SSE annotations and comparisons in scavenging toxic compounds such as organophosphorus (OP) nerve agents. a simple visual manner. For commercial production of BChE, it is practical to synthesize BChE in non-human expression systems, such as plants or animals. However, the glyco- 243-Pos Board B13 sylation profile in these systems is significantly different from the human glyco- Interactive 3D Macromolecular Structure Data Mining with MolQL and sylation profile, which could result in changes in BChE’s structure and Litemol Suite function. From our investigation, we found that the glycan attached to David Sehnal1,2, Mandar Deshpande3, Alexander Rose4, Lukas Pravda1,2, ASN241 is both structurally and functionally important due to its close prox- Adam Midlik1,2, Radka Svobodova´Varekova´1,2, Saqib Mir3, Karel Berka5, imity to the BChE tetramerization domain and the active site gorge. To inves- Sameer Velankar3, Jaroslav Koca1,2. tigate the effects of populating glycosylation site ASN241, monomeric human 1Central European Institute of Technology, Brno, Czech Republic, 2National BChE glycoforms were simulated with and without site ASN241 glycosylated. Centre for Biomolecular Research, Brno, Czech Republic, 3Protein Data Our simulations indicate that the structure and function of human BChE are Bank in Europe (PDBe), Hinxton, United Kingdom, 4RCSB Protein Data significantly affected by the absence of glycan 241. 5 Bank, San Diego, CA, USA, Palacky´ University, Olomouc, Czech Republic. 246-Pos Board B16 Macromolecular 3D structure is critical in understanding the function and Accurate Prediction of Forster Resonance Energy Transfer during mechanism of biomacromolecules. Recent advances in 3D structure determina- Co-translational Folding with Coarse-Grained Molecular Dynamics tion techniques have facilitated the study of large macromolecular machines, Simulations leading to a rapid increase in the number, size, and complexity of bio- Daniel A. Nissley, Edward P. O’Brien. macromolecular structures available in the Protein Data Bank (PDB). Facili- Chemistry, The Pennsylvania State University, University Park, PA, USA. tating data mining of this large data set will not only help in addressing the Protein folding, the assembly of a protein molecule or domain into a tertiary challenges in translational research but also allow improved understanding of structure, can occur as a protein is being synthesized by the ribosome in a pro- biological systems. The LiteMol suite offers an innovative approach to data cess referred to as co-translational folding. Forster resonance energy transfer archiving (BinaryCIF), data delivery (CoordinateServer and DensityServer), has recently emerged as a technique for monitoring co-translational folding and 3D molecular visualization (LiteMol Viewer). The newly developed in vitro and probing its pathways. Holtkamp et al. 2015 found using FRET as- Molecular Query Language (MolQL) enables describing substructures in says that the N-terminal domain of the E. coli protein Hemk folds co- macromolecules using a wide range of expressions, including the support for translationally via a compact state. Our computational study addresses key Jmol, PyMol, and VMD selections; and the ability to extract data based on questions about the co-translational folding of the Hemk N-terminal domain. structural relationship between individual parts of the molecule. Together, We will determine if coarse-grained molecular dynamics simulations with these technologies allow users to mine the 3D structural data in the PDB explicit representations of FRET dyes are able to accurately reproduce exper- (e.g., ligand binding sites) by providing a user friendly web based interface imental FRET curves. We will also ascertain by what pathways the co- to aid translational research and make it easier for non experts to assess and translational folding of HemkNTD occurs and to what extent FRET can serve exploit 3D macromolecular structure information. Visit litemol.org and as an order parameter for folding. This project will also experimentally verify molql.org for more information. our approach to modeling co-translational protein folding. 244-Pos Board B14 247-Pos Board B17 The Protein Recycling Machine of the Cell - Insights through a Novel Flexibility of Free and AcrB-Bound AcrA in the AcrAB-TolC Multidrug Hybrid Integrative Modeling Approach Efflux Pump of Escherichia coli Determined using 3D PMFS Till Rudack1,2. Anthony Hazel, James C. Gumbart. 1Department of Biophysics, Ruhr University Bochum, Bochum, Germany, Physics, Georgia Institute of Technology, Atlanta, GA, USA. 2NIH Center for Macromolecular Modeling and Bioinformatics, University Key components of the emerging global epidemic of antibiotic resistance are of Illinois at Urbana Champaign, Urbana, IL, USA. the multidrug efflux pumps of pathogenic, Gram-negative bacteria, which Protein recycling is a key process crucial to a wide spectrum of regulatory pro- span the periplasmic region between the inner and outer membranes of the cesses within living cells. The executive player in this process is an ATP hydro- cell. These protein complexes are an innate resistance mechanism, removing lysis driven molecular machine called 26S proteasome, that recruits, unfolds, harmful antibiotics from bacteria. Until 2014 many aspects of the mechanisms and degrades poly-ubiquitin tagged proteins through a complex interaction for pump activity remained elusive due to the lack of structural data. Since then clockwork of 33 different protein subunits. Given its critical role, the protea- several increasingly detailed electron microscopy maps of an entire efflux some is involved in multiple human diseases, and it serves as a perfect target pump complex, AcrAB-TolC, have been resolved, resulting in atomic-level for a plethora of different drugs, most prominently, those commonly used in structural models. Using these new models, we performed molecular dynamics chemotherapy of cancer. Despite its substantial role in the cell’s life cycle, simulations to study one of the key components of the protein complex, AcrA, the proteasome is one of the last key molecular machines, which detailed which connects the inner-membrane-bound AcrB to the outer-membrane- atomic mechanism still remains elusive. bound TolC. We determined the flexibility of free AcrA by calculating a Driven by the revolutionary advance of electron microscopy I developed an three-dimensional potential of mean force (PMF) focused on three angles integrative modeling approach to derive structural models from 3 to 12 A˚ res- that govern AcrA’s conformational dynamics. AcrA shows a wide range olution cryo-EM densities. My approach combines molecular dynamics flex- of accessible orientations, with two main energy basins separated by a low

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(< 4 kT) barrier, consistent with previously reported equilibrium simulations. of desmoplakin is composed of multiple tandem spectrin repeat (SR) domains, The conformation in one of the basins is similar to the AcrB-bound state, sug- with a single SH3 domain positioned on top of one of the SR domains. Previ- gesting that assembly proceeds by conformational selection. Additional PMF ously published studies suggest that this SH3 domain may be a hotspot for var- calculations of AcrA conformation when bound to AcrB further elucidate iants linked to arrhythmogenic cardiomyopathies (AC). While some of these how binding to TolC occurs. The 3D PMF calculations required petascale variants are associated with decreased amounts of desmoplakin protein, their supercomputing resources and tens of microseconds in aggregate to carry out. molecular mechanism of action remains undefined. Here, we examine these specific variants in silico and in vitro. CD and fluorescence analysis show 248-Pos Board B18 that these mutations do not significantly perturb the global desmoplakin struc- Identifying a Conformational Transition Critical for Carbapenem Drug ture and stability. However, MD simulations suggest significant changes to Resistance local stabilizing interactions within the SH3 domain. Thus, these studies pro- George A. Cortina, Peter Kasson. vide a compelling molecular mechanism of action for at least a subset of AC Biomedical Engineering, University of Virginia, Charlottesville, VA, USA. cases. The KPC-2 carbapenemase enzyme is responsible for extreme drug resistance in the majority of carbapenem-resistant gram-negative bacterial infections in 251-Pos Board B21 the US. A better understanding of what permits KPC-2 to hydrolyze carbape- How Lymphoma Mutation Disrupts Functional Conformation of IKK2 nem antibiotics and how this might be inhibited is thus of great importance Under the Lens of Computational Microscope to the infectious disease community. By correlating molecular dynamics sim- Thuy Tien T. Nguyen, Jamie Schiffer, Gourisankar Ghosh, Rommie Amaro. ulations with experimental enzyme kinetics, we have identified conformational Chemistry and Biochemistry, University of California, San Diego, La Jolla, changes that control KPC-2’s ability to hydrolyze carbapenem antibiotics. CA, USA. Beta-lactamases similar to KPC-2 interconvert between catalytically permis- IKK complex is the critical component of the NF-kB pathway which is respon- sive and catalytically nonpermissive forms of an acylenzyme intermediate in sible for proinflammatory gene expression and cellular response. The IKK hydrolysis, the critical point at which non-resistant enzymes arrest. Using mo- complex is an important drug target to develop treatments for chronic inflam- lecular dynamics simulations, we have identified a similar equilibrium in KPC- matory disorders as well as an array of different cancers. Mutated lysine residue 2 and analyzed the determinants of this conformational change. Because the numbered 171 to glutamic acid (K171E) in IKK2, the main subunits of IKK conformational dynamics of KPC-2 are complex and sensitive to allosteric complex, presents in lymphomas. This mutation constitutively activates the changes, we developed an information-theoretic approach to identify key deter- pathway. Due to IKK’s large multimeric structure and highly variable dy- minants of this change. We measured unbiased estimators of the reaction coor- namics, experimental studies encounter many difficulties; the protein-protein dinate between catalytically permissive and nonpermissive states, performed full signalosome complex is still not well understood. Using biochemical visu- information-theoretic feature selection, and then validated the top changes alization and molecular dynamics software, structures of IKK2 in both mutated via restrained molecular dynamics simulations. From this process, we identified and nonmutated forms have been simulated with molecular dynamics (MD). two binding-pocket residues that control the conformational transitions be- The aims of this research are looking at clustering with RMSD (root-mean- tween catalytically active and inactive forms of KPC-2. Mutations to these res- square-deviation) and small molecule docking. These results provide chemical idues both lower the stability of the catalytically permissive state in simulations composition of the pocket openings together with concrete potential drug struc- and have reduced experimental kcat values. This understanding can be lever- tures targeting this mutant. aged to predict the drug resistance of new KPC-2 mutants and also help design inhibitors to combat extreme drug resistance. 252-Pos Board B22 Rigid Rod Model for the Disordered Domains of Ribosomal Stalk Proteins 249-Pos Board B19 P1P2 Simulations Suggest a Structural Basis for Nicotinic Receptor Activation Simon Kit Sang Chu, Yi Wang. by Agonists Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong. Sushree Tripathy1, Wenjun Zheng1, Anthony Auerbach2. In eukaryotes, the ribosomal stalk proteins, which are responsible for the 1Physics, University at Buffalo, Buffalo, NY, USA, 2Biophysics and recruitment of transcription factors, consist of two P1P2 heterodimers attached Physiology, University at Buffalo, Buffalo, NY, USA. onto one copy of P0. Recent NMR structure of full-length P1P2 reveals that Nicotinic acetylcholine receptors (AChRs) are allosteric protein ion channels their C-terminals are disordered in nature and can extend up to 125A away that switch between resting and active conformations under the influence of from the center of the heterodimer. Here, we report a rigid rod model aimed agonists. Fetal endplate nAChRs have 2 agonist binding sites located at a-d at sampling the structures of the disordered C-terminal tails of P1P2. With and a-g subunit interfaces. Single-channel analyses show that for a series of sequence-dependent angles and dihedrals, our model is constructed from all- agonists that are related structurally to ACh, the binding energy to active sites atom (AA) molecular dynamics (MD) simulations of short P1P2 segments. is approximately double of that to resting sites. To understand the physical basis Despite its apparent simplicity, the end-to-end distance and radius of gyration of this binding energy ratio, we performed detailed structural and energetic an- computed with our model are found to be in decent agreement with extensive alyses of resting vs. active sites (a-d and a-g) occupied by ACh, TMA, CCh or AA simulations of the entire disordered C-terminals of P1P2. Using our model, choline. Homology models were built (Modeller), ligands were docked (Auto- we further investigate the experimentally observed correlation between the dock) and approximate energies were calculated from 50 ns MD trajectories recruitment efficiency of the ribosome inactivating proteins and the length of (Charmm36). The resting and active binding energies (and their ratio) esti- P1P2 C-terminal tails. Overall, results of our calculation are in good agreement mated in silico were in good agreement with those obtained by using electro- with experimental measurements based on truncation and insertion studies of physiology. In the simulations, at both sites agonists were positioned more P1P2, indicating that our rigid rod model offers an efficient and promising deeply inside a more-compact aromatic binding pocket, active versus resting. approach in structural and functional study of the disordered domains in the The distance between the quaternary nitrogen of the agonist and the volumetric ribosome stalk proteins. centroid of the pocket correlated with the binding energy ratio. Other structural parameters, including a differential occupancy of water in the pocket, are 253-Pos Board B23 candidates for contributing to this ratio, which is the driving force for receptor Molecular Dynamics of Streptococcus pnuenomae and Corynebacterium activation by agonists and the cellular response. diphtheriae Pili Emmanuel Naziga, Jeff Wereszczynski. 250-Pos Board B20 Illinois Institute of Technology, Chicago, IL, USA. Desmoplakin AC Mutations’ Affect on Structure and Stability of Its NH2- Pili are long filamentous protein structures that protrude from bacterial surfaces Terminus and increase their virulence. Structurally they are polymers built from mono- Taylor Albertelli1, Heather R. Manring2, Stuart Campbell3, meric pilin constituents via intermolecular isopeptide bonds by the action of Maegen A. Ackermann2, Nathan Wright1. cysteine transpeptidase sortases enzymes. Pili are constructed to withstand hy- 1Department of Chemistry and Biochemistry, James Madison University, drodynamic forces of the order of hundreds of piconewtons in the cell to help Harrisonburg, VA, USA, 2Department of Physiology & Cell Biology, The bacteria attach to surfaces, therefore, it is important to understand the mecha- Ohio State University, Columbus, OH, USA, 3Department of Biomedical nism by which they overcome such external perturbations since this is pertinent Engineering, Yale University, New Haven, CT, USA. to their function as virulence factors. To combat external forces pili are struc- Desmoplakin is a large (260 kD) protein in the desmosome, a subcellular struc- turally reinforced by additional intramolecular isopeptide bonds which have ture that links the cytoskeleton of one myocyte to that of its neighbor. In the been shown to be important in their mechanical stability. In this work, we heart, the desmosome works to propagate the contractile force and allows for use all atom molecular dynamics simulations to study the conformational dy- the synchronized, strong contractions of the human heart. The N-terminal third namics of monomeric and dimeric forms of the pilins of streptococcus

BPJ 8556_8558 Sunday, February 18, 2018 49a pnuenomae (RrgB) and corynebacterium diphtheriae (spaA) in the presence 256-Pos Board B26 and absence of intramolecular isopeptide bonds. The results from conventional, Conformational Dynamics of Dopamine b-Hydroxylase by Computer Sim- accelerated and steered molecular dynamics calculations show that the isopep- ulations tide bonds restrict pili conformational dynamics and limit structural change in Alida Besch, Alessandro Cembran. the presence of external forces. Our results agree with the proposal that isopep- University of Minnesota Duluth, Duluth, MN, USA. tide bonds provide mechanical stability and elucidates the molecular basis for Dopamine b-hydroxylase (DBH) is an enzyme that catalyzes the conversion of this observation. dopamine into norepinephrine. An imbalance of these neurotransmitters can lead to many neurological and psychiatric disorders. A key step in the mechanism 254-Pos Board B24 involves an electron transfer between two copper centers. Based on the structure of Modeling and Conformational Analysis of Cyclotides, a Class of Macrocy- other hydroxylases, mechanisms invoking organized water networks have been clic Disulfide Bonded Plant Peptides proposed, where the electron is transferred over a distance greater than 10 A˚ . Neha V. Kalmankar1,2, P. Balaram2,3, Sowdhamini Ramanathan2, X-ray crystallography has recently shown that DBH is a homodimer in which Radhika Venkatesan2. ˚ 1 the distance between the two copper centers can span both short (5 A) and long The Institute of TransDisciplinary Health Sciences and Technology (TDU), (15 A˚ ) distances. Our hypothesis is that the protein can switch between open Bengaluru, India, 2National Centre for Biological Sciences, Bengaluru, India, 3 and closed states at a rate that is compatible with catalysis. In the closed state, Indian Institute of Science, Bengaluru, India. the short-distance between the two-copper center is exploited to transfer the elec- Cyclotides are a novel class of disulfide-rich macrocyclic peptides (26-31 res- tron, whereas the open state facilitates substrate binding and release. To test this idues), formed by cyclization of a gene encoded, linear precursor in specific hypothesis, we used molecular dynamic simulations. We have developed the pa- plant species. In addition to the circular backbone they form a cyclic cystine rameters necessary to describe the copper centers with the CHARMM molecular knot (CCK) arrangement formed by a conserved six cysteine framework mechanics force field, reconstructed regions missing in the structure, and refined (Cys I-IV, II-V, III-VI). They can be divided into two subfamilies i.e Mo¨bius the structure to better account for disulfide bond formation and hydrogen bond net- and Bracelet, based on presence or absence of a cis-proline residue in loop 5, works. Finally, we used free energy perturbations of Naþ,Kþ,Mg2þ,andCa2þ to respectively, that creates a twist in the peptide backbone. We have analyzed determine the most stable ion configuration in the putative ion binding site in the 38 X-ray/NMR structures from PDB, from both subfamilies, for structure DOMON domain. The results from our initial computer simulations are compat- and sequence signatures. Using peptidomics and transcriptomics reported se- ible with the proposed hypothesis. If our hypothesis stands further testing, it would quences ( 70) from Clitoria ternatea plant, based on size of interlinking loops provide an alternative mechanism that can explain the electron transfer between between two adjacent Cys residues, ‘cliotides’ may be characterized into two the copper centers without the intervention of organized water networks. broad subclasses: 33 sequences of ‘3-4-4-1-4-{4-8}’ motif (predominantly Mo¨bius kind) and 36 sequences of ‘3-4-6/7-1-4-{4-7}’ motif (all Bracelet); 257-Pos Board B27 where the numbers represent the length of the intervening peptide segments. Remarkable Similarity in Plasmodium falciparum and Plasmodium vivax Thus far only one crystal structure (PDB 3E4H) of a cyclotide (Mo¨bius) has Geranylgeranyl Diphosphate Synthase (GGPPS) Dynamics and its Impli- been reported, while several NMR derived structures are available for the cation for Anti-malarial Drug Design Bracelet conformation. The relative paucity of high resolution structures Aishwarya Venkatramani1, Clarisse Gravina Ricci1, Eric Oldfield2, further calls for development of improved computational methods for predict- J. Andrew McCammon1. ing disulfide-rich peptide structures and interactions, using only sequence infor- 1Pharmacology, UCSD, San Diego, CA, USA, 2Chemistry, University of mation. We have used the random conformation generation algorithm Illinois at Urbana-Champaign, Urbana-Champaign, IL, USA. (RANMOD), which builds Ramachandran allowed conformations for peptides Malaria, mainly caused by Plasmodium falciparum and Plasmodium vivax, has of defined length by assigning stereochemically accessible local conformations been a growing cause of morbidity and mortality, globally. While P. falciparum at each residue, in conjunction with MODIP disulfide modelling algorithm to is more lethal than is P. vivax, there is a vital need for effective drugs against both generate linear precursors with three disulfide bridges, followed by an energy species. Geranylgeranyl diphosphate synthase (GGPPS) is an enzyme involved minimization routine to form cyclizable conformations. ESI-MS analysis of in the biosynthesis of quinones and in protein prenylation, and has been proposed Clitoria ternatea extracts reveal the presence of masses corresponding to to be a malaria drug target. However, the structure of P. falciparum GGPPS dimeric and trimeric species of cyclotides, which may arise from intermolec- (PfGGPPS) has not been determined, due to difficulties in crystallization. ular disulfide bond formation. Here, we created a PfGGPPS model using the homologous P.vivax GGPPS X-ray structure as a template. We simulated the modeled PfGGPPS as well as 255-Pos Board B25 PvGGPPS using conventional and Gaussian accelerated molecular dynamics Hydrophobic Effect: The Entropic Structure of the Protein Hydration in both apo- and GGPP -bound states. The MD simulations revealed a striking Interface similarity in the dynamics of both enzymes with loop 9-10 controlling access Guillermo Ibal, Brian Oye, Hyun Joo, Jerry Tsai. to the active site. We also found that GGPP stabilizes PfGGPPS and PvGGPPS University of the Pacific, Stockton, CA, USA. into closed conformations and via similar mechanisms. Shape-based analysis of Characterizing the interactions between protein and water is quintessential to the binding sites throughout the simulations suggest that the two enzymes will be protein folding and dynamics. As the driving force of protein folding, the hy- readily targeted by the same inhibitors. Finally, we produced three MD-validated drophobic effect is mainly characterized by burial of non-polar surface area conformations of PfGGPPS to be used in future virtual screenings for potential that indirectly measures the increase in water entropy. A more direct approach new anti-malarial drugs acting on both PvGGPPS and PfGGPPS. is to understand how polar and nonpolar residues arrange on the protein surface in a way to maximize the entropy of solvating water, which minimizes the 258-Pos Board B28 chemical potential of overall folded protein system. At the protein-water inter- pH Sensitive Conformational Changes Responsible for the Anomalous face, distribution of hydrophilic and hydrophobic groups determines the Behavior of Ionizable Residues in the Hydrophobic Interior of SNase arrangement of water molecules, which directly relates to the water entropy. Ankita Sarkar1, Pancham Lal Gupta2, Adrian E. Roitberg2. In maximizing water entropy, proteins need to minimally disturb water’s 1Department of Physics, University of Florida, Gainesville, FL, USA, hydrogen bonding network by presenting interfaces very close to water: the po- 2Department of Chemistry, University of Florida, Gainesville, FL, USA. lar group distribution on the protein surface should mimic the bulk water struc- Internal ionizable residues in certain proteins are responsible for crucial ture. To investigate water entropy, many near native protein structures in water biological processes like energy transduction and enzyme catalysis. These were simulated using molecular dynamics. A dynameomics library of over 400 residues display anomalous pKa values. An atomistic understanding of protein domains were run using the CHARMM force field implemented in the factors determining the anomalous pKa values of internal ionizable residues NAMD package. The distribution of pairwise polar atom distances connected is important in getting insights into the mechanisms driving these important through the hydrogen bonding network of waters were collected and analyzed. biological processes. We study the diversity of the pH sensitive conforma- The polar group distribution holds a striking resemblance to the radial distribu- tional changes undergone by different mutants of staphylococcal nuclease tion function of bulk water. The distribution of polar groups are also classified (SNase), driven by the presence of internal ionizable residues. We carried depending upon the secondary structure, and are also categorized according to out constant pH replica exchange molecular dynamics simulations the nature of the polar group atoms such as side chain atoms and backbone (pH-REMD) in explicit solvent using the AMBER suite, implemented to atoms. Because these results imply that the entropy of water can be directly run in Graphics Processing Units (GPUs). Our calculations show that the calculated from the distribution of polar groups on the protein surface, this pKa values of these internal residues are significantly deviated from their research provides a stepping-stone in developing more accurate potential func- intrinsic pKa values and are mostly in good agreement with the experiments. tions to describe solvation free energies of proteins. We present thermodynamic models to calculate the ‘conformation-specific

BPJ 8556_8558 50a Sunday, February 18, 2018

pKa’ of the water-exposed and buried conformations of the internal residues open and closed state. In the ribosome-selB complex, our method further re- and explain it in association with the observed ‘apparent pKa’. The present fines structural transitions for GTPase activation. computational study provides an atomistic understanding in terms of the In our work, the free energy for each molecule conformation under a cryo-EM structural determinants of the anomalous pKa of internal ionizable residues, experiment is derived from a probabilistic formulation of the complete cryo- besides reinforcing the latest experimental findings. EM measurement process. It is shown why a set of reconstructed densities is a good approximation to the rigorous but computationally challenging calcula- 259-Pos Board B29 tion of conformation weights from all cryo-EM images. It is found that morph- Molecular Dynamics Simulation of the KaiC Clock Protein ing densities and refining structures into cryo-EM maps is tightly coupled Theo Crouch, Andy LiWang, Michael E. Colvin. through the measure of goodness-of-fit. University of California, Merced, Merced, CA, USA. With these insights, we asess how much of all underlying configurations are The protein KaiC is the central player the well-characterized circadian clock represented by single densities and sample pathways between densities, that system of cyanobacteria. KaiC functions as a homohexamer that forms an over- represent physical intermediate states of the ribosome-selB complex upon all structure that looks like two stacked rings, one on top of the other. The two GTPase activation. domains are called the C1 and C2 ring. The clock’s 24-hour period is driven by sequential phosphorylation of residues in the C1 ring and the choreographed Posters: Protein Stability, Folding, and binding of KaiA and KaiB the C1 and C2 rings. Although the specific steps in the KaiABC clock have been mapped, the structural changes causing, and Chaperones I caused by, the phosphorylation and protein binding are not fully understood. We have performed 500ns all-atom Molecular Dynamics (MD) simulations 262-Pos Board B32 on a set of 5 different KaiC hexameric structures: wild type, S431E, T432E, Flexibility of the Global Protein Structure Defines the Multiple Conforma- S431E þ T432E, and a 10 C-terminal AA truncated structure, all including tions of the Lactate Dehydrogenase at the Stage Preceding Irreversible 12 bound ATPs. The glutamic acid mutations have been shown to functionally Thermal Inactivation mimic the effect of phosphorylation and the truncation mutant mimics the bind- Sergei Khrapunov, Eric P. Chang, Robert H. Callender. ing of KaiB to the C2 ring. Our simulations show a number of differences be- Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA. tween these different structures. The truncation mutation shows increased The thermodynamics of the apoenzyme, holoenzyme (LDH-NADH) and flexibility in distinct regions of the protein and allows much greater access to ternary (LDH-NADH-oxamate) complex of the glycolytic enzyme lactate de- waters to the ATPs bound in the C2 ring. The observed changes near the hydrogenase (LDH) from porcine heart, phLDH (mesophilic Sus scrofa) and from mackerel icefish, cgLDH psychrophilic Chamapsocephalus gunnari) ATP-binding pockets can affect the exchange of the ATPs and the rate that 1 the ATPs phosphorylate nearby residues. Additionally, sodium ions are found have been investigated. A novel fluorescence assay elaborated in was uti- in close proximity to the ATPs of the structures with glutamate mutations, but lized that simultaneous monitors changes to the global protein structure, not in the wild type or truncation mutant. We are also beginning coarse-grained structural changes near the active site, and aggregation of the enzyme in MD simulations of the KaiC hexamer using Marrink’s Martini force field which response to increasing temperature. The reverse changes of stability and af- will allow much longer simulation timescales. finity for oxamate were established for both, phLDH and cgLDH. A revers- ible low-temperature (pre-denaturation) structural transition that precedes the 260-Pos Board B30 high-temperature (denaturation) transition was found for both Michaelis CASTp 3.0: Computed Atlas of Surface Topography of Proteins and complexes. This transition was found to coincide with a marked change in Beyond enzymatic activity for both LDHs. An observed lower substrate binding af- Wei Tian, Chang Chen, Jie Liang. finity for cgLDH compared to phLDH was accompanied by a higher contri- Univ of IL at Chicago, Chicago, IL, USA. bution of entropy to G which reflects a higher functional plasticity of the The concave surface regions such as pockets and cavities in the three- psychrophilic cgLDH compared to the mesophilic phLDH. The comparative dimensional structures of proteins with specific physicochemical texture pro- study of the apoenzyme and holoenzyme has shown that the basis for the pre- vide the microenvironment for biochemical reactions to occur. Computed denaturation transition of the Michaelis complex is the flexibility of the Atlas of Surface Topography of proteins (CASTp) has been a useful resource global protein structure. for locating, delineating and measuring these concave surface regions, and The hypothesis is expressed that the multiple active and inactive along with in- for providing information of amino acid residue sites of functions and termediate sub-state conformations of the enzyme exist in equilibrium at the diseased-related single nucleotide polymorphisms (SNPs). We developed a stage preceding irreversible thermal inactivation. This equilibrium is an essen- new version of CASTp (3.0) that contains significantly enriched information. tial selective factor for the adaptation of an enzyme to the environmental In addition to overall geometry, the new CASTp constructs explicit solid temperature. model of imprints of pockets and cavities, with further metric information [1] Khrapunov, S., Chang, E., and Callender, R. H. (2017) Thermodynamic and of pocket/cavity wall atoms bounding the imprints provided. This provides Structural Adaptation Differences between the Mesophilic and Psychrophilic useful information in assessing how these regions interact with other mole- Lactate Dehydrogenases, Biochemistry 56, 3587-3595. cules. Furthermore, we provide mapping of the enlarged SNP and other variant information from the Exome Aggregation Consortium (ExAC) onto 263-Pos Board B33 a surface pockets and voids in CASTp 3.0, enabling detailed analysis of vari- Conformational Changes of -Crystallin Proteins Induced by Heat Stress Ming-Tao Lee1,2, Yu-Yung Chang1, Wei-Chin Hung3. ants at single amino-acid level in the geometric context of protein local sur- 1 face regions. Life Science Group, National Synchrotron Radiation Reasearch Center, Hsinchu, Taiwan, 2Department of Physics, National Central University, 261-Pos Board B31 Jhongli, Taiwan, 3Department of Physics, R. O. C. Military Academy, Molecule Mechanics from Cryo-EM Images and Multiple Reconstructed Kaohsiung, Taiwan. Densities The a-crystallin is a major structural protein in the eye lens of vertebrates, Christian Blau, Erik Lindahl. composed of two relative subunits, aA and aB crystallin for maintaining the Theoretical and Computational Biophysics, Stockholms Universitet, Solna, lens transparency over a lifetime. As a member of small heat shock protein fam- Sweden. ily (sHsp), a-crystallin exhibit chaperone-like activity in response to prevent After the resolution revolution, cryo-EM moves from sets of individual struc- the misfolding or aggregation of critical proteins in lens, associated with tures, to understanding properties of biological samples as a whole, like path- cataract disease. In this study, high purity aA and aB crystallin proteins ways between resolved structures and binding affinities. Extracting these were expressed from E.coli and purified by affinity and size-exclusion chroma- molecule mechanics as probability distribution of arbitrary observables, tography. The experiment of size-exclusion chromatography shows that both like open and close configuation, or small molecule binding probabilities aA and aB crystallin exhibited oligomer complex in solution. Here, we present however, is still challenging despite increasing resolution and numbers of the structural characteristics of a-crystallin proteins from low to high temper- resolved structures of a single complex, due to the non-trivial connection ature by combining circular dichroism (CD) and small-angle X-ray scattering three dimensional structures and all the possible molecule conformations (SAXS). Not only CD data but also SAXS data show that a-crystallin proteins they represent. exhibit the transition behavior on conformation with temperature increasing. Here, we present how to resolve all molecule conformations that are repre- Although protein sequence is highly conserved, the analysis of thermal stability sented by three dimensional structures and pathways between multiple recon- showed different properties in aA and aB crystallin. In this study, taken structed densities. The opening and closing dynamics of adenylate kinase are together the data were discussed to give more insights on the chaperone-like used to verify our the method, using ensemble-averaged density maps of activity of a-crystallin proteins.

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264-Pos Board B34 surfaces, the way it alters the different states on a protein’s folding pathway Denatured State Conformational BIAS in a 3-Helix Bundle are not well understood. Our goal is to resolve how TMAO changes the Moses Leavens, Bruce Bowler, Melisa M. Cherney. different ensembles along the folding pathway. To this end, we combine University of Montana, Missoula, MT, USA. long timescale simulations with kinetic and thermodynamic experiments of Previous work with the four-helix bundle protein cytochrome c’ from Rhodop- the folding process of the well characterized, fast folding Protein B. Addition- seudomonas palustris using histidine-heme loop formation thermodynamic ally, we measure pressure unfolding of PRB in presence of TMAO to charac- methods revealed fold-specific deviations from random coil behavior in its de- terize changes in the hydration shell of the protein. The simulations show an natured state ensemble. To examine the generality of this finding, we extend increased stability of PRB in the presence of TMAO because of a change in this work to a three-helix bundle polypeptide, the second ubiquitin- the population of intermediate ensembles, leading to an overall smoothening associated (UBA) domain, UBA(2), of the human DNA excision repair protein. of the protein folding landscape. The major factor behind this was found to We use yeast iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain to be the composition of the hydration shell of PRB in presence of TMAO. By the N-terminus of iso-1-cytochrome c. Using site-directed mutagenesis, we combining experimental and computational approaches we are able to investi- have engineered histidine into residue positions with high solvent accessible gate how osmolytes affect specific ensembles along the protein folding pathway surface area within the all alpha-fold, creating six single histidine variants. at the atomistic level of detail. Isothermal equilibrium denaturation studies by guanidine hydrochloride titra- tion reveal this fusion protein unfolds in a 3-state process, commencing with 267-Pos Board B37 iso-1-cytochrome c followed by UBA(2). Thus, the engineered histidine resi- Microscale Foldamer Production and Characterization dues in UBA(2) strongly destabilize iso-1-cytochrome c. Histidine-heme Roxanna Kiessling, Katherine Snell, Collin Barraugh, Samuel J.S. Rubin, loop formation equilibria measurements in the denatured state in 4 and 6 M Babak Sanii. guanidine hydrochloride show that loop stability decreases as the size of the Keck Science Department, Claremont, CA, USA. histidine-heme loop increases, consistent with loop entropy being an important We present an experimental platform for studying foldamers on an easily factor in the loop equilibria. Comparing the degree of deviation of loop stability observable scale. It is composed of elastomers extruded into filaments, versus loop size using the Jacobson-Stockmayer relationship, which assumes 100mm in diameter and up to 0.5m long, via a two-step curing technique. that loop equilibria in a random coil are due solely to the entropy of loop for- These filaments are large enough for convenient experimental observation, mation, we observe a significant deviation near one of the reverse turn se- and flexible enough to access a large number of folded conformations on a quences within this three-helix bundle. This result is consistent with reasonable timescale. We acoustically excite the filaments at the air-water observations for the four-helix bundle, cytochrome c’. Loop breakage kinetic interface and optically image them to quantify conformational parameters experiments demonstrate a specific reverse turn sequence persists in the dena- and dynamics. The conformational end-to-end distances are statistically tured state compared to other sequences. Therefore, this reverse turn sequence consistent with a worm-like chain model of semi-flexible polymers, with a may bias the conformational search for the UBA(2) domain, and would be well-defined persistence length. The choice of polymer (PDMS, polydimethyl- important in setting up the overall topology of the fold. siloxane) enables hydrophobicity patterning along the length of the filament with UV light or corona discharge, which could be used to tune folded struc- 265-Pos Board B35 tures. We characterize the elastic modulus and hydrophobic switchability of The Polydispersity Problem: Investigating the Effect of Crowding Agent the filaments, and demonstrate that the hydrophobicity patterning of the fila- Polydispersity in Protein Stability ment has an impact in its folding conformational statistics. We envision this Alan van Giessen, Anastasia Osti. system as a tunable experimental means of determining design rules for com- Chemistry, Mount Holyoke College, South Hadley, MA, USA. plex folding pathways, with possible applications to molecular (bio)polymer The dense, heterogeneous cellular environment is known to affect protein sta- folding problems. bility through interactions with other biomacromolecules. The effect of 268-Pos Board B38 excluded volume due to these biomolecules, also known as crowding agents, Molecular Evolution of L-PGDS: Substrate Recognition Mechanism of has long been known to increase the stability of a test protein. The cellular envi- Medaka L-PGDS ronment is heterogeneous not only in terms of its chemical composition, but Kimi Torii, Yuji Hidaka, Shigeru Shimamoto. also in terms of the sizes of the biomacromolecules present. It has been shown Kindai University, Higashi-osaka, Japan. experimentally that the presence of polydisperse or mixed crowding agents has The mammalian form of lipocalin-type prostaglandin D synthase (L-PGDS) is a a non-additive effect, i.e. that there is an optimal mixing ratio where the effect bifunctional protein that participates, not only in prostaglandin D2 (PGD2) syn- of the crowding agents is larger than that of monodisperse systems of each thesis, but also serves as a carrier protein for small lipophilic molecules. How- crowder. Here we investigate the role of polydisperse crowding on a small ever, a gene analysis of L-PGDS revealed that medaka L-PGDS does not 15-residue helical test protein through computer simulation. Unlike most pre- possess a catalytic Cys residue. In addition, chicken and xenopus L-PGDS mol- vious work, our crowding agents are themselves helical proteins; however, ecules, which contain the catalytic Cys residue, showed only weak enzymatic the crowding agents only interact with the test protein through excluded volume activity. Therefore, we hypothesized that L-PGDS originally carried small lipo- interactions. The test protein is simulated in two different systems; one system philic molecules, such as prostaglandin, and happened to acquire the enzymatic with crowding agents that are 10 and 20 residues and another where they are 6 activity during its molecular evolution. To validate this hypothesis, we pre- and 24 residues. For each system, simulations were run with different mixing pared recombinant medaka L-PGDS and compared its substrate binding ability ratios, ranging from all small crowders to all larger crowders. The density to that of mouse L-PGDS using isothermal titration calorimetry (ITC). In addi- was constant at 300 mg/mL for all simulations. We relate the thermal stability tion, circular dichroism (CD) measurements of the proteins were also carried and thermodynamic stability, as measured by the change in folding temperature out to elucidate structure-function relationships. The recombinant proteins and the change in the free energy of unfolding respectively, to the mixing ratio. were expressed using an E. coli expression system as GST-fusion proteins. In particular, we relate the non-additivity to the excluded volume of the crowd- To obtain structural information and confirm their correct folding, structural an- ing agents. We show that crowder polydispersity does not introduce significant alyses by CD measurements of the recombinant medaka and mouse L-PGDS non-additivity with respect to the test protein stability. In addition, we discuss were carried out. The results suggested that medaka L-PGDS consists of a the impact of crowder polydispersity on the free energy landscapes. b-sheet structure, as well as a typical L-PGDS. Substrate binding experiments of medaka L-PGDS by ITC were also carried out. The results revealed that the 266-Pos Board B36 recombinant medaka L-PGDS binds PGD2 and PGF2a with Kd values of 8.2 and The Role of TMAO in Protein Folding: A Joint Experimental and Simula- 5.9 mM, respectively. To evaluate the prostaglandin binding mode of medaka tion Study L-PGDS, we further compared the thermodynamic binding properties of Mayank M. Boob1, Shahar Sukenik2, Taras V. Pogorelov2, medaka L-PGDS with that of mouse L-PGDS. Martin Gruebele1. 1Center for Biophysics and Quantitative Biology, University of Illinois 269-Pos Board B39 Urbana-Champaign, Urbana, IL, USA, 2Chemistry, University of Illinois Disulfide-Coupled Folding of Prouroguanylin on Molecular Evolution Urbana-Champaign, Urbana, IL, USA. Kenta Mori, Saya Nishihara, Shigeru Shimamoto, Yuji Hidaka. Osmolytes are ubiquitous in the cellular environment, and they play an impor- Kindai University, Higashi-osaka, Japan. tant role in controlling protein stability under stress. Trimethylamine N-oxide The folding of uroguanylin is assisted by the pro-peptide region, which func- (TMAO), a natural osmolyte, is used by marine animals to counteract the effect tions as an intra-molecular chaperone. Our previous study of the disulfide- of pressure denaturation at large depths. While the stabilizing effect of TMAO coupled folding of prouroguanylin suggested that a mis-bridged disulfide on proteins has been well studied, and is linked to its exclusion from protein isomer is produced at the early stage of folding and the molecule is then shifted

BPJ 8556_8558 52a Sunday, February 18, 2018 to the native conformation, accompanied by a secondary structural change from calorimetry demonstrated that solvent reorganization upon ligand binding show an alpha-helix to a beta-sheet structure at the processing site and the C-terminal large differences between the two variants. The heat capacity change (DCp) region, respectively. Thus, the necessity of a folding intermediate being pro- also show antibiotic-dependent differences between the two variants. The duced suggests that the mechanism responsible for the chaperone-assisted X-ray structures of both variants show that the aminoglycoside neomycin is folding of prouroguanylin includes quality control in constructing the native positioned differently at the active site of T130K as compared to D80Y, in conformation expressly through the formation of a non-native disulfide isomer which the tyrosine substitution alters the geometry of binding site. This struc- as the folding intermediate on molecular evolution. To validate this hypothesis, tural difference explains why we observe differences in the thermodynamics of the folding of eel uroguanylin and prouroguanylin was compared to that for hu- the ligand binding properties between these variants. Furthermore, temperature man uroguanylin and prouroguanylin, respectively. affects the monomer-dimer equilibrium of these enzymes differently. Thus, For this purpose, a series of N-terminal extended eel uroguanylin analogs was data shown in this work suggest that dynamic properties of proteins, thermody- chemically synthesized. And a recombinant eel prouroguanylin was expressed namics of ligand-protein interactions, solvent effects and monomer-dimer equi- as a thioredoxin-fused protein in E. coli cells because the expression efficiency librium may be among the molecular parameters that separate thermophilic of the recombinant eel prouroguanylin was quite low. The fusion protein was proteins from simply those that are thermostable but otherwise identical to purified by Ni-affinity chromatography and treated with PreScission protease the mesophilic counterparts. to release the eel prouroguanylin. The refolding reactions of eel uroguanylin analogs and eel prouroguanylin were then examined and the folding mecha- 272-Pos Board B42 nism of eel prouroguanylin was compared to that for human prouroguanylin. Observation of the Cooperative Collapse in the Spontaneous Folding Process of Cytochrome C by Two-Dimensional Fluorescence Lifetime 270-Pos Board B40 Correlation Spectroscopy 1 2 2 1,3 Comparative Refolding of Guanidinium Hydrochloride Denatured Serum Miyuki Sakaguchi , Masaru Yamanaka , Shun Hirota , Kunihiko Ishii , 1,3 Albumin Assisted by Surfactants via Artificial Chaperone Protocol: Tahei Tahara . 1RIKEN, Wako, Japan, 2Grad. Sch. Mat. Sci., NAIST, Ikoma, Japan, Biophysical Insight 3 Mohd Ishtikhar, Nand Kishore. RIKEN Center for Advanced Photonics, RIKEN, Wako, Japan. Chemistry, Indian Institute of Technology-Bombay, Mumbai, India. The folding dynamics of cytochrome c (cytc) has been extensively studied by a Here, we have report the cooperative refolding/renaturation behaviour of variety of experimental methods. However, site-specific folding dynamics on guanidinium-hydrochloride (GdCl) denatured serum albumins in the presence the short time scale is still controversial. In this work, the microsecond folding of cat-anionic mixture of the cationic surfactant cetyltrimethylammonium bro- dynamics of cytc under the equilibrium condition was investigated by two- mide (CTAB) and the anionic surfactant sodium dodecyl sulphate (SDS) in dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). For the phosphate buffer solution of pH 7.4, using artificial chaperone assisted two clarifying the site-specific kinetics, we prepared three samples which have step method. We have also performed the similar experiment in the presence different labeling sites of the FRET donor. It is known that cytc is composed of gemini surfactants, SDS and CTAB lonely at different concentration. We of several structural units having different stabilities, which are called foldons. Therefore, the three labeling sites were designed to be located at different fol- have employed biophysical techniques such as dynamic light scattering, circular th rd th dichroism, extrinsic and intrinsic fluorescence and synchronus fluorescence dons, i.e., 50 ,83 , and 104 positions in the sequence. In 2D FLCS measure- methods to investigate and characterize the refolding mechanism of denatured ments at pH 2.5, the structural heterogeneity induced by acid denaturation was serum albumin after 200 times of dilution with/without methyl-b-cyclodextrin detected as two species for all the three samples. These two species were as- which act as artificial chaperone. We found maximum refolding of diluted albu- signed to the unfolded and a partially folded intermediate state based on their mins in the presence of gemini surfactant and minimum with mixture (CTAB/ fluorescence lifetimes. The interconversion timescale between the two species SDS=50/50) at physiological condition in the aqueous solution, it may be due was obtained by the filtered FCS method. The rate constants were determined as 26.251.0, 33.250.3, 34.4521.6 ms for the samples having the donor at to the micelles formation which is responsible for ordered/unordered aggregate th rd th microstructure formation. Other mixtures (CTAB/SDS=20/80 and 80/20) were 50 ,83 , and 104 positions, respectively. The observed timescale is very slightly play an effective role in the refolding process in the presence of similar for the three samples and is in good agreement with the timescale of methyl-b-cyclodextrin. But, exclusively these surfactants are more effective the initial collapse of cytc which was observed in nonequilibrium conditions then the mixtures these surfactants and CTAB shows the greater renaturation ten- with mixing methods. This result indicates that the collapse of cytc occurs dency as compare to SDS in the presence of methyl-b-cyclodextrin. Obtained re- cooperatively with little site-specificity. sults ascribed the presence of charge head group and length of hydrophobic tail 273-Pos Board B43 of the CTAB-surfactant that play an important task during electrostatic and Is Hydroydrodynamic Interaction Important to Protein Folding? hydrophobic interactions. Finding suggest that, CTAB and gemini surfactant Dirar M. Homouz1,2, Fabio C. Zegarra2,3, Yossi Eliaz2,3, assisted artificial chaperone protocol may be utilized in the protein renatur- Margaret S. Cheung2,3. ation/refolding studies, which may address the associated problems of biotech- 1Physics, Khalifa University of Science and Technology, Abu Dhabi, United nological industries for the development of efficient and inexpensive folding Arab Emirates, 2Physics, University of Houston, Houston, TX, USA, 3Center aides, which may also be used to produced genetically engineered cells related for Theoretical Biological Physics, Rice University, Houston, TX, USA. diseases, resulting from protein misfolding/aggregation. Hydrodynamic interaction (HI) arises by the solvent flow generated by the movement of a particle that affects other particles. However, it is controversial 271-Pos Board B41 about the extent of impact from HI on the kinetics of protein folding. Using a Investigation of the Molecular Mechanisms which Result in Aminoglyco- 0 minimalist coarse-grained computer model and the theoretical framework of side Nucleotidyltransferase 4 (ANT4) Variants with Different Levels of the Energy Landscape Theory (ELT) for protein folding, we revealed the effects Thermostability of HI on the folding rates of two proteins with distinctive topologies: a 64-res- 1 2 1 2 Seda Kocaman , Brinda Selvaraj , Edward Wright , Matthew Cuneo , idue a/b chymotrypsin inhibitor 2 (CI2) protein, and a 57-residue b-barrel 1,3 Engin Serpersu . a-spectrin src-Homology 3 domain (SH3) protein. At a temperature greater 1Biochemistry, Cellular and Molecular Biology, University of Tennessee at 2 than the folding temperature, HI dragged a polymeric chain from collapsing, re- Knoxville, Knoxville, TN, USA, Oak Ridge National Laboratory, Oak sulting in a retarded folding rate. At a temperature lower than the folding tem- Ridge, TN, USA, 3National Science Foundation, Alexandria, VA, USA. 0 perature, HI facilitates folding rates depending on the topology of a protein. The aminoglycoside nucleotidyltransferase 4 (ANT) is a homodimeric enzyme For a protein like CI2 where its folding nucleus is rather diffuse in a transition that detoxifies a large number of aminoglycoside antibiotics by nucleotidylat- 0 state, HI channels the formation of contacts, thus accelerating folding. For a pro- ing them at the C4 -OH site. Two thermostable variants for this enzyme show tein like SH3, where its folding nucleus is specific, HI matters less. Our investi- only single amino acid changes in their primary amino acid sequences (T130K, gation provides a general explanation of the impact of HI on protein folding. D80Y). D80Y has a higher melting temperature compared to T130K. Our pre- vious studies indicate that T130K mimics the WT enzyme in terms of protein 274-Pos Board B44 dynamics and thermodynamics of enzyme-ligand interactions while the D80Y SOD1 Folding Modulation in the Crowded Cell exhibits different behaviors in both aspects. This led us to hypothesize that the David Gnutt1, Jonas Ahlers1, Benedikt Ko¨nig1, Matthias Heyden2, T130K is just a thermostable version of the mesophilic WT exhibiting the Simon Ebbinghaus1. same behaviors while the D80Y is the true thermophilic variant with distinct 1Physical Chemistry II, Ruhr University Bochum, Bochum, Germany, properties. Our recent work shows that the thermodynamic parameters of the 2School of Molecular Sciences, Arizona State University, Tempe, AZ, USA. binary enzyme-aminoglycoside complexes of these variants show highly sig- Proteins experience a complex and crowded environment inside the living nificant differences. The data, acquired in H2O and D2O by isothermal titration cell. Nonspecific interactions and excluded volume effects play a major role

BPJ 8556_8558 Sunday, February 18, 2018 53a in modulating biomolecular stability in such conditions. Studying folding (HXMS). Less is known, however about the tertiary structure of such partially directly inside the cell is thus necessary to elucidate the native behavior of folded conformations. To study the change in solvent accessibility of individ- the biomolecule. We used a combination of laser induced temperature jumps ual residues in ecRNH* we have used a pulsed-labeling chemical modification and a FRET labelled folding reporter based on a superoxide dismutase 1 technique, x-ray footprinting/mass spectrometry (XFMS). When aqueous so- (SOD1) variant (SOD1-barrel) to investigate folding in vitro, in artificially lutions are exposed to x-rays, hydroxyl radicals are generated, which can react crowded systems or directly inside the cell. The results revealed that a single with amino acid side chains. These modifications are detected, identified, and point mutation in SOD1-barrel can influence the stability modulation by the quantified using LC-MS/MS. Synchrotron beams generate a sufficient number cell: While most mutants were destabilized by the cell and by protein crowd- of radicals to result in a detectable number of modifications on the millisecond ing, replacement of a single histidine lead to an increase in stability in such timescale. We have used XFMS to probe regional solvent accessibility of environments. Further, SOD1-barrel was targeted to subcellular structures to ecRNH* residues under acidic, denaturing, and native conditions. This tech- probe folding in specific subcellular regions, e.g. the cytoskeleton or the his- nique allows for the quantification of changes in side chain solvent accessi- tones. Our results show that destabilizing interactions between the protein and bility of unfolded and partially folded conformations reporting on their the cell environment depend on the protein sequence as well as its subcellular tertiary structure. This technique will allow for the observation of protein con- localization. formations populated during various biological processes in complex mixtures. 275-Pos Board B45 Aggregation and Stability of Proteins in Water: A Computational Study 278-Pos Board B48 Valentino Bianco. Spectra and Simulation of Model Beta-Sheets and Hairpins. Impact of University of Vienna, Vienna, Austria. Turn Sequences and Aromatic Contacts on Equilibria and Dynamics Proteins are molecules made of a sequence of amino acids that fold into the Timothy A. Keiderling1, Heng Chi2, Dan McElheny1, David Scheerer3, native structures. Such a structure is usually stable within a certain range of Ayesha Samer1, Karin Hauser3, Frank Vazquez1. temperatures and pressures, beyond which a protein denaturate. Such a phe- 1Chemistry, University of Illinois at Chicago, Chicago, IL, USA, 2Chemistry, nomenon is well known at higher temperatures, where the thermal fluctuations Jiangsu Food and Pharmaceutical College, Huai’an, China, 3Chemistry, disrupt the native conformation. However, similar phenomena are observed by University of Konstanz, Konstanz, Germany. decreasing the temperature or by increasing the pressure, respectively known as b-sheets can be modeled as antiparallel strands coupled by turns. Isolated cold- and pressure-denaturation. Moreover, in order to guarantee the correct model systems are less stable, due to amide solvation), but can be induced biological functions, proteins have evolved to have a low enough propensity to fold into sheet-like structures by restricting the conformational space of to aggregate within a range of protein expression required for their biological turn residues, such as with DPro-Gly sequences, or by inclusion of cross- activity, but with no margin to respond to external factors increasing/decreasing strand hydrophobic interactions, especially aromatic contacts, which tend their expression/solubility. Indeed, protein aggregation is mostly unavoidable to desolvate parts of the structure. Expanding to multistrand structures can when proteins are expressed at concentrations higher than the natural ones. provide different characteristics for study of physical properties. Larger Here, using a coarse-grain model on lattice, which includes the protein effects structures have strand differentiation, since the center strand of a three- on the water properties in the hydration shell explicitly, we investigate the strand sheet has a different H-bonds than the outer ones. In larger structures, folding, the stability and the aggregation of proteins. integral hairpin-like sequences of either the first two or the second two 276-Pos Board B46 strands might have different stabilities and folding mechanisms, if it is over- all a multistate process. To explore these factors, we have prepared a series Thermodynamically Coupled Unfolding Transitions in Dystrophin ABD1 D Christian Coffman, Robert Miller, Victoria Fringer, Erin Groth, of three-stranded sheet structures that are stabilized by either Pro-Gly or by Adewale Adeyemi, Alexis Doucette, Michelle Botts, Michael Fealey, Aib-Gly turns and contain various Trp-Tyr side-chain contacts. To assess the Jessica Sieber, Anne Hinderliter. impact of the third strand we also separately synthesized the component University of Minnesota Duluth, Duluth, MN, USA. hairpins and studied their relative properties with temperature dependent Dystrophin, a 427 kDa protein located on one of the largest genes in the hu- circular dichroism, fluorescence, infrared (IR) and vibrational circular di- man genome, has been implicated in myocytes’ ability to dissipate mechan- chroism for evaluation of global thermodynamic equilibria as well as IR–de- ical forces transduced between cytoskeletal and membrane features. Single tected temperature jump relaxation dynamics of selected components point mutations in Dystrophin can result in myocyte membrane shearing un- (strands, turns). Where possible, NMR structures were obtained for compar- der normal muscular flexion and is diagnosed as Becker’s Muscular Dystro- ison and MD simulations of unfolding were used to aid interpretations. phy. Given point mutations outside of direct binding sites can render While all the sequences showed evidence of b-structure formation, the extent and stability varied markedly. In terms of equilibrium properties, Dystrophin at a loss of function, the amino acids within each domain are D likely stabilized or destabilized allosterically. Our research aims to deter- the turn residues (especially Pro-Gly) seemed to have the strongest influ- mine how secondary and tertiary structures of Dystrophin can effectively ence, while the aromatic contacts had minor effects. However in terms of dy- couple during unfolding transitions within and between protein domains. namics, the aromatic effects differentiated the structures more, suggesting a Here, we study the actin binding domain (ABD1) of Dystrophin which con- variation in the folding mechanisms. tains the highest frequency of point mutations resulting in Becker’s muscular dystrophy. In order to gain insight as to how ABD1 may couple 279-Pos Board B49 within itself, we designed a series of thermodynamic analyses to describe Effects of Aggregating Agents in Protein Misfolding. An Infrared ABD1 unfolding semi-mechanistically. We use differential scanning calo- Spectroscopy Study rimetry as well as additional spectroscopic techniques (CD and FLT) to Jose Luis R. Arrondo, Laura Aguirre Araujo, Igor De, la Arada. monitor ABD1 unfolding energetics and determine how energy may be Biochemistry, University of Basque Country, Bilbao, Spain. stored in ABD1’s secondary structure or in its buried hydrophobic residues. Protein misfolding, which include the formation of amyloid aggregates, Relating these approaches provides a comparative approach as to how insoluble aggregates resistant to degradation, are related to a large number different structural features may affect ABD1’s ability to dissipate mechan- of different diseases, mostly neurodegenerative. In this work, hen egg white ical stress through folding and unfolding thermodynamically favorable lysozyme has been used as model because it is a good characterized protein folding features. with ability to form this kind of aggregates if it is exposed to extreme con- ditions. Usually, the in vitro studies are done in a diluted medium, and the 277-Pos Board B47 action of the protein at these concentrations differs from what happens inside Probing Regional Solvent Accessibility of Molten Globules and Folding the cell, mainly because the internal concentration is crowded by numerous Intermediates using X-Ray Footprinting/Mass Spectrometry macromolecules. The purpose of this research is to study what occurs if a Shawn M. Costello1, Sayan Gupta2, Corie Y. Ralston2, Susan Marqusee3. protein forms amyloid aggregates in a crowded medium made by different 1Biophysics Graduate Program, University of California Berkeley, Berkeley, agents (Dextran 40, Dextran 70 and Ficol 70) at different crowder concen- CA, USA, 2Physical Biosciences Division, Lawrence Berkeley National trations (5%, 10% and 20%). In order to characterize what happens with Laboratory, Berkeley, CA, USA, 3Department of Molecular and Cell lysozyme when it is forced to form this kind of aggregates, infrared spectros- Biology, University of California Berkeley, Berkeley, CA, USA. copy has been used not only because it is a useful technique for this kind of The refolding pathway of Escherichia coli ribonuclease HI* (ecRNH*) has studies, but also due to the fact that analysis time is short. Moreover, little been studied in detail using various techniques. For example, the secondary quantity of protein is needed and also the technique has a high sensitivity structure of conformations populated during ecRNH* refolding has been to b structures which are characteristic of amyloid fibers. As it is shown, determined with pulsed-labeling hydrogen exchange/mass spectrometry lysozyme aggregates are formed different in a diluted medium or in a

BPJ 8556_8558 54a Sunday, February 18, 2018 crowded one because the fiber quantity formed decreases, and the kinetic 283-Pos Board B53 formation differs. These changes arise from the different effect of crowders Identification and Characterization of an Inside-Out Intermediate in the at distinct concentrations. Folding Pathway of Bacteriophage Sliding Clamp Manika I. Singh, Vikas Jain. 280-Pos Board B50 Biological Sciences, Indian Institute of Science Education and Research b Molecular Dynamics Investigations of -Sheet Stability and Folding Bhopal, Bhopal, India. Pathways Protein folding process involves formation of transiently-occurring interme- Anthony Hazel1, Chris Rowley2, James C. Gumbart1. 1 2 diates that are difficult to isolate and characterize. It is both necessary and Physics, Georgia Institute of Technology, Atlanta, GA, USA, Chemistry, interesting to characterize the structural conformations adopted by these in- Memorial University of Newfoundland, St. John’s, NL, Canada. termediates, also called molten globules (MG), to understand protein Molecular dynamics (MD) simulations of peptides and proteins offer folding. Here, we investigated an equilibrium (un)folding intermediate state atomic-level detail and unique insight into many biological processes. Pro- of T4 phage gp45 sliding clamp (a protein family present in all the domains tein folding is one of the many complex biological problems for which of life) obtained during chemical denaturation. The T4 gp45, besides func- MD has improved our understanding. We used all-atom MD simulations tioning as a DNA polymerase processivity factor, also moonlights as the late to study the folding properties of the C-terminal b-hairpin of protein G, promoter transcription determinant. Chemical denaturation of gp45 follows GB1, a model peptide used in the study of b-sheet folding. Using replica- a three-state transition, wherein the protein displays substantial conforma- exchange umbrella sampling simulations, we examined the folding free- tional rearrangement during unfolding and forms an expanded dry-MG energy of two all-atom CHARMM force fields, C36 and C22*, as well as (DMG). By monitoring the fluorescence of tryptophans that were strategi- the CHARMM Drude polarizable force field. Although they follow roughly cally introduced at various sites, we demonstrate that the urea-treated mole- the same folding pathway, the three force fields show surprisingly distinct cule has its surface residues ‘flip’ inside the core, and closely placed folding free-energy landscapes. While C36 produced a completely stable, residues move farther. We were also able to isolate and purify the MG fully folded b-hairpin and C22* produced a floppier, but still partially folded form of gp45 in native condition (i.e. non-denaturing buffer, physiological b-hairpin, the b-hairpin was unstable in Drude. Similar results were found pH and temperature); characteristics of this purified molecule substantially for the a-helical peptide, Ala10,whichis 50% folded in C36 and C22*, match with urea-treated wild-type gp45. Ours is one of the few reports but is less folded in Drude. Examination of backbone and side-chain solva- that demonstrates the isolation and purification of a protein folding interme- tion free energies, hydrogen bonding and hydrophobic packing, and b-turn diate in native condition. Further biophysical dissection of the two domains structure and interactions reveals key differences between the three force of gp45 reveals contrasting properties. While the C-terminal domain shows fields and offers some insights into the general folding mechanisms of stability and rigidity, we find that the N-terminal domain is unstable and b-sheets. Finally, we apply these techniques to study the in vitro and in flexible. We believe that the asymmetric characteristics of the two domains vivo folding mechanisms of the b-helical passenger domain of the autotrans- and the interface they form hold significance in gp45 structure and function. porter, pertactin, in order to elucidate the export mechanism of virulence We envisage that our work will help in the designing of specific inhibitors factors in infectious, Gram-negative bacteria. against sliding clamps to treat a wide variety of diseases from bacterial 281-Pos Board B51 infection to cancer. Folding Thermodynamics of a Three-Helix Bundle Protein and Its 284-Pos Board B54 Engineered Thermostable Variant Monitoring the Folding Pathway of a Protein Over Evolutionary Time Emily K. Hamlin, Srivarchala Chandu, Michelle E. McCully. using Hydrogen Exchange - Mass Spectrometry (HX-MS) Biology, Santa Clara University, Santa Clara, CA, USA. Eric Bolin, Shion Lim, Susan Marqusee. The Engrailed Homeodomain (EnHD) is a three-helix bundle transcription fac- University of California, Berkeley, Berkeley, CA, USA. tor natively found in D. melanogaster. It was used previously as a backbone Previous studies of homologous proteins have provided much information on template to test a side chain repacking algorithm. The resulting protein, how drastically different biophysical properties can arise in proteins with UVF, not only folded as expected but was extremely thermostable (Tm >95 very few sequence changes and similar structure; however the recent intro- vs. 52 C in EnHD). Based on subsequent molecular dynamics studies, we duction of ancestral sequence reconstruction (ASR) allows one to probe hypothesized that a smaller loss of entropy upon folding was the main contrib- how evolution has shaped these properties over time. While protein folding uting factor to the increased thermostability observed in UVF relative to EnHD. is known to proceed through transient partially folded structures, how these Here, we used equilibrium unfolding to derive thermodynamic properties for intermediates change over evolutionary time is unclear. We have applied a the folding of both proteins. pulsed labeling hydrogen-exchange method with detection by mass spec- 282-Pos Board B52 trometry to characterize the structure of several folding intermediates in pro- Folding Analyses of a de novo Designed Prouroguanylin teins from an ancestral reconstruction of the RNase H family. This approach Yuji Hidaka, Saya Nishihara, Kenta Mori, Shigeru Shimamoto. allows us to follow the formation of hydrogen-bonded structure at near Kindai University, Higashi-Osaka, Japan. amino acid resolution. We find that the essential core intermediate that forms Peptide hormones are typically produced in the form of a prepro-peptide, pre- before the rate limiting step of folding is conserved over three billion years cursor molecule, which is then processed into the biologically active mature of evolution, despite having as little as 50% sequence identity, suggesting an form. However, the role of the pro-peptide region, especially its role in the cor- evolutionary pressure acting upon the intermediate structure. Interestingly, rect folding which is absolutely required for biological activity, has not been we find that the very early events in folding - formation of the initial stable explained in detail. helices are not conserved and show trends along the phylogenetic tree. Uroguanylin folds into its native conformation with the assistance of the pro- Directed mutagenesis reveals that relatively few sequence changes can result peptide region, which functions as an intra-molecular chaperone. We recently in the switch, and these changes can be predicted by simple intrinsic aspects applied its chaperone function to construct a de novo designed peptide, which is of the sequence such as helical propensity. This suggests that while some as- able to fold into only a bioactive conformation via the fusion of the propeptide pects of protein folding are robust and evolutionarily important enough to be region of uroguanylin. In addition, our previous studies of the disulfide-coupled preserved for billions of years, other aspects are easier explained by local ef- folding of a series of Gly mutants of prouroguanylin suggested that only a few fects arising from minor sequence changes. amino acid residues of prouroguanylin showed critical effects in the formation Posters: Protein-Small Molecule Interactions I of the native conformation. To further investigate the mechanism for the propeptide-mediated folding of peptide hormones, the several amino acid res- 285-Pos Board B55 idues of the de novo fusion protein were mutated to Gly residues and the refold- Mapping Ligand Binding Landscapes with WExplore ing reactions of these proteins were examined. Alex Dickson. The mutant proteins were prepared using an E. coli expression system. The Biochemistry and Molecular Biology, Michigan State University, East mutant proteins were produced in the form of inclusion bodies and then solu- Lansing, MI, USA. bilized in 8 M urea containing dithiothreitol. The reduced forms of the mutant Ligand binding is often thought of in analogy to protein folding, where the free proteins were purified by RP-HPLC and the structures confirmed by MALDI- energy landscape has a funnel-like shape, encouraging folding (or binding) TOF/MS analyses. The oxidative refolding reactions of the proteins were car- through successive steps where lower entropy is compensated by lower energy. ried out in the presence or absence of glutathione. The results of our studies will However, ligands can often bind in more than one stable pose. Docking algorithms be discussed in this paper. predict sets of stable poses, although their predicted free energies are often

BPJ 8556_8558 Sunday, February 18, 2018 55a unreliable, especially for flexible targets. Molecular dynamics can be used to test mations during conformational space annealing (CSA) global optimization. the stability of predicted binding modes, but this is difficult due to the long time- With this approach, higher-quality initial conformations were obtained and scale of diffusion on the ligand binding landscape. Enhanced sampling methods the binding pose sampling of large flexible ligands was improved compared such as steered molecular dynamics and metadynamics have been used to rank to the previous version of GalaxyDock. poses, but these approaches are generally unable to discover new binding modes. Using WExplore we have mapped the binding landscapes of inhibitors of two hu- 288-Pos Board B58 man bromo-domains BAZ2 and the first bromodomain of BRD4, which are both Blind Prediction of Protein-Peptide Complex Structures: A Novel Method anti-cancer drug targets. Starting from a set of two docked poses in each case, we and a Web Server Xianjin Xu1,2, Chengfei Yan1,2, Xiaoqin Zou1,2. not only rank which pose is more favorable, we predict alternative states - using 1 only free energy - that agree with crystallographic and NMR data in both cases. Dalton Cardiovascular Research Center, Department of Physics and Astronomy, University of Missouri-Columbia, Columbia, MO, USA, We then combine our sampling results to view the binding landscape, and deter- 2 mine now interconversion events take place between stable bound poses. For Department of Biochemistry, Informatics Institute, University of Missouri- BAZ2 we find that stable states interconvert using the unbound state as a mediator, Columbia, Columbia, MO, USA. while for BRD4 we find that the ligand ‘‘crawls’’ between binding poses about Protein-peptide interactions play a crucial role in a variety of cellular pro- 50% of the time. These observations of the binding landscape can have implica- cesses. In silico prediction of protein-peptide complex structure is highly tions for inhibitor design, particularly design efforts based on ligand binding ki- desirable for mechanistic investigation of these processes and for therapeutic netics. This work suggests that WExplore can be used together with docking to design. However, predicting all-atom structures of protein-peptide com- predict stable binding poses in cases where structures are difficult to obtain, plexes without any knowledge about the peptide binding site and the bound such as membrane proteins. peptide conformation remains a big challenge. Here, we present a docking- based method for predicting protein-peptide complex structures, referred to 286-Pos Board B56 as MDockPeP, which starts with the peptide sequence and globally docks the Rational Design of AGO-Allosteric Small Molecule of GLP-1R all-atom, flexible peptide onto the known protein 3D structure. MDockPeP Tejashree Redij1, Rajan Chaudhari2, Zhiyu Li3, Zhijun Li2. was tested on the peptiDB benchmarking database using both bound and un- 1Biological Sciences, University of the Sciences in Philadelphia, bound protein structures. The results show that MDockPeP successfully Philadelphia, PA, USA, 2Chemistry and Biochemistry, University of the generated near-native peptide binding modes in 95.0% of the bound docking Sciences in Philadelphia, Philadelphia, PA, USA, 3Pharmaceutical Sciences, cases and in 92.2% of the unbound docking cases. MDockPeP is computa- University of the Sciences in Philadelphia, Philadelphia, PA, USA. tionally efficient and suitable for large-scale applications. The method has Glucagon Like Peptide-1 Receptor (GLP-1R) is a major drug target in the treat- been implemented in a publicly accessible web server, which can be used ment of Type II Diabetes (T2D). The failure of injectable insulin or Glucagon for both protein-peptide complex structure prediction and initial-stage sam- Like Peptide 1 (GLP-1) mimetic to cure T2D is believed to underlie the high pling of the protein-peptide binding modes for other docking or simulation T2D incidences worldwide. Moreover, the discovery of small molecule drugs methods. remains a challenge due to the nature of the orthosteric binding site in GLP- 289-Pos Board B59 1R. Targeting allosteric sites of GLP-1R represents a novel approach to the dis- Preferential Binding of Flavonoids with Bovine Serum Albumin: In-Silico covery of anti-diabetic agents. Allosteric sites in G-Protein Coupled Receptors and Spectroscopic Insight into Cytotoxic Competence (GPCRs) are less conserved than orthosteric site, show spatiotemporal speci- Bhumika Ray. ficity, saturation and non-competitive inhibitory effect. We hypothesize that Physico Mechanical Metrology Division, CSIR-National Physical if we design small molecule positive allosteric modulator (PAM) targeting Laboratory, New Delhi, India. GLP-1R, then the combined effect of PAM and endogenous GLP-1 would Flavonoids are polyphenolic secondary metabolites with numerous pharma- enhance GLP-1R signaling. For this study, in-house homology modeling cological advantages including their competence as anticancer molecules. method and SiteMap was used to predict the GLP-1R model and the allosteric Flavopiridol (FLAP) and riviciclib (RCB) are semi-synthetic analogs of a site in the model respectively. Next, we generated a library of 5689 compounds natural flavonoid rohitukine, isolated from the stem bark of an Indian me- and docked in the predicted allosteric site. Based on the docking scores, top 9 dicinal plant, Dysoxylum binectariferum. These are cyclin-dependent kinase molecules were screened in vitro using luciferase assay. From the preliminary inhibitors (CDKIs), however, their specificity for biomolecules and mech- screening, we found two novel GLP-1R agonists. Furthermore, synergistic anism of inhibition inside cell is not well deciphered. Considering the sig- studies confirmed that one of the two GLP-1R agonists bind to the allosteric nificance these inhibitors in cancer therapeutics, herein, we explored the site in GLP-1R. In future, we will perform rational design to improve the bind- molecular interaction mechanism between FLAP/RCB and bovine serum ing affinity and absorption, distribution, metabolism and excretion (ADME) albumin (BSA) using various spectroscopic and molecular docking properties of the hit compound. The innovative aspect of this work is the methods. Surface enhanced Raman, circular dichroism and absorption spec- structure-based design of small molecule, allosteric compounds for GLP-1R troscopy have been employed to understand the structural-conformational activation. The significance of this work to public health is getting an anti- aspects and other binding parameters between FLAP/RCB and BSA. Vibra- diabetic oral molecule that would enhance insulin production in the presence tional spectroscopic studies reveal that interacts with the amino acid resi- of low levels of endogenous GLP-1 peptide in T2D patients. dues of BSA without interrupting with the microenvironment of around the residue Trp213. Further, the FLAP-BSA interactions are stabilized by 287-Pos Board B57 hydrophobic and electrostatic forces, whereas, hydrophobic interactions Improving Docking Performance of Large Flexible Ligands using Hot Spot are observed for binding between RCB and BSA. Comparative experi- Information Predicted by Fragment Docking mental results illustrate that the primary binding sites for FLAP and Minkyung Baek, Chaok Seok. RCB on BSA are sites II and III. Conformationally, there is reduction in Chemistry, Seoul National University, Seoul, Republic of Korea. alpha-helical content of BSA, when complexed with FLAP/RCB indicating Docking large flexible ligands having many rotatable bonds is important not partial unfolding of BSA and increase in the turns and random coil struc- only for understanding biological function of natural ligands but also for tures of protein. The binding constant (Ka) estimated for the complexation designing putative drug compounds as in peptidomimetics. Although many is of the order of 105 and 104 for FLAP/RCB-BSA complexes respectively, protein-ligand docking programs have been developed, it is still challenging specifying moderate to strong binding affinity. Further, molecular docking to predict the correct binding poses of large flexible ligands as the dimen- simulations have been performed, which are in corroboration with spectro- sionality of the conformational space increases. Here, we introduce a new scopic outcomes. Hence, the findings concluded here, paves a way in delin- approach to improve the sampling ability of GalaxyDock protein-ligand eating the action mechanism of flavonoids that may lead to rational docking program inspired by pharmacophore-based docking and fragment- designing of flavonoid based new therapeutics and recognizing BSA as mo- based drug discovery methods. We assumed that binding ‘‘hot spots’’ of a lecular target. specific receptor protein could be reasonably predicted for each fragment of a given ligand. Utilizing the predicted hot spot information might lead 290-Pos Board B60 to efficient conformational search by reducing the conformational space Computing Protein-Ligand Binding Association Rate Constants by that should be sampled. To identify ‘‘hot spots’’ for each fragment, target Combining Brownian Dynamics and Molecular Dynamics Simulations ligands were fragmented into rigid fragments, and up to five fragments S. Kashif Sadiq, Rebecca C. Wade. were docked onto the receptor protein using fast Fourier transformation Heidelberg Institute for Theoretical Studies, Heidelberg, Germany. (FFT)-based rigid-body docking. The detected fragment binding hot spot in- Understanding the detailed molecular kinetics of protein-ligand association formation was used to generate initial conformations and further trial confor- is of fundamental importance in biomedical research - increasingly so, as

BPJ 8556_8558 56a Sunday, February 18, 2018 the kinetic characteristics of drugs become more widely appreciated as key 293-Pos Board B63 to their efficacy. Conventional unbiased all-atom molecular dynamics Modelling Interactions of Urokinase Plasminogen Activator with (MD) simulations can now accurately sample the millisecond timescale al- Amiloride and Its Derivatives lowing detailed kinetic properties of binding, conformational changes and Peggy Palsgaard1, Fredric A. Gorin2, Igor Vorobyov3. protein folding to be reconstructed from large ensembles of simulations. 1Department of Biomedical Engineering, University of California, Davis, However, it still remains a major computational hurdle to calculate Davis, CA, USA, 2Dep. of Neurology, School of Medicine and Dep. of protein-ligand binding kinetics for even moderately sized drugs and/or Molecular Biosciences, School of Vet. Medicine, University of California, large receptor targets, especially if the binding pathway is coupled to Davis, Davis, CA, USA, 3Department of Physiology and Membrane Biology conformational changes in either, or if the kinetics are too slow. Binding and Department of Pharmacology, School of Medicine, University of timescales for clinically relevant ligands usually extend far beyond those California, Davis, Davis, CA, USA. that can be simulated by conventional MD methods. Implicit solvent, Subpopulations of persistently hypoxic cancer cells play a key role in tumor recur- rigid-body Brownian dynamics (BD) methods are promising because calcu- rence and drug resistance. These cancer cells express high levels of urokinase lations are computationally feasible. However, whilst such methods have (uPA) and its serpin inhibitor PA-1 with the protein complex uPA-PAI-1 being been successful for computing diffusional association rate constants, a highly predictive of cancer recurrence and metastasis. The binding of uPA and rigid-body approach cannot capture all the various ligand/protein confor- PAI-1 is via a salt bridge between an aspartic acid on uPA and an arginine on mations that are often involved along binding pathways. Here, we develop PAI-1. Our studies focus on amiloride and related analogs that disrupt intracellular a multiscale method that integrates conventional MD with rigid-body BD uPA-PAI-1. These compounds, containing a guanidine group and a ring structure, and thereby allows target-ligand flexibility to be integrated into calcula- are potentially able to bind to the active site of uPA and inhibit its activity. tions of association rate constants (kon). Our approach involves pre- Moreover, some amiloride derivatives were shown to cause endosomal computing the conformational kinetics of the apo-protein ensemble using ‘mis-trafficking’ of the uPA/PAI-1 complex and associated proteins, leading to MD simulations and Markov state models and then integrating a set of irreversible cancer cell demise. Using molecular docking calculations with Auto- kinetically distinct conformers within the framework of the BD calcula- Dock Vina and Rosetta-Ligand as well as atomistic molecular dynamics (MD) tions. Based on this approach, we compute kon values for a set of inhibitors simulations, we modelled the binding of these compounds to both the isolated with known experimental kinetics, that bind to the conformationally flex- uPA protein and uPA/PAI-1 complex using available crystal structures. We ible protein HIV-1 protease. The method also allows us to compute and observed that, while AutoDock Vina can provide reasonable predictions of exper- analyze ligand gating effects mediated by the major conformational imental uPA binding affinities, Rosetta-Ligand more accurately predicts con- changes in flexible proteins. formers of drug binding to the uPA active site. All-atom MD simulations were found to be instrumental for testing stabilities of drug - protein complexes pre- 291-Pos Board B61 dicted by the molecular docking analyses. Molecular docking and MD simulations MDockServer: An Efficient Docking Platform for Inverse Virtual of drug binding to the uPA/PAI-1 complex was used to identify molecular sites of Screening uPA-PAI-1 that correspond with drug-induced endosomal mis-trafficking and Zhiwei Ma1, Xianjin Xu2, Xiaoqin Zou2,3. 1 cancer cell death. These calculations are helping us identify the molecular deter- Department of Physics and Astronomy, Dalton Cardiovascular Research minants of novel small molecules that kill hypoxic cancer cells independent of Center, University of Missouri-Columbia, Columbia, MO, USA, their proliferative status by triggering selective endosomal relocation. 2Department of Physics and Astronomy, Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO, USA, 3Department of 294-Pos Board B64 Biochemistry, Informatics Institute, University of Missouri-Columbia, Molecular Mechanism of Resistance to Kinase Inhibitors Clarified by a Columbia, MO, USA. Binding Free Energy Computation Method and Its Improvement by The prediction of the most likely protein target for a known ligand from thou- Incorporating Protein Flexibility sands of protein candidates is a challenging task but a useful strategy to inves- Mitsugu Araki1,2, Yasushi Okuno1,2. tigate novel targets to combat diverse diseases. Recently, we have 1Kyoto University, Kyoto, Japan, 2RIKEN, Kobe, Japan. implemented an improved version of MDock that includes an effective statis- Accurate prediction of protein-compound binding free energy (DG) is strongly tical potential-based scoring function ITScore developed in house as a web required for not only efficiently optimizing drug candidates but also elucidating server for the inverse virtual screening. MDockServer is a user-friendly molecular mechanisms of their pharmacological activities. In recent years, a ligand-protein docking server to predict the potentially promising therapeutic number of DG computation methods based on the molecular dynamics (MD) target provided in our built-in protein database for a query compound of in- simulation have been developed. In this study, based on an alchemical free energy terest. In our current version of the protein target database, we have included perturbation method, MP-CAFEE (Fujitani et al., Physical Review E (2009), 79, 3146 protein-ligand complexes, each with the searchable disease association 021914), we approached molecular mechanism of drug resistance of Anaplastic information. Our achieved competitive success rates and efficiency allow lymphoma kinase (ALK) inhibitors. We constructed structural model of drug- the retrieval of useful protein-ligand binding conformations associated with resistant mutants of ALK, and their binding affinities with three ALK inhibitors relevant disease information, making MDockServer an effective tool for ther- were predicted using MP-CAFEE. The predicted DGcorrelatedwellwithdrugef- apeutic intervention. ficacy, suggesting that decreased binding affinity is a major contributor of drug resistance. We further characterized structural differences between wild-type 292-Pos Board B62 ALK and its drug-resistant mutants by analyzing MD trajectories. (Katayama Kinetic Machine Learning Unravels Ligand-Directed Conformational et al., Clin. Cancer Res. (2014) 20(22), 5686-5696). Whereas our MD simulations m Change of Opioid Receptor correctly captured structural dynamics of the drug-binding site on ALK, generally, Evan N. Feinberg1, Vijay S. Pande1, Amir Barati Farimani2, accurate DG prediction remains challenging because of protein flexibility. In case Carlos X. Hernandez1. 1 2 of CDK2 and ERK2 kinases, conformational flexibility of the glycine-rich loop in Biophysics, Stanford, Stanford, CA, USA, Chemistry, Stanford, Stanford, the drug-binding site was identified using MD simulations. DGoftwenty-four CA, USA. ATP-competitive inhibitors toward these kinases was assessed. However, large The m Opioid Receptor (mOR) is a G-Protein Coupled Receptor (GPCR) that calculation errors of 2-3 kcal/mol were observed when using MP-CAFEE, where mediates pain and is a key target for clinically administered analgesics. The the free energy simulation starts from a single equilibrated conformation. Here, we current generation of prescribed opiates - drugs that bind to mOR - engender developed a new DG computation method, where the starting structure was set to dangerous side effects such as respiratory depression and addiction in part by multi-conformations to cover flexibility. The calculation accuracy was success- stabilizing off-target conformations of the receptor. To determine both the fully improved, especially for larger molecular size compounds, leading to reli- key conformations of mOR to atomic resolution as well as the transitions be- able prediction of a broader range of drug candidates (Araki et al., J. Chem. Inf. tween them, long timescale molecular dynamics (MD) simulations were con- Model., (2016) 56(12), 2445-2456). ducted and analyzed. These simulations predict new and potentially druggable metastable states that have not been observed by crystallography. 295-Pos Board B65 We applied cutting edge algorithms (e.g., tICA and Transfer Entropy) to guide Hydrogen Bond Surrogate Beta-Hairpins to Inhibit Protein-Protein our analysis and distill the key events and conformations from simulation, pre- Interactions senting a transferrable and systematic analysis scheme. Our approach provides Nicholas Sawyer, Paramjit S. Arora. a complete, predictive model of the dynamics, structure of states, and structure- Chemistry, New York University, New York, NY, USA. ligand relationships of mOR with broad applicability to GPCR biophysics and Beta-hairpins are fundamental units of protein structure and play a critical role medicinal chemistry. in many protein-protein interactions (PPIs). Current strategies for stabilization

BPJ 8556_8558 Sunday, February 18, 2018 57a of isolated b-hairpin peptides have focused on stabilizing three elements of arsenate. These properties makes PBPs a prime candidate for phosphate recov- b-hairpin structure: individual b-strands, cross-strand interactions, and the ery in a biomimetic application. In order to utilize and improve these proteins reverse turn. Though these strategies have produced a variety of stable, well- for practical applications, we need to understand the molecular mechanisms by folded b-hairpins, the stabilizing moieties often occupy potential functional which these proteins bind and release phosphate. sites that might interfere with the design of b-hairpin PPI inhibitors. PBPs have previously been extensively studied by experimental methods, with To overcome this limitation, we evaluated the hydrogen bond surrogate (HBS) several identified crystal structures available in both phosphate-bound and un- approach as a sequence-independent strategy to generate stable b-hairpin PPI bound conformations. Yet little is known about the dynamical changes that inhibitors. The HBS approach replaces a structural main-chain hydrogen result from binding and release, thus making it difficult to use molecular engi- bond with an isosteric covalent bond and has been particularly successful for neering to control these processes. stabilization of peptides in the a-helical conformation. We have recently shown We have therefore used molecular dynamics simulations as a tool to probe that HBS mimicry of the N-to-C-terminal hydrogen bond of a model b-hairpin protein-ligand interactions, and the resulting changes in the structure and leads to a thermally and proteolytically stable b-hairpin. dynamics of the PBPs. In particular, we have performed both unbiased and Here we show that the HBS approach is generalizable to other b-hairpins and metadynamics-based, enhanced sampling molecular dynamics simulations apply it to the design of b-hairpin PPI inhibitors. Specifically, we evaluated a of a PBP. We have investigated the free-energy landscape of domain- critical b-hairpin fragment in the NS1 monobody, which inhibits Ras dimeriza- movements, phosphate binding and solvation of the binding pocket. Together tion and signaling. We show that NS1-derived HBS b-hairpins bind to Ras with the known crystallographic-states of the protein these simulations provide in vitro and affect cell proliferation in Ras-dependent cell lines. These NS1 new insights into the molecular mechanisms by which PBPs recognize, bind and HBS b-hairpins will be useful as research tools to study Ras signaling. We envi- release phosphate ions. With the information gained by this study we look further sion that the HBS approach will be useful in the generation of additional into various options for bioengineering of the PBPs for phosphate recovery. b-hairpin PPI inhibitors and will provide a ‘‘stepping stone’’ for the creation of stable, sequence-independent b-sheet PPI inhibitors. 298-Pos Board B68 Binding Free Energy Calculation of Protein-Carbohydrate Complexes: 296-Pos Board B66 Learnings so Far CANDOCK: Conformational Entropy Driven Analytics for Class-Specific Sushil K. Mishra1, Jaroslav Koca2, Yoshiki Yamaguchi1. Proteome-Wide Docking 1Structural Glycobiology Team, Systems Glycobiology Research Group, Jonathan A. Fine, Gaurav Chopra. RIKEN, Wako-351 0102, Japan, 2Central European Institute of Technology Chemistry, Purdue, West Lafayette, IN, USA. (CEITEC), Masaryk University, Kamenice-5, 625 00 Brno, Czech Republic. Entropy, a driving ‘‘force’’ relating microscopic behavior to macroscopic Protein glycosylation, a most common protein modification, has consequent phenotype, specifies the direction of time. Biophysical processes, like implications in several pathological events like promoting protein folding, sta- protein-ligand binding, occur due to rearrangement of atoms guided by inter- bilizing cell-surface glycoproteins and malignancy. Non-covalent interactions atomic interaction energy and entropy. In the context of protein-ligand docking, of these glycans with other proteins regulate several important biological func- little attention has been given to incorporate the effect of entropy. In drug dis- tions like cell adhesion, recognition, differentiation, metastasis, microbial path- covery, docking is traditionally used to virtually screen large compound li- ogenesis, immunological recognition etc.1 During these processes, information braries against one or a few disease targets, compared to evaluating global encoded in glycans is recognized, deciphered and put into biological action by effects on the proteome necessary to translate docking methods to medicine. special proteins called lectins.2 Lectin-glycan binding is, usually, determined We have developed CANDOCK for proteome-scale docking guided by by polar interactions between the polar carbohydrate group and the polar pro- changes in conformational entropy to determine the native ligand conformation tein functional groups. Such interactions are often enhanced by ion or water in complex chemical environments including solvent and cofactors. Our bridging. Moreover, recent observations demonstrate that solvent-exposed aro- method identifies native ligand conformations by hierarchically building li- matic residues frequently engage in very strong stacking interactions with the gands from atom types specific for a chemical environment, and incorporates carbohydrates.3 The above facts make lectin-carbohydrate interactions study dynamics using generalized statistical potentials to estimate intermolecular more difficult by computational methods, especially when the situation is energy and computes changes in conformational entropy. CANDOCK uses: further complicated by the anomeric effect and inherent flexibility along the (i) Entropy: binding sites and hierarchies of ligand atoms possess regions of glycosidic linkages. high and low structural stability to guide binding mode selection, (ii) Sampling: Understanding the atomic basis of lectin-carbohydrate interactions is of interest atom hierarchies with correlated motions are computed using induced fit, and not only for theoretical reasons, but also for the design of glycan-based thera- (iii) Context: intermolecular chemical complementarity of protein-ligand peutics. We have been accessing the performance of several free-energy calcu- conformational degeneracy for binding strength prediction, and the similarity lation approaches on various lectin/glycan complexes. In this presentation, we of binding interaction networks of ligands with proteome(s) for drug discovery. will show the current capabilities of the Linear Interaction Energy, MM/PBSA, Parameters were optimized in a class dependent manner for proteome-scale MM/GBSA and Thermodynamic Integration approaches. Finally, we will docking applications. We obtain a class average accuracy of 83% (class discuss various lessons we have learned so far and key recommendations.46 average improvement of 13%) to predict accurate binding modes in PDBBind References: and DUD-E datasets, retrospectively. Previously, we have shown most human [1] Karlsson. Trends Pharmacol. Sci. 12 (1991) 265–272. approved drugs interact with proteins from different druggable classes. These [2] Gabius. Sci. 40 (2015) 341. druggable networks were used to experimentally verify new drug repurposing [3] Wimmerova et al., PLoS ONE. 7 (2012) e46032. leads spanning nine different diseases, prospectively. We conclude incorpo- [4] Mishra et al., J. Comput. Chem. 33 (2012) 2340–2350. rating entropy is essential for modeling dynamical biophysical processes. [5] Mishra et al., J. Chem. Theory Comput. 11 (2015) 3333–3345. [6] Nagae and Mishra et al., Glycobiology (2017) cwx081. 297-Pos Board B67 Towards Biomimetic Phosphate Recovery: Molecular Dynamics 299-Pos Board B69 Simulations of Phosphate Binding Proteins All-Atom Study of the Interactions of the Neurotransmitters Dopamine Sigurd F. Truelsen1, Yong Wang2, Kresten Lindorff-Larsen2, and Norepinephrine with a-Synuclein Oligomers and the Effects on Claus Helix-Nielsen1,3. Nucleation 1Department of Environmental Engineering, Technical University of Yu Zou1, Junhang Hu2, Qingwen Zhang1. Denmark, Kgs. Lyngby, Denmark, 2Department of Biology, University of 1College of Physical Education and Training, Shanghai University of Sport, Copenhagen, Copenhagen, Denmark, 3Faculty of Chemistry and Chemical Shanghai, China, 2Centre for Quality Science Research, Henan Quality Engineering, University of Maribor, Maribor, Slovenia. Polytechnic, Pingdingshan, China. Phosphorous is a scarce and biologically-essential resource for sustaining the The accumulation of self-assembled a-synuclein (aS) aggregates in the sub- global food supply. Excess use, however, leads to eutrophication of rivers, stantia nigra is regarded as the principal factor that leads to the Parkinson’s dis- lakes and oceans, and harms the natural environment. Thus, it is important to ease. Experimental studies have shown that the neurotransmitters dopamine develop technologies for the extraction of phosphorous from e.g. waste water, and norepinephrine could inhibit the formation of these aggregates and disag- enabling its reuse while limiting the environmental impact. gregate existing aggregates. Otherwise, they could also stabilize the aS oligo- Employing highly specialized proteins could provide an efficient and effective mers. In this work, we studied the influence of dopamine and norepinephrine starting point to develop a technology for phosphorous recovery. Phosphate molecules on aS dimer and trimer by performing atomistic molecular dynamics Binding Proteins (PBPs) from Escherichia coli have an intrinsically high simulations of 7.2 ms in total. We also conducted the aS tetramer and pentamer affinity and selectivity for phosphate over similar compounds such as the toxic to show the influence of small molecules on the nucleation of aS. The results

BPJ 8556_8558 58a Sunday, February 18, 2018 suggest that the minimum nucleus size of aS consists of four peptide chains, CaMBR, which is believed to control CaN autoinhibition, spontaneously forms and the molecules could affect the nucleation via stabilizing the aS dimer an amphipathic a-helix. BD simulations of DH binding to CaM suggest that the and trimer. By comparing the simulation results of aS dimer and trimer in interaction interface may involve CaM surface residues W99, D133 and E139. the presence and absence of molecules, we found that the binding of molecules These findings provide structural and kinetic insights into the molecular mech- prevents the b-sheet structure from converting into disordered conformation, anism of CaM/CaN activation. protects the inter-peptide backbone hydrogen bonds from being destroyed, 1. Szabo, A., Shoup, D., Northrup, S. H., & McCammon, J. A. (1982). Stochas- and stabilizes both interchain and intrachain salt bridges. The results from tically gated diffusion-influenced reactions. Journal of Chemical Physics, our study can not only uncover the nucleation of aS, but also reveal the specific 77(9), 4484-4493. interactions of dopamine and norepinephrine molecules with b-sheet-rich oligomers of aS, which may be helpful to the design of drug candidates against 302-Pos Board B72 Parkinson’s disease. Boltzmann Docking Identifies Allosteric Small Molecule Modulators of Protein Activity 300-Pos Board B70 Thomas E. Frederick, Kathryn M. Hart, Katelyn E. Moeder, Chris M.W. Ho, Prediction of Binding Hot Spots in Cyclooxygenase and Their Implication Maxwell I. Zimmerman, Gregory R. Bowman. to Protein-Protein and Protein-Ligand Interactions Biochemistry and Molecular Biophysics, Washington University School of Inseok Song. Medicine, St. Louis, MO, USA. University of Seoul, Seoul, Republic of Korea. Computational approaches can facilitate the design of clinically relevant small Many studies suggest that some individual residues in the binding region molecule inhibitors. Classical drug design focuses primarily on targeting a pro- contribute more than others to the free energy of binding and hence these tein’s active site based on its x-ray crystal structure. One drawback to this small regions are defined as binding hot spots, which are often utilized as approach is crystal structures do not report on protein dynamics and do not the starting point for function identification and drug discovery. Binding capitalize on the presence of hidden cryptic pockets. Cryptic pockets are tran- hot spots are identified by several analytical methods experimentally, but sient allosteric sites invisible to conventional structural biology techniques and computational solvent mapping can also be exploited for the same purpose present new opportunities for drug design. Here we introduce Boltzmann dock- and assist complementarily in characterizing binding properties of a target ing as a method to identify small molecules that bind to cryptic pockets and protein. Binding pattern of cyclooxygenase (COX), also known as prosta- allosterically modulate enzymatic activity. We leverage knowledge of protein glandin endoperoxide H synthase, has been intensively investigated, which dynamics from molecular dynamics simulations and Markov State Models deepens substantially our understanding about COX at the protein level. (MSMs) to identify cryptic pockets. MSMs are a network representation of a COX catalyzes both the oxygenation of arachidonic acid (AA) to a bicyclic protein’s free energy landscape that provides representative states and their endoperoxide and the reduction to the corresponding alcohol, which is the populations. Boltzmann docking screens a library of small molecules against precursor of biologically active prostanoids. In addition to AA, various satu- multiple MSM states containing a target cryptic pocket and generates a new rated or unsaturated fatty acids, mostly classified as non-substrates, can bind Boltzmann score that is a population weighted average of the individual states’ COX and affect the Vmax and KM of AA to a different extent, which unveils docking scores. We successfully applied this approach to identify multiple allo- the way of communications between COX subunits. This long-range interac- steric modulators of TEM b-lactamase activity, and are currently looking to tion passes through the dimeric interface region of COX and binding hot identify novel inhibitors of the Ebola viral protein VP35. Boltzmann docking spot(s) inside or near which can be predicted by computational protein map- should provide a general framework to identify allosteric modulators of protein ping. This potentially valuable information suggests a clue for conforma- activity, especially for targets widely considered ‘‘undruggable’’ owing to the tional change upon binding of COX substrate(s) and substrate-analogs lack of an obvious binding site in their crystal structures. including inhibitory ligands even though the physiological importance of al- losterism of COX protein remains to be resolved. 303-Pos Board B73 The Impact of Alternative Binding Site on HCT Substrate Permissiveness 301-Pos Board B71 from Numerical Analysis CaMBR Conformational Flexibility on CaM-CaN Association Rate and Chun Kei Lam, Ying-Chih Chiang, Yi Wang. Distal Helix’s Interaction Surface with CaM: A Computational Study Department of Physics, The Chinese University of Hong Kong, Shatin, Hong Bin Sun, Peter M. Kekenes-Huskey. Kong. Chemistry, University of Kentucky, Lexington, KY, USA. Substrate permissiveness is recognized as a key mechanism of evolution that Calcineurin (CaN) is widely involved in many physiological and pathophysio- facilitates the development of new enzymatic functions. Using extensive all- logical processes such as nervous system development and immune system atom molecular dynamics simulations, we have examined the alternative, activation. CaN is activated upon Calmodulin’s (CaM) binding to the CaM weak binding site for its non-native substrate in hydroxycinnamoyltransferase binding region (CaMBR) in CaN’s regulatory domain. Due to the intrinsically (HCT), a plant enzyme involved in the phenylpropanoid metabolism. By disordered feature of CaMBR, the accessibility of CaMBR to CaM is deter- numerically solving the Smoluchowski equation, we further investigate the mined by the conformational flexibility. We explored CaMBR’s conforma- role of such alternative binding site in facilitating the reaction of non-native tional flexibility on CaM/CaN association rates via microsecond MD and substrate. Our analysis begins with a general model of a catalytic center and Brownian Dynamic simulations on three CaMBR peptides with varying lengths an alternative binding site, each represented as a sphere. Properties of the sites and charge mutations. We characterized the switching kinetics of these peptides such as their relative affinities for the substrate, the reactivity of the catalytic between ‘‘open’’ (suitable for CaM’s binding) and ‘‘closed’’ (unsuitable for center, as well as their distance of separation, are varied so factors affecting binding) states based on a Markov State Model (MSM). We found that, for the reaction rate can be determined quantitatively. The effect of an alternative all three CaMBR peptides, the switching rates are all faster than the diffusional binding site on non-native substrate reaction in HCT is then studied with the encounter rates between CaMBR and CaM calculated by BD, implying that the software Smolfin, which enables us to determine rate constant even for compli- conformational flexibility of CaMBR does not impede diffusion limited CaM/ cated biomolecular geometries. Together, our calculations shed light on the po- CaN association per stochastic gating arguments (Szabo et. al.1). Further, addi- tential role played by alternative binding sites in the development of new tional simulations of distal helix (DH, residues 441 to 459) C-terminal to metabolic functions in substrate-permissive enzymes.

BPJ 8556_8558 Sunday, February 18, 2018 59a

Posters: Protein Assemblies I 306-Pos Board B76 A Novel Drp1 Interface Specifically Governs Mff Interactions 304-Pos Board B74 Ryan W. Clinton, Jason A. Mears. Supramolecular Assembly of Calsequestrin Is Stabilized by Multivalent Pharmacology, Case Western Reserve University, Cleveland, OH, USA. Interactions of the N-Terminus and N-Linked Glycans In eukaryotic cells, mitochondria comprise a dynamic network of organelles Joseph M. Autry1, Bengt Svensson1, Ke Shi1, Thomas E. Bohl1, that undergo cycles of fission and fusion. Among many other functions, these Steven E. Cala2, John K. Lee1, David D. Thomas1, Hideki Aihara1. opposing processes allow for the quality control of the cellular mitochondria 1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, pool by allowing for the rescue and/or disposal of dysfunctional mitochon- Minneapolis, MN, USA, 2Physiology, Wayne State University, Detroit, dria. Therefore, mitochondrial fission is an essential cellular process. The MI, USA. dynamin family GTPase, dynamin-related protein 1 (Drp1), initiates fission We have used x-ray crystallography to determine the structure of calseques- by translocating from the cytosol to the mitochondrial outer membrane trin isoform 1 (CSQ) from rabbit muscle, identifying a novel conformation at (MOM), where it forms a helical polymer circumscribing the mitochondria. 3.0 A˚ resolution. CSQ is a highly-acidic 42 kDa glycoprotein that reversibly This initial recruitment of Drp1 to the mitochondria is a highly regulated binds/releases 60 Ca ions per molecule, undergoing a Ca-induced disorder- process that requires interaction with one or more MOM-anchored partner to-order transition (protomer) and extensive self-association (polymer). CSQ proteins including mitochondrial fission 1 protein (Fis1), mitochondrial functions as the high-capacity calcium-storage protein in the sarcoplasmic re- fission factor (Mff), and the mitochondrial dynamics proteins of 49/51 ticulum lumen. Localization of CSQ near the Ryanodine Receptor Ca channel kDa (MiD49/51). In metazoans, Mff is a key partner protein for Drp1, so un- facilitates rapid Ca release from the SR to generate muscle contraction. Dur- derstanding the nature of Drp1 interaction with Mff may yield significant ing myocyte Ca cycling, Ca ions on CSQ are dynamically exchanged with insight to the mechanisms underlying mitochondrial fission in higher eukary- potassium ions and protons. Here we report the x-ray structure of CSQ crys- otes. Given the absence of Mff in lower eukaryotes, we compared regions of tallized in the potassium/proton-bound state, finding that CSQ assembles as a low sequence conservation between human and yeast Drp1 homologues to slightly-asymmetric dimer which forms the core structural unit of long linear identify putative Drp1-Mff interaction interfaces. Using biochemical and polymers within the crystal lattice. This structure reveals the assembly of structural studies, we identified one region in particular where mutations CSQ polymers is stabilized by multiple distinct protomeric contacts: N-termi- ablate Mff-stimulated Drp1 activities. Importantly, no effect on intrinsic nal, C-terminal, and lateral. The mature N-terminus of CSQ (signal-sequence Drp1 behaviors including GTPase activity, cardiolipin interaction, and removed) is tucked into a groove on the adjacent protomer (iþ1), with abun- several modes of self-assembly were observed. Identification of this region dant side-chain interactions stabilizing dimer formation. The CSQ glycan provides a novel tool to selectively interrogate the role of Drp1-Mff interac- (GlcNAc2-Man2) hydrogen-bonds with the C-terminus of its own protomer tions in diverse cellular functions. þ (i), and the terminal mannose extends across to the i 2 protomer, thereby 307-Pos Board B77 forming a bridging hydrogen-bonded network running in parallel spirals Molecular Basis for the Hierarchical Dependency of Collagen Mechanics along the linear polymer. The protein matrix within our CSQ crystal exhibits Sameer Varma1, Joseph P.R.O. Orgel2, Jay D. Schieber2. repeating lateral contacts of neighboring protomers between tightly-packed 1University of South Florida, Tampa, FL, USA, 2Illinois Institute of adjacent polymers. Mapping of five recently-discovered disease-causing mu- Technology, Chicago, IL, USA. tations onto our CSQ structure predicts that human mutations disrupt either Type I collagen is the predominant collagen in mature tendons and ligaments, dimer contacts associated with high-capacity Ca binding, or lateral branches where it gives them their load-bearing mechanical properties. Fibrils of type I suggested by classic electron microscopy studies. Structural dynamics of collagen are formed by the packing of polypeptide triple helices. Higher-order myopathic CSQ mutants are being examined by crystallography and spectros- structures like fibril bundles and fibers are assembled from fibrils in the pres- copy. Acknowledgments: NIH grants to DDT (R01GM027906) and HA ence of other collagenous molecules and noncollagenous molecules. Experi- (R35GM118047). ments, including ours, show that the assembly of collagen molecules into fibrils/fibril-bundles makes them less resistant to axial stress — the Young’s 305-Pos Board B75 moduli of fibrils/fibril bundles are an order-of-magnitude smaller than the Modeling Reaction-Triggered Infectivity during Retroviral Assembly and Young’s moduli of triple helices. Here we will present results from our ongoing Maturation molecular dynamics studies (Proteins 83:1800-1812, 2015; Biophys J 111:50- S. Kashif Sadiq. 56, 2016) that provide key insight into this hierarchical dependency. As such, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany. these studies recommend a paradigm shift in our understanding of how collagen Retrovirus particle (virion) maturation is a remarkable example of macromo- fibrils bear stress — we find that it is not the fibril core that bears the initial lecular assembly coupled to chemical reactions. Infectivity usually requires strain, as was thought, but it is the portion of the fibril exposed to the solvent clustering of multiple transmembrane envelope proteins (Env3) on the virion and/or fibril-fibril interface that bear the initial strain. This has direct implica- exterior, yet is only triggered by protease-dependent degradation of a tion on understanding how various biological factors, including disease- membrane-bound Gag polyprotein lattice on the virion interior through a mech- causing congenital mutations and post-translational modifications, affect anism which is unclear. High resolution cryo-electron microscopy has revealed collagen structure, assembly and mechanics. the architecture of the outer shell of the immature virion, but alone does not explain maturation pathways. Modeling could provide insight but current ap- 308-Pos Board B78 proaches are unable to account simultaneously for both assembly and reactions Knowledge-Based Coarse-Grained Model for Simulating Multi-protein at this scale. Recently, interaction particle-based reaction diffusion (iPRD) ap- Complexes proaches have emerged. These combine space-excluded particle-based diffu- Youngchan Kim1, Jeetain Mittal2. sion with state-changing phenomenological chemical reactions, including the 1Naval Research Laboratory, Washington DC, USA, 2Department of assignment of inter-particle and particle-geometry interaction potentials and Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, make a range of reaction-coupled clustering problems accessible at the required PA, USA. spatiotemporal scale. Here, I develop an approach that integrates cryo-EM and A residue-level coarse-grained model suitable for simulating multi-protein crytallographic data with iPRD models to provide insight into the onset of HIV- complexes is developed in this work. Contact potentials and distances between 1 infectivity from an immature virion. The model implements an ultra-coarse- all pairs of residues are constructed via Boltzmann inversion of inter- graining (UCG) scheme based on the structural dimensions of each molecule to molecular contacts of more than 700 protein complexes. It is shown that the treat entire proteins at near-single particle resolution. It includes binding stoi- contact potentials between hydrophobic pairs are less sensitive to the residue chiometry and lattice symmetries and can follow transmembrane Env3 diffu- identities when compared to the Miyazawa-Jernigan (MJ) contact potentials sion in the presence of a monotopic truncated Gag lattice composed of derived from intra-molecular contacts. On the other hand, the contact poten- membrane-bound matrix (MA) proteins linked to capsid (CA) subunits and tials between hydrophilic pairs depend more strongly on the residue identities. freely diffusing protease (PR) enzymes. Simulations suggest that initial immo- These potentials and the contact distances are fitted with the so-called bility of Env3 is conferred through lateral caging by matrix trimers vertically Lennard-Jones (LJ)(m,n) potentials (where m and n are the exponents of repul- coupled to the underlying hexameric capsid layer. Gag cleavage by PR verti- sive and attractive parts, respectively) for the short-range van der Waals (vdW) cally decouples the matrix and capsid layers, induces both MA and Env3 diffu- interactions for the purpose of simulating thermodynamic, kinetic and struc- sion, and permits Env3 clustering. Spreading across the entire membrane tural properties of protein complexes. Note that the softness of the repulsive surface reduces crowding, in turn, enhancing the effect and suggests how infec- part and the range of the attractive part of the vdW interactions can be tivity can be achieved. controlled by varying the exponents m and n. Upon parameterizing to fit the

BPJ 8559_8561 60a Sunday, February 18, 2018 second virial coefficient of lysozyme and the binding affinity of the ubiquitin- as a function of time, binding and activation energies as well as apparent diffu- CUE complex, we show that the LJ(m,n) potential with softer repulsion sion coefficient were estimated and the equilibrium species were imaged by (m=10) and shorter-range attraction (n=12) performs best in predicting the cryo-transmission electron microscopy. Surprisingly, substituting synthetic binding affinities and native bound structures compared to the standard polyelectrolyte to genome lowered the energy barrier to filled capsids. We pro- LJ(12,6) potential. Furthermore, the new model yields thermodynamic and pose a schematic free energy landscape that accounts for the observed path- structural properties of the ubiquitin and its various binding domains much ways. Our results further point to the NPCs as checkpoints in the assembly closer to the experimental data compared to the previous models based on pathway. MJ-type potentials. Our new protein binding model can easily be implemented to many existing molecular dynamics and Monte Carlo simulation packages. 311-Pos Board B81 We apply this new model to study the effects of crowding on protein- Podocin Oligomerization Revealed by FRET Analysis: Sites of Interallelic binding thermodynamics and kinetics aimed at providing a better understand- Interactions 1 2 3,4 5 ing of biological processes inside a cell. Gusztav Schay ,Pa´l Stra´ner , Eszter Balogh , Christelle Arrondel , A´ gnes Miko´3,4, Gerda L’Aune4, Alexandre Benmerah5, Andra´s Perczel2, 309-Pos Board B79 Do´ra K. Menyha´rd2, Corinne Antignac5,Geraldine Mollet5,Ka´lma´n Tory4,5. A Investigation on the Interaction between Viral Subunits 1Biophysics and Radiation Biology, Semmelweis University, Budapest, Jingzhi Chen1, Maelenn Chevreuil1, Yves Lansac2, Guillaume Tresset1. Hungary, 2MTA-ELTE Protein Modeling Research Group and Laboratory of 1Laboratoire de Physique des Solides, CNRS, Univ. Paris-Sud, Universite Structural Chemistry and Biology, Eo¨tvo¨s University, Budapest, Hungary, Paris-Saclay, Orsay, France, 2GREMAN, Universite Franc¸ois Rabelais, 31st Department of Pediatrics, Semmelweis University, Budapest, Hungary, CNRS, Tours, France. 4MTA-SE Lendulet€ Nephrogenetic Laboratory, Semmelweis University, Assembly and disassembly are crucial stages in the life cycle of viruses. The Budapest, Hungary, 5Laboratory of Hereditary Kidney Diseases, Imagine stability of simple viral particles mostly relies on a delicate balance between Institute, Institut national de la sante et de la recherche medicale, Paris, three interactions: (1) a short-range attraction due to hydrophobic and/or van France. der Waals forces; (2) a long-range electrostatic repulsion; (3) a long-range Podocin, a membrane-anchored component of the slit diaphragm, is encoded electrostatic attraction between subunits and the negatively charged genome. by NPHS2, the major gene mutated in hereditary podocytopathies. We A good knowledge of the interactions at work is essential to understanding formerly showed that its most frequent non-silent variant, R229Q, is only the disassembly of viral particles. However, the subunit interactions is pathogenic when trans-associated to specific, dominant negative 3’ mutations poorly documented. A common method used by Ceres and Zlotnick is based giving the first example of clinically important interallelic interactions in hu- on the analysis of self-assembly kinetics. Recently, we proposed another man genetics. We suggested the causal role of an abnormal oligomerization. approach for estimating the interaction strength between subunits. Instead Here we aimed to determine the oligomerization sites of podocin and the ef- of studying the self-assembly process of viral capsid upon a change of pH fect of dominant negative substitutions. We show in a series of FRET spec- or ionic strength, we dissociated cowpea chorotic mottle viruses (CCMV) troscopy experiments that podocin oligomerization occurs exclusively by heating up the solution and we monitored the melting temperature by through the C-terminal tail (residues 283-382): through the first C-terminal fluorescence thermal shift assay. A lattice model was proposed to refine helical region (H1, 283-313), which forms a coiled coil, and through the the interaction between subunits from the measured melting temperatures. 332-348 region. We found amino acid substitutions with a dominant negative To have further understanding on the interaction of subunits, the potential effect on R229Q to cause a significant change in FRET efficiency, suggesting of mean force between the dimers of CCMV was determined by all-atom an altered conformation of pathogenic heterooligomers. Coexpressed podocin molecular dynamics (MD) simulations. Their overall association energy variants with a dimerization capacity strongly influenced each other’s local- was found to be 11 kBT, which is higher than those determined by exper- ization, indicating the primary role of oligomerization in the mediation of iments and may be corresponding to the maximum interaction energy be- interallelic interactions. Interestingly, we found oligomerization to also tween dimers. However, in multi-dimers MD simulations, we did not mediate interallelic complementation: oligomerization with membranous observe the formation of regular subassemblies, which was probably due podocin variants with an intact C-terminal tail rescues the endocytosis of to the high association energy and the dense concentration of viral capsid podocin mutants lacking the region distal the second oligomerization site. subunits. Thus, C-terminal oligomerization of podocin mediates interallelic interac- tions, potentially modifying the pathogenicity of trans-associated NPHS2 310-Pos Board B80 alleles. Nonequilibrium Self-Assembly Dynamics of Icosahedral Viral Capsids Packaging Genome 312-Pos Board B82 Maelenn Chevreuil1, Didier Law-Hine1, Jingzhi Chen1, Self-Association Mechanism of E. coli ClpA Walker B Variants Stephane Bressanelli2, Sophie Combet3, Doru Constantin1,Jeril Degrouard1, Elizabeth C. Duran, Aaron L. Lucius. Johannes Mo¨ller4, Mehdi Zeghal1, Guillaume Tresset1. Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA. 1Laboratoire de Physique des Solides, Universite Paris-Saclay, Orsay, Energy-dependent molecular chaperones are ubiquitous molecular machines France, 2Institute for Integrative Biology of the Cell (I2BC), Universite Paris- involved in protein degradation and remodeling processes essential to cellular Saclay, Gif-sur-Yvette, France, 3Laboratoire Leon Brillouin, CEA, Gif-sur- vitality. E. coli ClpA is an AAAþ (ATPase Associated with diverse cellular Yvette, France, 4European Synchrotron Radiation Facility (ESRF), Grenoble, Activities) chaperone that catalyzes the ATP-dependent unfolding and translo- France. cation of substrate proteins targeted for degradation into a protease, ClpP. The simplest icosahedral viruses can be viewed as nanometer-scaled protein ClpA, like many other AAAþ proteins, assembles into a hexameric ring shells called capsids encasing the genome. The packaging of genome is a competent for binding polypeptide substrate clients in the presence of ATP. crucial step for the survival of viruses, and it has to occur in a fast and accurate Each ClpA protomer contains two nucleotide binding domains, NBD1 and fashion. Surprisingly, little is known about the nonequilibrium dynamics that NBD2. Hydrodynamic studies have established that ClpA resides in a distribu- lead to infectious virions from their building blocks. Cooperative and tion of oligomers in the absence of nucleotide and addition of nucleotide pop- nucleation-growth pathways have been identified in coarse-grained simula- ulates the hexameric state. However, our work shows that a distribution of tions, but even for the simplest viruses, experiments have failed so far to pro- ClpA oligomers persists in the presence of excess nucleotide, suggesting that vide a clear-cut description of these phenomena. macromolecular assembly is thermodynamically linked to nucleotide binding. Here, we probed the self-assembly dynamics of icosahedral viral capsids pack- Lacking a model to quantify the distribution of ClpA oligomeric states, it is aging RNA genome or synthetic polyelectrolyte using time-resolved small- currently not possible to predict the concentration of ClpA hexamers available angle X-ray scattering with synchrotron source. We used the cowpea chlorotic for any given nucleotide and ClpA concentration. In this work, sedimentation mottle virus, a single-stranded RNA plant virus, whose capsid (28 nm in diam- velocity studies are used to quantitatively examine the ClpA self-assembly eter) can self-assemble from and disassemble into dimeric subunits through mechanism in the absence and presence of ATP using variants deficient in long-lived intermediate species. By tuning the subunit-genome interactions ATP hydrolysis at either one or both NBDs. The stoichiometry and affinity through ionic strength, we found out that subunits and genome formed amor- of nucleotide binding to NBD1 and NBD2 are revealed by examining the phous nucleoprotein complexes (NPCs) of 30 nm in size via a cooperative dependence of the apparent association equilibrium constants on nucleotide pathway. These NPCs in turn relaxed into virions via a nucleation-growth concentration. This analysis is the first step in a detailed quantitative under- pathway by strengthening the subunit-subunit interactions through pH. The standing of how the twelve nucleotide binding and hydrolysis sites within binding of subunits on genome could be as fast as 30 ms and the structural the hexametric ring coordinate ATP hydrolysis and coupling to polypeptide relaxation into virions could last several hours. Mean number of bound subunits translocation.

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313-Pos Board B83 the proteins in these complexes. These structural equivalence restraints can Reconstitution of the Liquid Liquid Phase Separation Underlying the supplement other information in integrative structure determination of one Microalgal Rubisco Supercharger or more of these n complexes, such as chemical cross-links and electron mi- Tobias Wunder, Le Hung S. Cheng, Oliver Mueller-Cajar. croscopy density maps. Optionally, integrative structure determination can Nanyang Technological University, Singapore, Singapore. iterate through alternative protein equivalence assignments, in the search for Global transformation of carbon dioxide to biomass is almost entirely depen- solutions that satisfy the structural equivalence restraints as well as other dent on the CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygen- data. The structural equivalence restraints are computed with the aid of ase (Rubisco). Paradoxically this critical catalyst displays a low carboxylation Bayesian inference, based on the detected evolutionary equivalence, the velocity and is prone to confuse CO2 with oxygen. To mitigate these properties sequence alignments, and metrics that quantify our confidence in these align- the chemo- and photosynthetic organisms relying on Rubisco activity have ments.We benchmarked the approach by modeling the structures of 16 sets of evolved elaborate machineries to assist this catalyst. Almost all eukaryotic mi- related protein complexes of known structure. In addition to structural equiv- croalgae utilize CO2- concentrating mechanisms (CCMs) that function as alence restraints, we used either simulated or actually measured chemical carbon dioxide superchargers for Rubisco. The heartpiece of this CCM is a cross-links and electron microscopy density maps. Structural sampling was liquid-like Rubisco-containing membraneless organelle known as the pyrenoid. performed with the Replica Exchange Gibbs method, based on the Metropolis Recently a putative Rubisco linker protein comprised of four highly conserved Monte Carlo algorithm. We demonstrate that structural equivalence restraints 60-residue repeats was identified in the green alga Chlamydomonas reinhardtii. significantly improve the accuracy and precision of the resulting models. The We report a reconstituted system using pure components that recapitulates modeled structures often correctly reproduce evolutionarily non-conserved properties of the pyrenoid in vitro. Addition of the linker protein to Rubisco re- features of the quaternary structure, in addition to generally recapitulating sults in a salt sensitive liquid-liquid phase separation. The two proteins rapidly the conserved features. demix from the bulk solution to form dense droplets that fuse and can be har- vested by centrifugation. The droplet composition is dynamic and both compo- 316-Pos Board B86 nents exchange rapidly with the bulk solution. Highly homologous Rubisco Real-Time Observation of the Assembly Dynamics of an Artificial Rod- enzymes from diverse organisms such as higher plants and cyanobacteria shaped Virus-Like Particle show a great variability in their tendency to demix with the algal linker. Linker Margherita Marchetti1, Douwe Kamsma1, Renko de Vries2, Wouter Roos3, protein variants containing fewer repeats can phase separate with Rubisco, but Gijs Wuite1. 1Vrije Universiteit Amsterdam, Amsterdam, Netherlands, 2Wageningen require higher protein concentrations. Our data permits first conclusions 3 regarding the interaction of Rubisco and the linker protein to be drawn. The University, Wageningen, Netherlands, Rijksuniversiteit Groningen, ability to dissect aspects of pyrenoid biochemistry in vitro will permit us to Groningen, Netherlands. inform and guide synthetic biology ambitions aiming to engineer microalgal Artificial virus-like particles (VLPs) have been demonstrated to be safe gene CCMs into crop plants. and drug delivery systems as well as potential antiviral tools. Nevertheless the design of effective and controlled artificial carriers is still a challenge. 314-Pos Board B84 One of the unsolved key steps in the VLPs development is the full understand- When an Enzyme Self-Assembles on a Membrane: Focal Adhesion Kinase ing of their assembly, as well as the reproduction of the natural viral coopera- Csaba Daday1, Iva´n Acebro´n2, Max Simon1, Ricardo Righetto3, tivity self-assembly behavior. In this study we investigate the assembly 2 € 4 Daniel Lietha , Frauke Grater . dynamics of a biosynthetic coat polypeptide (CS10B) designed to self- 1Molecular Biomechanics, HITS, Heidelberg, Germany, 2Centro Nacional de assemble into a rod-shaped virus-like particle on double stranded DNA. By em- Investigaciones Oncolo´gicas, Madrid, Spain, 3Biozentrum, University of ploying three different single-molecule techniques we can directly observe the Basel, Basel, Switzerland, 4Interdisciplinary Centre for Scientific Computing, simultaneous pathways during peptides-nucleic acid interactions. By virtue of Heidelberg University, Heidelberg, Germany. optical tweezers combined with confocal fluorescence microscopy we present How enzymatic and scaffolding functions can be coupled within the same pro- local and specific information on the binding kinetics of single peptides. tein is an intriguing question. Focal adhesion kinase (FAK) is a membrane- Complementarily acoustic force spectroscopy provides the VLP real-time bound protein found in focal adhesion sites, the kinase domain of which is compaction dynamics. We found that diverse peptides oligomers can directly autoinhibited by the so-called FERM domain. FAK binding to the membrane bind the DNA among which pentamers represent the minimum size required is mediated by a specific interaction between a basic patch of four cationic for the formation of an active nucleus required to trigger the particle growth. residues and PIP2, a highly negative lipid, and leads to specific conforma- The DNA condensation process proceeds in a step-wise fashion (steps tional changes of FAK which along with pulling forces is required for 30nm). The rod-shaped particles mean diameter obtained with atomic force activation. microscopy and the observation of an increase of the DNA flexibility upon pep- How does self-assembly on the PIP2-rich membrane alter the enzymatic func- tides binding, suggest that the regular compaction might be associated with tion of FAK? We examined FAK self-assembly and dynamics in both simu- loops formation. This work opens the doors for new insights into the assembly lations and experiments. We performed large-scale coarse-grained (CG) process of rod-shaped artificial and natural viruses. simulations on 25 copies of FAK on a PIP2-enriched membranes. We find spontaneous FAK oligomerization into chiefly fibril-like structures. Our CG 317-Pos Board B87 simulations, reproducing previously known binding poses and showing partial Role of the Lipid Membrane on the Oligomeric Assembly and Function of kinase-FERM interdomain opening, capture the first steps of FAK clustering. Proteorhodopsin We structurally examined finally assembled FAK using cryo electron Chung-ta Han1, Sunyia Hussain1, Matthew N. Idso1, Sirish Narayanan2, microscopy (EM) of 2D crystals. Clustering involves a strikingly large struc- Tristan Chan2, Songi Han1,2. 1Chemical Engineering, University of California, Santa Barbara, Santa tural rearrangement of FAK, whereupon the kinase domain is fully released 2 and activated and interacts with the membrane and neighboring protein Barbara, CA, USA, Chemistry and Biochemistry, University of California, molecules. Santa Barbara, Santa Barbara, CA, USA. Overall, our combined results propose FAK as a prime example of how self- Transmembrane proteins are biomolecules in the lipid bilayers of cells that organization into large and periodic assemblies impacts enzymatic function. accomplish critical tasks such as sensing, transport, and enzymatic catalysis. Previous studies indicated that transmembrane proteins could tune their func- 315-Pos Board B85 tion by forming oligomeric complexes. However, it remains unclear whether Integrative Structure Determination of Protein Complexes by Inferred oligomerization modulates function significantly more than other factors in Structural Equivalence native-like lipid bilayer environments. In this study, we aim to elucidate how Ignacia Echeverria, Andrej Sali. protein oligomerization and other environmental factors tune the function of Department of Bioengineering and Therapeutic Sciences, University of transmembrane proteins in lipid membrane systems. Proteorhodopsin (PR), a California, San Francisco, San Francisco, CA, USA. model transmembrane protein, was used to examine this functional modulation Protein complexes are functional modules responsible for performing much of because of its assessable light-responsive proton transferring functions. Our re- the work in cells. Different organisms often contain similar protein com- sults showed that oligomerization remained an important factor in tuning PR’s plexes. For example, recent studies have shown that over 70% of protein pairs functions in a liposome environment. Analyses of PR photocycle kinetics sharing at least 30% sequence identity also occur in a similar quaternary struc- measured by a time-resolved optical absorbance technique indicated that faster ture contexts. We propose a method for determining the structure of proteins photocycle kinetics can be observed with the monomeric PR E50Q mutant than complexes based on identifying topologically equivalent proteins across n with the pentameric/hexameric wild type PR in a liposome environment. These complexes from different species and computing distance restraints between results concur with what has been observed for surfactant-solubilized PR.

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Interestingly, the pKa of the PR D97 residue that determines the pH at which results are compared to dynamic light scattering approaches which are limited proton transport switches direction was insensitive to oligomeric distribution to solutions of pure protein. This software is also applicable to experiments in the lipid bilayer environment, but was sensitive to changes in the hydration performed with band-forming centerpieces. (Supported by Boehringer- gradient across lipid bilayers. Therefore, our work suggested that besides Ingelheim and UMC AUC Facility). oligomerization, the lipid hydration gradient could play a role in modulating PR’s function. The new understanding developed here is expected to yield 320-Pos Board B90 guidelines for functional studies of other therapeutically relevant transmem- Interplay between Components of Efflux Pump Machinery brane proteins. Cesar A. Lopez Bautista, Timothy Travers, S. Gnanakaran. Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los 318-Pos Board B88 Alamos, NM, USA. Human Inosine Monophosphate Dehydrogenase 2: Cryo-EM of Highly Antibiotic efflux is one of the most important mechanisms of bacterial multi- Flexible Filaments to Near Atomic Resolution drug resistance. Antibiotics are pumped out of the bacterial cell by the efflux Matthew C. Johnson1, Anika Burrell1, Sajitha Anthony2, Jeffrey Peterson3, pumps that are assembled from outer-membrane, periplasmic, and inner- Justin Kollman1. membrane protein components. The structure-function-mechanism relationship 1Biochemistry Department, University of Washington, Seattle, WA, USA, between the components of this molecular machinery still remains elusive. Us- 2Fox Chase Cancer Center, Philadephia, PA, USA, 3Biochemistry ing a high-throughput pipeline for reconstructing the atomistic models with Department, Fox Chase Cancer Center, Philadephia, PA, USA. exhaustive cryo-EM map fitting followed by all-atom molecular dynamics Inosine monophosphate dehydrogenase 2 (IMPDH2) is a constitutively ex- (MD) simulations, we characterize the interface between the components of pressed enzyme responsible for the rate-limiting step in guanine synthesis the MexAB-OprM efflux pump from Pseudomonas aeruginosa. The interfacial with homologues in all forms of life. We are becoming increasingly aware contacts identified from our approach were corroborated by an independent that many metabolic enzymes, including IMPDH2, form inducible large- analysis based on sequence covariation. Our resulting structural model was scale assemblies as a regulatory mechanism. Human IMPDH2 consists of in good agreement with the recent high-resolution cryo-EM structures. Then, two domains, a catalytic domain and a regulatory Bateman domain. We have we carry out all-atom and coarse-grained MD simulations of this entire efflux previously demonstrated that hIMPDH2 forms micron-scale flexible filaments pump complex in a gram-negative membrane mimic with inner and outer mem- whose assembly is regulated by purine nucleotides binding allosteric sites in the branes and periplasm compartment to deduce the mechanism of drug transloca- Bateman domain, and that these filaments can assume two distinct conforma- tion. These analyses suggest a plausible mechanism by which the drug is tions: ATP plus either of the substrates IMP or NADþ promotes extended fil- pumped out of the outer membrane component via opening of its periplasmic aments with activity comparable to the unassembled enzyme, whereas GTP helices through a concerted interaction with the periplasmic component. In promotes filaments that are compressed and inactive. We present here a 3.6 addition, the developed methodology can be used as a high-throughput tool A˚ cryo-EM structure of hIMPDH2 filaments in the extended, active conforma- for extracting atomistic structures of macromolecular complexes for which tion formed in the presence of ATP and NADþ. Solving this structure required only low-resolution cryo-EM density maps are available. the development of a novel image processing pipeline to identify the coordi- 321-Pos Board B91 nates of D4 symmetry centers along the flexible filaments and to refine to Automatically Building Multi-conformer Ligand Models in Electron Den- high resolution by iterative signal subtraction and focused refinement. This sities with qFit-Ligand structure reveals that polymerization of hIMPDH2 tetramers is mediated by Gydo van Zundert. two interfaces. ATP-driven Bateman domain dimerization forms a ‘‘face-to- Schrodinger, New York, NY, USA. face’’ interface, and a ‘‘back-to-back’’ interface is formed by the N-terminus, X-ray-crystallography is generally the method of choice for resolving the struc- (unresolved, deleted or in a different conformation in homologous crystal struc- ture of macromolecules and investigating the binding pose of ligands. While tures), which binds a pocket in the neighboring catalytic domain, in part due to the electron density represents a time-averaged representation of the underlying specific interactions between residues Y12 and R356. We have also determined conformational ensemble, in the majority of cases the data are interpreted to an intermediate resolution structure of the compressed, inactive filament represent a single conformation at unit occupancy. Temperature factors inade- formed in the presence of ATP and GTP. These data demonstrate that quately account for atom position ambiguity and thermal motion from their hIMPDH2 filaments can adopt multiple conformational states, a feature unique averaged positions, especially in the presence of discrete conformational het- among the enzyme filament structures determined to date. erogeneity. Moreover, the impact of multiple alternative ligand conformations remains underexplored, even though the presence of different binding poses for 319-Pos Board B89 ligands would have important consequences for rational drug design and a AUC Measurements of Diffusion Coefficients of Monoclonal Antibodies in fundamental understanding of the underlying binding mechanisms. Based on the Presence of Human Serum Proteins our earlier work on conformational heterogeneity in proteins, we hypothesize 1 1 2 3 John J. Correia , Robert T. Wright , David Hayes , Peter J. Sherwood , that alternative ligand conformations are currently under-represented in Walter F. Stafford4. 1 2 the PDB. Biochemistry, Univ Miss Medical Center, Jackson, MS, USA, Boehringer- Here, we show that evidence for discrete alternative ligand poses is common in Ingelheim, Ridgefield, CT, USA, 3Interactive Technology, Oakland, CA, 4 the PDB, including for pharmaceutically highly relevant targets. In addition, we USA, Department of Systems Biology, Harvard Medical School, Boston, introduce a fast, automated method for building multi-conformer ligand models MA, USA. in electron densities by hierarchically sampling the ligand’s degrees of The goal of this work is to develop methods for quantitative hydrodynamic and freedom, called qFit-ligand. We rely on powerful, state-of-the-art solvers to thermodynamic analysis of therapeutic proteins in crowded environments like identify a minimal set of conformations to collectively explain the density human serum. These measurements are consistently complicated by weak and for determining the individual occupancies. We benchmarked and opti- mAb-IgG interactions. In an attempt to explore different approaches we mized our approach on a diverse set of deposited multiconformer models have investigated measurements of diffusion coefficients by a traditional syn- curated from the PDB. We prospectively applied qFit-ligand on the D3R and thetic boundary experiment (3 mm SB cells from Spin Analytical by absor- Twilight structural databases, uncovering new alternate, near iso-energetic bance and Aviv-FDS). Here we present a new software technique ligand states. Our user-friendly tool provides a much needed unbiased view incorporated into SEDANAL that can globally analyze the full time course on the ligand’s structural heterogeneity, while paving the way for a deep inves- of synthetic boundary (SB) experiments. This approach also utilizes F-mAb tigation of its impact on rational drug design. against a high concentration of unlabeled carrier protein (like mAb, HSA or IgG). In principle both diffusion and sedimentation coefficient information 322-Pos Board B92 can be extracted including Kij and BijMj estimates. The method can be per- Binding Pathway of Opiates to m-Opioid Receptors Revealed by Machine formed at a traditional low speed (5-7 K rpm) or at high centrifuge speeds Learning (s10). The high speed method can also be used to measure D and s for small Amir Barati Farimani, Evan Feinberg, Vijay Pande. molecules like fluorescein (often contaminants of F-mAb or F-HSA). Dapp(c) Chemistry, Stanford, Stanford, CA, USA. values can be corrected for Kij effects by plotting Dapp*(1þKijcj) vs total car- Significant analgesics relieve pain by binding to the m-Opioid Receptor (mOR), rier concentration. The slope of the fitted data (Do*(1þ2BijMjcj)) allows an and are therefore among the most important proteins in G Protein Coupled Re- alternate approach to determining self- and cross-term thermodynamic noni- ceptor (GPCR) family. Despite studies on the activation pathways of the deality. This method can also compare F-mAb diffusion done in a constant GPCRs, the mechanism of opiate binding and the selectivity of mOR are largely concentration of carrier protein with F-mAb diffusion in a gradient of unknown. We performed extensive molecular dynamics simulation and anal- concentrations, and this explores cross-term diffusion coefficient effects. These ysis to find the (selective) allosteric binding sites of the mOR and also the

BPJ 8559_8561 Sunday, February 18, 2018 63a path opiates take to bind to the orthosteric site. In this study, we discovered the 326-Pos Board B96 allosteric site responsible for the attraction and selection of Opiates. Using Evaluating Protein - Protein Interactions in Chemokine - Inhibitor Com- Markov State Models and machine learning, we traced the pathway of opiates plexes using MD Simulation in binding to the orthosteric site, the main binding pocket. Our results have Lauren E. Stark, Patricia J. LiWang, Michael E. Colvin. important implications in designing novel analgesics. School of Natural Sciences, University of California, Merced, Merced, CA, USA. 323-Pos Board B93 Chemokines are signaling proteins that play many roles, including the recruit- Molecular Self-Assembly of the a-Carboxysome ment of immune system cells to infections. Pathogens have evolved chemo- Luke M. Oltrogge, Thawatchai Chaijarasphong, David F. Savage. kine inhibitors that show promise as anti-inflammatory drugs. The poxvirus Molecular and Cell Biology, University of California, Berkeley, Berkeley, family produces a viral CC chemokine inhibitor (vCCI) which suppresses im- CA, USA. mune response by sequestering CC chemokines and thus prevents signaling to The carboxysome (CB) is a proteinaceous bacterial organelle that plays a cen- immune cells. The herpesvirus family produces a CC chemokine analog, viral tral role in the CO2 concentrating mechanism and the initial step of carbon fix- macrophage inflammatory protein (vMIP-II), to compete with other CC che- ation in cyanobacteria. The CB is a complex assembly of approximately 10,000 mokines at receptors to prevent immune response. In this study, we have used individual proteins that self-organize into a large virus-like capsid containing a computer modeling to study the binding vCCI to three chemokines, human dense enzyme cargo. The mechanisms whereby this assembly process achieves macrophage inflammatory protein-1b (MIP-1b), vMIP-II, and the human highly regular icosahedral particles with well-defined internal cargo composi- CC chemokine Eotaxin to compare the structural basis for their binding. tions are poorly understood. We focus on a large repetitive intrinsically disor- Molecular Dynamics simulations allow for atomistic level detail of potential dered protein called CsoS2 that is a major CB constituent and appears to play a interactions between the chemokines and vCCI. Solvent accessible surface pivotal role in the assembly and cargo encapsulation process. Guided by area analysis, as well as hydrogen bond count and persistence throughout protein-protein interaction studies, the effects of CsoS2 truncations of CB the length of a simulation, provide a means of comparing the strength morphology, and the cysteine disulfide properties of the CB cargo proteins, and types of interactions between vCCI and each of the chemokines. In addi- we propose new models of CB nucleation and assembly and of a possible redox tion, evaluating these interactions is key to determining which are critical to driven error-checking mechanism. binding chemokines. vMIP-II, which has experimentally been shown to bind vCCI more strongly, has more overall hydrogen bonds with vCCI and 324-Pos Board B94 maintains these hydrogen bonds through more of the simulation. We have A Tale of Two CRYS: Identifying the Biochemical Determinants of Thier also studied the effects of vCCI mutations on chemokine binding. Experi- Differential Regulation of Circadian Timekeeping mental studies of vCCI have shown that the Y80A mutation, predicted to in- 1 2 1 Jennifer L. Fribourgh , Alicia K. Michael , Leslee T. Nguyen , crease binding strength to chemokines, instead, disables protein function. 1 Carrie L. Partch . Simulations of vCCI Y80A reveal that the mutation on the microsecond 1Chemistry, University of California Santa Cruz, Santa Cruz, CA, USA, 2 time scale allows for the flexible loop of vCCI to collapse on the beta sheet Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. docking site for chemokines, blocking key interaction sites for chemokine The circadian clock links our behavior and physiology to the daily light-dark binding. cycle, providing a timekeeping system that ensures cellular processes are per- formed at the optimal time of day. Disruption of the circadian clock has adverse 327-Pos Board B97 effects on human health, increasing the risk of metabolic syndromes, cardiovas- A Molecular Dynamics Study on the Self-Assembly of Supramolecular cular disease, and cancer. Circadian rhythms are driven by a set of interlocked Nanotubule transcription-based feedback loops that take 24 hours to complete. The basic YoungBeom Jo, Jeseong Yoon, Seokmin Shin. helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) domain-containing tran- Chemistry, Seoul National University, Seoul, Republic of Korea. scription factor CLOCK:BMAL1 forms the positive arm of the core feedback Self-assembly of peptide-based building blocks has been shown to be useful loop. Of the 40% of the genome that is rhythmically driven each day by in constructing artificial bionanostructures. Recently, a rational approach to the circadian clock, several of these target genes (Per1, Per2, Cry1, Cry2) construct aˆ-barrel protein mimics from the self-assembly of peptide-based constitute the negative components that close the core feedback loop. We building blocks has been demonstrated. In recent experiments, diverse recently identified the biochemical determinants of how CRY1 binds to novel supramolecular nanotubules are prepared by adopting non-covalent CLOCK:BMAL1 to repress transcriptional activation. CRY1 makes distinct, bonding amphiphiles as the building block to mimic these biological struc- multivalent interactions with both CLOCK and BMAL1 that allow it to serve tures. We performed MD simulations on the proposed nanoring structures as a potent repressor even when expressed at near stoichiometric levels with formed by the self-assembly of aˆ-sheet forming peptides. The stability of its target. In this study, we reveal distinct differences in how the structurally the nanoring structure with respect to the size is investigated and several similar repressor, CRY2, interacts with CLOCK:BMAL1. While both CRY1 factors contributing to the stability are examined. We have also demonstrated and CRY2 can sequester the transactivation domain of BMAL1 in the presence that the sizes of nanorings can be controlled by introducing selective or absence of their partner PER2, CRY2 does not form a stable complex mutations. We also performed molecular dynamics simulation on self- with the CLOCK:BMAL1 tandem PAS domains, leading to a more transient assembled supramolecular nanotubule constructed by bent-shaped rod amphi- complex and generally weaker activity as a circadian repressor in vivo. Identi- phile as a building block. By systematically examining from small aggregate fying these differences between CRY1 and CRY2 at the biochemical level is composed of several molecules to fully constructed toroidal shape, we beginning to help explain how these similar proteins play discrete roles in identified optimal structure consistent with experimental data. We illustrated the clock. that cooperative contributions of various interactions play a role as a driving force to construct a nanotubule from small fragmental intermediates. 325-Pos Board B95 Understanding strategies for rational designs of such structures will be All-Atom Molecular Dynamics Simulations of Beta-Solenoid Protein Self- discussed. Assembly Amanda Parker, Daniel Cox. 328-Pos Board B98 Physics, University of California, Davis, Davis, CA, USA. The Knob-Socket Code to Quarternary Interactions Reveals the Specificity Beta-solenoid proteins show promise in bottom-up engineering applications of Pro-Survival BCL-2 Binding due to their nano-scale size, functionalizability, stability in extreme environ- Hyun Joo, Shivarni Patel, Nathaniel Chien, Vivian Kellner, Jerry Tsai. ments, and ability to self-assemble. Understanding the mechanisms of this Chemistry, University of the Pacific, Stockton, CA, USA. self-assembly is crucial in order to design them for certain functions. We pre- Cancer has been a pervasive and deadly problem for many years. A primary sent of a study of the self-assembly of beta-solenoid antifreeze proteins goal in cancer treatment is to effectively destroy cancer cells while also keeping (SBAFP and RiAFP) using molecular dynamics simulations with a structure- healthy cells safe. One current strategy is to interrupt cancer cells ability to based, all-atom potential model. The structure-based model is generated using avoid apoptosis via expression of pro-survival BCL-2. The Knob-Socket model SMOG and the molecular dynamics simulations are done in GROMACS. We allows an exact characterization of the packing surfaces involved in quarternary simulate large systems with atomic resolution in which nano-scale monomers packing based on a repeated tetrahedral motif. More importantly, instead of self-assemble into multi-protein complexes due to attractive interactions at treating residues individually, this model identifies the codependent relation- their interfaces which mimic hydrogen bonding. We report on the mechanisms ship of packing groups. A knob-socket analysis of the quarternary packing and timescales of assembly, as well as the technical aspects of doing this type of structure maps the key protein interactions between a pro-survival Bcl-2 family simulation. protein and its ligand, specifically with the protein Mcl-1. To identify which

BPJ 8559_8561 64a Sunday, February 18, 2018 interactions were important for packing strength and specificity, the interac- template-directed assembly of complex biomolecules and provides insight tions between Mcl-1 and 8 BH3 ligands were mapped and compared with 16 into mechanisms of antibiotic interference to assembly. Approved for unlimited other interactions including different pro-survival proteins from the BCL-2- release, LA-UR-17-29000. like protein family. Comparison of maps revealed which packing interactions were core to the affinity of ligand binding and which provided specificity for 331-Pos Board B101 Mcl-1 recognition. Specifically, the BH3 a-helix ligand contains a central A Cylindrical Assembly Model and Dynamics of the Ebola Virus VP40 glycine that was found in nearly every interaction that can be thought of as ori- Elumalai Pavadai, Bernard S. Gerstman, Prem P. Chapagain. enting the binding interaction. For this reason, this glycine and was defined as Physics, Florida International University, Miami, FL, USA. position 0. All other positions in the BH3 a-helix were labeled in relationship to The Ebola virus, a member of the filovirus family, causes a severe hemorrhagic this glycine. Other residues also make up the core affinity binding residues fever with a high fatality rate in humans. Of the seven proteins that are encoded interact with specific groups of residue on the packing surface of Mcl-1. The by the Ebola virus, VP40 is the primary matrix protein and exists in different conformational and oligomeric states. VP40 plays crucial roles in viral assem- two P-12 and E3 residues are found to determine specificity, and these 2 residues packed into specific surface groups on the surface of Mcl-1. These findings bly and budding at the plasma membrane of the infected cells. This matrix pro- directly inform the design of a a-helices specific to Mcl-1 binding for cancer tein is capable of forming virus-like particles without the need for other Ebola therapeutics. proteins. The budding and formation of new virus-like particles requires VP40 hexamers to arrange side-by-side into a cylinder, as well as end-to-end to 329-Pos Board B99 extend the cylinder to a long filament. However, no experimental three- Identification of PCNA Binding Sites on Chromatin Assembly Factor 1 dimensional structure for any filovirus VP40 cylindrical assembly matrix is Molly Carrig1, Jacquelyn Ho1, Nicholas Kuttner1, Kurt Shaffer1, currently available. Here, we developed cylindrical assembly models of 42 Robyn Scott1, Hunter VanDolah1, Lynne Dieckman2. nm and 92 nm diameters with varying numbers of VP40 hexamers using a 1Creighton University, Omaha, NE, USA, 2Department of Chemistry, protein-protein docking approach. These models match well with the 2D aver- Creighton University, Omaha, NE, USA. ages of cryo-electron tomograms of authentic virions. In addition, our models Proper organization and maintenance of genomic DNA are important for hu- form a multilayered matrix that is in good agreement with the experimental ob- man health. Immediately after replication, newly synthesized DNA is orga- servations. Using a pair of hexamer from the models, we studied the energetics nized into structures called nucleosomes. Nucleosomes make up the basic of the interactions between the hexamers for different side-by-side arrange- structure of chromatin, and their relative compaction influences the level of ments, which indicated the most important residues in the protein-protein bind- expression of genes within a particular region of DNA. This selective gene ing site responsible for the cylindrical matrix assembly. We also investigated expression is necessary for cells to grow and adapt to changes in their environ- the stability and dynamics of the cylindrical hexamer models using implicit ments. As a result, it is important that the level of DNA compaction is preserved all-atom molecular dynamics simulations, which revealed the flexibility of through DNA replication. The process of replication-coupled nucleosome as- the cylinders. Our models will provide helpful information to better understand sembly is mediated by two key proteins: chromatin assembly factor 1 (CAF- the assembly processes of filoviruses and such structural studies could also lead 1), the heterotrimeric protein complex that deposits histone proteins onto newly to the design and development of antiviral drugs. synthesized DNA, and proliferating cell nuclear antigen (PCNA), a homotri- 332-Pos Board B102 meric sliding clamp that recruits and regulates proteins during replication Understanding the Assembly and Disassembly Kinetics of Streptococcus and repair. However, how these two proteins interact and function together pneumoniae FtsZ to couple replication to nucleosome organization is not well understood. In Hemendra Pal Singh Dhaked, Shashikant Ray, Anirban Banerjee, yeast, CAF-1 is composed of three subunits - Cac1, Cac2, and Cac3 - and Dulal Panda. studies have shown that CAF-1 interacts with PCNA via the largest subunit, Biosciences and Bioengineering, Indian Institute of Technology Bombay, Cac1. In particular, PCNA is predicted to interact with Cac1 via a PCNA inter- Mumbai, India. acting peptide (PIP) motif. Here, we used enzyme-linked Immunosorbent as- Here, we have analyzed the assembly dynamics of Streptococcus pneumoniae says and other in vitro protein-protein binding experiments to identify new FtsZ, an essential protein for the division of Streptococcus pneumoniae cells functional motifs within Cac1 that interact with PCNA. We are currently car- using several biophysical techniques including tryptophan fluorescence, light rying out mutational analysis of these motifs to map the specific residues scattering, atomic force microscopy and electron microscopy. The critical involved in the CAF-1-PCNA interaction. concentration for the polymerization of FtsZ was determined to be 2.4 5 0.1 and 2.2 5 0.2 mM by precipitating FtsZ polymers and by monitoring 330-Pos Board B100 the GTPase activity of FtsZ, respectively. Using intrinsic tryptophan fluores- High-throughput Mapping of Cotranscriptional Assembly of the 30S Ribo- cence of FtsZ, GTP was found to bind to FtsZ with a dissociation constant of somal Subunit 653 5 192 nM and the binding of GTP to FtsZ induced conformational Feng Yang, Yuliya A. Kunde, Scott P. Hennelly, Karissa Y. Sanbonmatsu, changes in FtsZ. Further, the assembly and disassembly kinetics of FtsZ Shawn R. Starkenburg, Peter M. Goodwin. were analyzed using the tryptophan fluorescence of the protein. In the pres- Los Alamos National Laboratory, Los Alamos, NM, USA. ence of 0.1 mM GTP, the assembly of FtsZ reached a plateau within 150 s The ribosome, assembled from RNA and protein, is a complex biomolecular and then, the disassembly of the FtsZ filaments occurred slowly in time machine that synthesizes all proteins in an organism. In bacterial cells, the ribo- dependent fashion. A high concentration GTP or GMPPNP, a non- some is also the major target of antibiotics. Extensive studies have provided in- hydrolyzable analogue of GTP, inhibited the disassembly of FtsZ filaments. sights into the assembly process of ribosome under equilibrium conditions However, GMPPNP did not induce bundling of FtsZ filaments. In the pres- using full length, folded ribosomal RNA (rRNA). However, the more realistic ence of divalent calcium, bundles of FtsZ filaments were observed by electron cotranscriptionally guided folding/assembly of the ribosome, as well as the microscopy and atomic force microscopy. Further, divalent calcium decreased mechanisms of antibiotic interference during this process, are still not well un- the GTPase activity of FtsZ and also increased the stability of FtsZ filaments. derstood due to the technical difficulty of simultaneously monitoring transcrip- Interestingly, tryptophan fluorescence assay showed that divalent calcium de- tion, folding/refolding of rRNA and the binding of ribosomal proteins. Here we layed the nucleation process of FtsZ assembly. The data provided a significant are developing a high-throughput, single-molecule method to study the cotran- new insight into the understanding of the assembly of Streptococcus pneumo- scriptional assembly of the bacterial 30S ribosomal subunit using the PacBio niae FtsZ and the mechanism through which divalent calcium enhances the RS II DNA sequencer. Over 10,000 individual cotranscriptional assembly pro- assembly and bundling of FtsZ filaments. cesses can be imaged inside of the zero-mode waveguides (100 nm apertures etched through a 100 nm thick aluminum layer bonded to a glass substrate) of 333-Pos Board B103 the PacBio RS II system. This will be accomplished by immobilizing single Pre-clinical Biophysical Characterization of Therapeutic Antibodies in RNA polymerase molecules in the zero-mode waveguides and monitoring Human Serum by Analytical Ultracentrifugation four different fluorescence signals simultaneously from the incorporation of Robert T. Wright1, Walter F. Stafford2, Peter J. Sherwood3, David Hayes4, labeled uridine nucleotides into the nascent 16S rRNA strand and multiple John J. Correia1. combinations of three (out of twenty in total) different fluorescently-labeled ri- 1Biochemistry, University of Mississippi Medical Center, Jackson, MS, USA, bosomal proteins that will bind to growing 16S rRNA strand to form 30S sub- 2Systems Biology, Harvard Medical School, Boston, MA, USA, 3Interactive unit during the in vitro transcription. The order of binding and binding kinetics Technology, Oakland, CA, USA, 4Boehringer Ingelheim, Ridgefield, CT, of individual ribosomal proteins can be correlated to the rRNA transcriptional USA. states to generate a detailed cotranscriptional assembly landscape of the 30S ri- The preclinical characterization of therapeutic proteins seeks to determine the bosomal subunit. This study contributes to the fundamental understanding of stability, state of aggregation, and interaction of the therapeutic with other

BPJ 8559_8561 Sunday, February 18, 2018 65a macromolecules in serum. Analytical ultracentrifugation (AUC) is one of the dimer and the p50/RelA heterodimer using hydrogen/deuterium exchange experimental methods best suited to determine and understand these factors. mass spectrometry. The results showed surprising differences in the dy- Sedimentation velocity, sedimentation equilibrium, synthetic boundary, and namics of RelA in the homo- vs. heterodimer. We also performed all-atom band sedimentation experiments are performed in order to determine the molecular dynamics (MD) simulations to study the differences in the two. behavior of therapeutic antibodies in highly concentrated environments. The Both our H/D exchange and MD-simulation results provide evidence for Aviv-FDS utilizes a single fluorescently labeled sedimenting species against an interaction between the DNA-binding domains in the homodimer that is a concentrated and heterogeneous background of serum proteins which results absent in heterodimer. We surmised that this interaction would need to be in nonideality. Nonideality results from excluded volume and charge effects. broken for DNA binding to occur and that this might be the cause of the This describes the hydrodynamic and thermodynamic nonideality of a single lower DNA binding affinity for the RelA homodimer as compared to the component as a function of high concentrations of other components in solu- p50/RelA. These results are supported by our stopped flow experiments tion. Thus, the aim of our current work is to study the behavior of a fluores- which showed a slower association rate for DNA binding to RelA homo- cently labeled therapeutic antibody in the presence of high concentrations of dimer. Further, these NFkBs showed distinct structural changes upon binding human serum proteins (HSA and human IgG) and in human serum. To study to the inhibitors. and understand the behavior of therapeutic antibodies in the presence of HSA, human IgG, and other serum components, tracer experiments are done 336-Pos Board B106 pairwise as a function of HSA, IgG and therapeutic protein concentration. Mapping Domain Interaction Networks in Signaling Proteins with Optical This generates a 3x3 matrix of data that describes self- and cross-term hydro- Tweezers dynamic and thermodynamic nonideality (Ks and BM1). The goal is to develop Rodrigo Maillard. a preclinical biophysical method for quantitative hydrodynamic and thermody- Chemistry, Georgetown University, Washington, DC, USA. namic analysis of therapeutic proteins in crowded environments like serum. Signaling proteins are dynamic molecular switches that sample multiple (Supported by Boehringer-Ingelheim.) conformational states. Such conformational plasticity allows these proteins to adapt and respond to different biological signals. Protein Kinase A (PKA) is a signaling protein complex composed of regulatory and catalytic Posters: Protein Dynamics and Allostery I subunits. The regulatory subunit harbors two cAMP binding domains (A and B) that oscillate between inactive and active conformations depending 334-Pos Board B104 on cAMP concentration. In this study, we use single molecule optical twee- Protein Dynamics Underlie Cre-loxp DNA Recombination zers to identify the networks of communication between cAMP binding do- Aparna Unnikrishnan, Carlos Amero, Mark Foster. mains that enable PKA to switch from inactive to active states. We show Chemistry and Biochemistry, The Ohio State University, Columbus, that in the inactive PKA conformation, domain B serves as an energetic OH, USA. hub, controlling the magnitude of interaction between domain A and the cat- Cre recombinase (38.5 kDa, 343 residues), belonging to the tyrosine recombi- alytic subunit, and whose mechanical perturbation triggers the highly cooper- nase family of enzymes, mediates site-specific recombination between specific ative and coordinated dissociation of the PKA complex. In the presence of loxP DNA recognition sequences. Two Cre molecules bind to a 34 bp inverted cAMP, domains A and B establish an intricate network of communication loxP sequence and further assemble with another Cre2-loxP complex to form an that involves direct, interfacial domain contacts as well as long-range interac- antiparallel tetrameric synaptic complex. Crystal structures of the synaptic tions between non-contiguous structural motifs. In contrast, without ligand the tetrad show that the DNA duplexes bend by about 80 facilitating the two domains behave as independent, non-interacting structures. Lastly, we protein-protein interactions between Cre protomers. While single molecule demonstrate the application of optical tweezers to dissect how loss-of- and ensemble kinetics data implicate protein conformational changes (i.e., dy- function mutations can disrupt very distinct communication networks in namics) in the progression of the Cre-loxP recombination pathway, the mo- PKA. The experimental approach based on optical tweezers should be readily tions, timescales, amplitudes, and their structural basis still remain poorly applicable to study disease mutations and the mechanism of action of small understood. We aim to characterize the dynamics of Cre recombinase at atomic molecules in PKA or other signaling proteins. resolution using solution NMR spectroscopy through measurements of back- 15 bone and side chain heteronuclear relaxation. Backbone N T1, T2 and {1H}-15N heteronuclear NOE relaxation measurements on the free catalytic 337-Pos Board B107 domain of Cre identified specific regions that exhibit dynamics on the ps-ns Functional Analysis of Antifreeze Proteins for Cold Tolerance Behavior b b a and X-Ray Single Molecule Observations in C. elegans timescales. Two of the flexible regions, the 2- 3 loop and helix N, form 1,2 1,2 3 part of the inter-protomer interface in the synaptic tetramer, while a third, Masahiro Kuramochi , Chiaki Takanashi , Hiroshi Sekiguchi , Motomichi Doi4, Sakae Tsuda5, Yuji C. Sasaki1,2. the aJ-aK loop, contacts the DNA phosphate backbone. These findings shed 1 2 light on the solution structure of unbound Cre recombinase and reinforce the University of Tokyo, Kashiwa, Japan, AIST-UTokyo Operando-Oil, Kashiwa, Japan, 3SPring-8/JASRI, Hyogo, Japan, 4AIST, Tsukuba, Japan, hypothesis that protein dynamics are significant for mediating DNA recogni- 5 tion and synaptic assembly. Progress towards characterizing dynamics in Cre AIST, Sapporo, Japan. recombinase, over a range of timescales, in the absence and presence of Antifreeze proteins (AFPs) can inhibit ice recrystallization in cells, and work as DNA substrate, will be presented. These studies provide new insights into biological antifreeze materials. However, it is not clear how the molecular dy- the mechanism of Cre-loxP DNA substrate recognition and Cre-mediated namics of AFP in living animal tissues function and work for the cold tolerance site-specific recombination. behavior. Here, we evaluated that the cold tolerance behavior in the transgenic C. elegans expressing AFP at cell-specifically. We found out that AnpAFP 335-Pos Board B105 (derived from a fungi) in body wall muscles works for cold tolerance behavior Role of Conformational Plasticity in Determining the DNA Binding Affin- in C. elegans. Although the muscle cells in C. elegans are not freezing at 0 de- ity of Different NFkB Dimers gree, AnpAFP works for cold tolerance behavior. The previous study also re- Dominic Narang. ported that an ability of hypothermic (þ4 degree) preservation of fish Chemistry and Biochemistry, University of California, San Diego, LA Jolla, antifreeze protein (AFP) against rat insulinoma cells (Kamijima et al., 2013). CA, USA. These results suggest AFPs protect an animal and cells under hypothermia. The Nuclear factor kappa B (NFkB) family proteins play a significant role in One possibility for this AFP function is that the internal motion of AFP protects the regulation of genes associated with immune response, cell growth and the intracellular damage from ice crystal. As supporting this possibility, our development. The activation of NFkB family proteins is tightly regulated previous work found out the internal motion of the extracted AFP (derive by the IkB inhibitor protein like IkBa and IkBb in cells and its misregulation from fish) increased approximately 0.4 rad/sec, when the temperature is can lead to diseases such as cancer. There are five members of the NFkB decreased from 18 to 4 degrees. To monitor this molecular motion in living an- family proteins: RelA (p65), RelB, c-Rel, p50, (NFKB1) and p52 imals, furthermore, we observe the AFP internal motion in C. elegans employ- (NFKB2). These proteins contain a conserved Rel Homology domain ing the Diffracted X-ray Tracking (DXT). DXT can monitor the molecular (RHD) responsible for DNA-binding and dimerization. Different NFkB pro- internal motion at picometre and nanosecond accuracy from records of X-ray tein homo or heterodimers work together to regulate distinct but overlapping diffraction spot from a gold nanocrystal attached to the single molecules. We set of genes. The NFkB dimers bind to 9 to 10 bp of DNA sites (kB sites) are now analyzing the AFPs molecular dynamics regulating cold tolerance be- with different binding affinities. Much of the structural information available haviors using DXT. Our integrated strategy will help to understand how AFP are from NFkBs either bound to DNA or an inhibitor IkB protein. To under- molecular dynamics do contribute to the cold tolerance behavior under the stand the dynamics of free NFkB proteins, we analyzed the free RelA homo- hypothermia.

BPJ 8559_8561 66a Sunday, February 18, 2018

338-Pos Board B108 docking guided by hydrogen/deuterium (H/D) exchange experimental data Simulation-Guided Selection of Spectroscopic Experiments to Refine showed that membrane phospholipids bind to allosteric sites located on the Highly Flexible Protein Structures interfacial surface of PLA2s, shifting their conformation from the ‘‘closed’’ 1 2 2 1,3 Jennifer M. Hays , Marissa Kieber , Linda Columbus , Peter M. Kasson . to the ‘‘open’’ state. This process enables PLA2s to extract and bind a phospho- 1Biomedical Engineering, University of Virginia, Charlottesville, VA, USA, lipid molecule in the active site where the hydrolysis occurs. This is a novel 2Chemistry, University of Virginia, Charlottesville, VA, USA, 3Molecular study addressing recognition between water-soluble enzymes and membranes Physiology and Biological Physics, University of Virginia, Charlottesville, as well as substrate specificity, binding and interactions using computational VA, USA. techniques guided by experimental data. PLA2s are implicated in chronic in- Determining the structure and function of highly flexible proteins is extremely flammatory diseases and understanding their association with membranes as difficult because many measurements that capture multiple conformational well as their interactions with substrates will allow us to identify potent and se- populations provide sparse rather than complete data on the conformational lective inhibitors that can be further developed as anti-inflammatory agents (J. ensemble. Therefore, a critical challenge lies in selecting an optimal set of Med. Chem. 2016, 59, 4403-4414). informative experimental measurements that efficiently refine the ensemble. Hybrid refinement techniques have been developed to combine molecular dy- 341-Pos Board B111 namics (MD) data and experimental data, and while many have leveraged Modeling Protein Conformational Changes with SAXS Profiles experimental data to restrain MD simulations, few have leveraged MD data Dina Schneidman. to guide experiments. In order to address the challenge of selecting optimal The Hebrew University of Jerusalem, Jerusalem, Israel. experiments, we have developed a model-free, information-theoretic approach Proteins generally populate multiple structural states in solution. Transitions for guiding double electron-electron resonance (DEER) experiments that 1) between these states are important for function, such as allosteric signaling uses a mutual information distance metric to rank pairs of residues based and enzyme catalysis. Structures solved by X-ray crystallography provide valu- on how well they refine a conformational ensemble and 2) identifies a set able, but static, atomic resolution structural information. In contrast, Small of highly informative pairs that perform well under this metric. To test this angle X-ray scattering (SAXS) profiles, while limited in resolution, contain in- formation about conformational and compositional states of the system in so- approach, we used simulations of the Opa60 protein to identify a set of maximally-informative and minimally-redundant (mRMR) pairs. We lution. Moreover, SAXS profiles can be rapidly collected for a variety of measured the distance distributions of those pairs using DEER and incorpo- experimental conditions, such as ligand-bound and unbound protein samples, rated the experimental distributions into restrained-ensemble MD simulations. different temperatures, or pH values. The challenge lies in data interpretation We demonstrate that the set of high-scoring mRMR pairs better reduce the since the profiles provide rotationally, conformationally, and compositionally conformational search space than a set of spectroscopist-selected pairs. This averaged information about protein shape in solution. We have developed a systematic approach provides a way to select optimal spectroscopic experi- novel computational method, MultiFoXS that simultaneously uncovers the ments, to use the data to efficiently refine structures of flexible receptor- set of structural states and their population weights for multiple input SAXS ligand complexes, and to elucidate fundamental physical principles of profiles. The input is a single atomic structure, a list of flexible residues, and receptor-ligand binding. one or more SAXS profile(s) for the protein. The method proceeds in two steps. In the first step, it samples the input structure by exploring the space of the 4 c 339-Pos Board B109 and main chain dihedral angles of the user-defined flexible residues with a Probing collective Motions of Proteins and Hydration Dynamics in Rapidly exploring Random Trees (RRTs) algorithm. In the second step a Aqueous Solutions by a Wide Range Dielectric Spectroscopy SAXS profile is calculated for each sampled conformation with FoXS, followed Ali Charkhesht, Djamila Lou, Nguyen Q. Vinh. by a branch-and-bound enumeration of the multi-state models that are consis- Department of Physics and Center for Soft Matter and Biological Physics, tent with the SAXS profile. The method was benchmarked on over 30 cases Virginia Tech, Blacksburg, VA, USA. with experimental SAXS profiles, including large multi-domain proteins and Conformational dynamics of proteins affect how biological molecules exert proteins with long disordered fragments. Moreover, comparison of conforma- their function and regulate their biological processes. Studying the dynamics tions and their weights between the ligand-bound and unbound SAXS profiles of proteins and their hydration allows us to analyze their functions in aqueous can help in determining the allosteric mechanism. The applicability of the solutions. However, the strong absorption of water makes tracking collective method extends beyond SAXS and it has been applied to datasets from Small mode changes challenging. Thus, we have developed a very sensitive dielec- Angle Neutron Scattering, Electron Microscopy, and residual dipolar tric megahertz-to-terahertz frequency-domain spectroscopy system to probe couplings. the hydration shells as well as large-scale dynamics of these biomolecules. 342-Pos Board B112 Using this system, we explore the real and imaginary dielectric response of Defining a Ligand-Binding Pocket in the Orphan Nuclear Receptor solvated biomolecules over a range of 50 MHz to 2 THz. We have chosen Nurr1 several proteins to provide a model system including bovine serum albumin Paola Munoz-Tello, Sarah Mosure, Patrick Griffin, (BSA), lysozyme, cytochome_c, and myoglobin proteins. We have compared Venkatasubramanian Dharmarajan, Ian de Vera, Douglas Kojetin. the dynamics of proteins as well as their hydration water. We have deduced The Scripps Research Institute, Jupiter, FL, USA. the dynamics and the hydration water of these proteins by comparing their Nuclear receptor related 1 protein (Nurr1/NR4A2), an orphan nuclear receptor real and imaginary dielectric response and hydration water relaxation times. with no known physiological ligands, is considered to function independent of Comparing these assets of various proteins in different classes helps us ligand. Compared to other non-orphan nuclear receptors with solvent accessible shed light on the macromolecular dynamics in a biologically relevant water apo-protein ligand-binding pockets, a crystal structure of the Nurr1 ligand- environment. binding domain (LBD) revealed with no physical space to bind ligand. Using solution structural methods, including NMR spectroscopy and hydrogen/deute- 340-Pos Board B110 rium exchange mass spectrometry, and atomic-level molecular simulations, we Allosteric Regulation by Membranes Controls Specificity of Lipolytic En- found that the putative ligand-binding pocket in Nurr1 is dynamic and can zymes through Recruitment of Unique Hydrophobic Binding Pockets expand to bind a natural ligand. Mutation of putative ligand-binding pocket res- Varnavas D. Mouchlis, J. Andrew McCammon, Edward A. Dennis. idues shows reduced Nurr1 cellular activity, indicating the integrity of the Pharmacology, and Chemistry and Biochemistry, University of California, pocket is important for function. Our data indicates the crystalized pocket San Diego, La Jolla, San Diego, CA, USA. conformation likely represent a collapsed conformation, but in solution the Connecting molecular structure with cellular function is fundamental to under- pocket is dynamic and can expand to bind ligand. standing the mechanism of action of water-soluble enzymes acting on cell membranes (PNAS 2015, 112, E516-E525). Cytosolic (cPLA2), calcium- 343-Pos Board B113 independent (iPLA2) and lipoprotein-associated (Lp-PLA2) provide an ideal Feedforward and Feedback Amplification in the Pink1-Parkin Pathway system for studying protein-lipid molecular recognition and membrane func- Generate a Binary Switch for Mitochondrial Quality Control in Parkinson tioning. cPLA2 is the main arachidonic acid provider for the eicosanoid Disease pathway, iPLA2 is involved in membrane phospholipid remodeling and Lp- Kalle Gehring. PLA2 was found to associate with LDL and HDL in human plasma to hydrolyze Biochemistry, McGill University, Montreal, QC, Canada. phospholipids containing short-chain and oxidized fatty acids. A lipidomics The Parkinson disease associated proteins, Parkin and PINK1, together mass spectrometric assay allowed us to define unique specificity of PLA2s to- comprise a mitochondrial quality control system that promotes neuronal sur- wards a wide variety of phospholipids that was not feasible with existing as- vival through autophagy of damaged mitochondria. In the pathway, PINK1 says. Computational techniques such as molecular dynamics simulations and acts as a sensor of depolarized mitochondria and phosphorylates ubiquitin to

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recruit and activate Parkin on the mitochondrial outer membrane. Parkin ubiq- tropy (TDSconf), however, has remained poorly understood. Recent develop- uitinates mitochondrial proteins, which leads to the removal of the damaged ments in nuclear magnetic resonance spectroscopy (NMR) have provided an mitochondria in a process known as mitophagy. We have carried out structural experimental way to quantify the contribution from TDSconf to binding through studies of Parkin to understand its regulation and mechanism of activation. Par- dynamics measurements of fast (ps-ns) motions in proteins. We apply this kin exhibits low basal activity as a ubiquitin ligase and requires activation by approach to barnase-barstar, one of the strongest protein-protein interactions PINK1 both in vitro and in cells. Two features inhibit activity: 1) the E2- known, to understand the role of TDSconf in achieving femtomolar (fM, DG binding site on RING1 is blocked, and 2) the catalytic site is partially occluded. 20 kcal/mol) affinity. Our results indicate that both proteins become globally The structures of activated states of Parkin show that phosphoubiquitin binding more rigid upon binding, resulting in an unfavorable TDSconf worth 5.4 5 1.3 and Parkin phosphorylation act as feedforward and positive feedback controls kcal/mol. The total binding entropy, TDStotal, of this complex is zero, suggest- to induce mitophagy in a binary all-or-nothing response. These structural ing a balancing contribution from TDSsolv. Interestingly, the crystal structure of studies have revealed how the Parkin/PINK1 switch functions in cells and pro- the complex shows an interface hydrated by over a dozen fully buried water vide a framework for the design of small molecules for treating Parkinson molecules. Rigidifying these waters represents a large entropic penalty. To disease. evaluate the presence of rigid waters in solution, we used nuclear Overhauser (NOE) NMR methods to detect magnetization transfer between amide protons 344-Pos Board B114 and water. The NOEs detected are consistent with the crystallographic waters Regulation of the Promoter of RNA Silencing observed, with an average NOE/ROE ratio of -0.26. Remarkably, similar NOEs 1 2 2 2 Suzanne Scarlata , Shriya Sahu , Leo Williams , Alberto Perez , were observed for the free state of barnase, suggesting that the interfacial wa- Finly Philip2, Giuseppe Caso2, Walter Zurawsky1. 1 2 ters are pre-rigidified. Order parameters of side chains directly hydrogen Worcester Polytechnic Institute, Worceseter, MA, USA, Stony Brook bonded to the rigid waters were consistent with a pre-organized interface. Sup- University, Stony Brook, NY, USA. port from NIH, NIH F32, The Mathers Foundation and NSF. RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (compo- 347-Pos Board B117 nent 3 of RISC). We have previously found that phospholipase Cb, which in- Evaluating How Binding Interactions for PARs Change as Prothrombin is creases intracellular calcium levels in response to specific G protein signals, Converted to Thrombin inhibits C3PO activity towards certain genes. Understanding the parameters Ramya Billur, Thomas Michael Sabo, Muriel C. Maurer. that control C3PO activity and which genes are impacted by G protein activa- Chemistry, University of Louisville, Louisville, KY, USA. tion would help predict which genes are more vulnerable to down-regulation. 18 Thrombin is generated during the final stages of blood coagulation from its Here, using a library of 10 oligonucleotides, we show that C3PO binds oligo- inactive precursor prothrombin (ProT). The resultant serine protease plays nucleotides with structural specificity but little sequence specificity. Alter- important roles in procoagulation, anticoagulation, and platelet activation. nately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to Thrombin’s specificity is regulated by its active site, surface loops, and regu- substrate stability. Importantly, we find that oligonucleotides with higher Tm latory Anion Binding Exosites I and II (ABE I and ABE II). Binding of phys- values are inhibited by bound PLCb. This finding is supported by microarray iological ligands to ABE I can allosterically control thrombin conformation analysis in cells over-expressing PLCb1. Taken together, this study allows pre- and activity. Protease Activated Receptors (PARs) function as tethered ligands dictions of the genes whose post-transcriptional regulation is responsive to the that elicit transmembrane signaling for platelet activation and aggregation. G protein / phospholipase Cb / calcium signaling pathway. Both PAR1 (49-62) and PAR3 (44-56) have been hypothesized to bind 56 59 345-Pos Board B115 thrombin ABE I with the help of a hirudin-like motif ( FEEI ). Unexpect- IDP Segment Conservation and Divergence in I-Domains of the Phage edly, some ligands can already bind to pro-ABE I of ProT. Ongoing NMR Lambda Supercluster studies are comparing the binding environments encountered as PAR1 (49- Andrei T. Alexandrescu1, Therese N. Tripler1, Anne R. Kaplan1, 62) interacts with immature pro-ABE I and mature ABE I relative to that of Kristin N. Parent2, Carolyn M. Teschke3. PAR3 (44-56). 1D proton line broadening confirmed that PAR1 and weaker 1Molecular & Cell Biology, University of Connecticut, Storrs, CT, USA, binding version PAR1G already exhibited line broadening with ProT and 2Biochemistry and Molecular Biology, Michigan State University, East such broadening increased when bound to thrombin. 2D tr-NOESY revealed Lansing, MI, USA, 3Molecular & Cell Biology and Chemistry, University of PAR1 (49-62) bound to pro-ABE I in an extended conformation with the pro- line adopting a trans conformation. 1H-15N HSQC titrations provide quantita- Connecticut, Storrs, CT, USA. 15 Viruses enclose their genomes in proteinacious shells called capsids, that are tive estimates of binding interactions for selectively N-labeled PAR assembled from multiple copies of one or more types of coat proteins. Phage aminoacids interacting with ProT vs Thrombin. Similar to PAR3, PAR1 pep- P22 has a T=7 icosahedral capsid comprised of 420 copies of a coat protein tides revealed the hydrophobic pocket (F34, L65, and I82) of pro-ABE I is based on the HK97-fold, which additionally contains a genetically inserted partially available for peptide binding. When bound to thrombin, the acidic domain (I-domain). The NMR structure of the P22 I-domain has a 6-stranded C-terminal tail of PAR1 (49-62) remains unresolved by crystallography. NMR titrations are making it possible, for the first time, to monitor binding b-barrel fold, flanked by two long disordered loops called the S and D-loops. 58 62 The loops maintain their disorder in the intact coat protein but become struc- of PAR1 ( DEEKN ) to pro-ABE I and ABE I. Knowledge gained from tured in the assembled capsid. The S-loop modulates the size of the capsid, this project may help develop drug candidates that target or avoid specific whereas the D-loop forms interactions between coat protein monomers that sta- hots spots on (pro)-ABE I. bilize the icosahedral capsid structure. For viruses built from multiple copies of a single coat protein, and icosahedral symmetry greater than T=1, the mono- 348-Pos Board B118 mers have to adopt pseudo-symmetric conformations dependent on their posi- A Single Mutation on a Surface Loop Alters the Key Dynamics of the tion in the capsid. Thus the intrinsic flexibility of the D-loop is likely to provide Distant Active Site: High Pressure and Relaxation Dispersion NMR an avenue for the conformational heterogeneity that allows coat proteins to Studies of E. coli Dihydrofolate Reductase adopt context-dependent conformations in the capsid, as well facilitating the Kazuyuki Akasaka. expansion form the procapsid to the dsDNA-packaged virus during phage Kyoto Prefectural University of Medicine, Otsu, Japan. maturation. We are currently extending NMR structural studies to the distantly If, at all, a single mutation on an exposed loop affects significantly the confor- related phages CUS-3 and sf6, to examine the extent to which the structure and mation and dynamics of a protein crucial to its function is a key question, in disorder of the I-domain is conserved in these representatives of phage super- relation to the neutral theory of molecular evolution. Here we examine how families. While the 6-stranded beta-barrel fold is conserved between P22, CUS- widely the effect of a mutation Gly67Val in the exposed CD loop of E-coli 3 and sf6 I-domains the dynamic loops show considerable divergence that may DHFR could be on the structure and dynamics of the whole protein, particularly account for the different surface morphologies of the capsids in cryo-EM data. at the distant active site, by applying high-pressure and relaxation-dispersion NMR spectroscopy on 15N-uniformly labeled wild-type and Gly67Val DHFR 346-Pos Board B116 in its binary complex with folate and in its ternary complex with NADPH Role of Conformational Entropy in Extremely High Affinity Protein Inter- and folate, respectively. We find that the surface mutation affects the average actions structure of the protein only locally around the mutation site, while it causes Jose A. Caro, Kathleen G. Valentine, A. Joshua Wand. significant changes in the conformational dynamics of the entire protein mole- University of Pennsylvania, Philadelphia, PA, USA. cule, particularly at the distant catalytic site. This work shows unequivocally Molecular recognition by proteins is fundamental to biology. Specific interac- the case that even a single mutation on a well-exposed surface loop can affect tions at the interface (DHbinding) and the release of solvating water (TDSsolv) are significantly the dynamics of the entire protein molecule and its catalytic acti- often assumed to govern binding energetics. The role of conformational en- vity.

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349-Pos Board B119 muscle myosin light chain kinase, were studied by inserting an artificial SCN Structural Insight into the Metal-Ion Mediated Modulation of the Cata- probe group at different sites along both the CaM and M13 chains. Compari- lytic Function of H. pylori Arginase sons between CN stretching frequencies of each mutant in the different binding Ankita Dutta1, Mohit Mazumder2, Mashkoor Alam1, Samudrala Gourinath2, states of the CaM/M13 complex showed correspondence between IR frequency Apurba Kumar Sau1. shift and changes in the solvatochromic environment upon binding. To gain a 1Immunoendocrinology, National Institute of Immunology, New Delhi, more quantitative understanding of the vibrational solvatochromism of the India, 2School of Life Sciences, Jawaharlal Nehru University, New Delhi, SCN probe in calmodulin ensembles, all-atom molecular dynamics (MD) sim- India. ulations in either the AMBER99sb or CHARMM36 force field were performed H.pylori arginase (RocF), a well-characterized virulence factor for acid- for conformational sampling. Infrared (IR) frequencies were calculated from protection and pathogenesis in the human stomach is a bimetallic enzyme the resulting trajectories, and transformed into IR lineshapes following the fluc- 2þ 2þ whose activity is dependent on either Co or Mn . The recombinant protein tuating frequency formalism. Two methods were attempted for IR frequency þ þ has 4-fold higher activity with Co2 than Mn2 . Also, bacterial extracts show calculations: a semi-empirical PM3-QM/MM method adapted from Layfield þ þ optimal activity with Co2 compared to Mn2 . These observations suggest that and Hammes-Schiffer and an ab-initio solvatochromic effective fragment po- þ Co2 is the preferred metal ion for higher catalytic activity and the metal- tential (SolEFP) method adapted from Blasiak and Cho. The SolEFP method induced active-site architecture of these two proteins could be different. To appeared advantageous, as the PM3 parameters for the QM/MM were not test this hypothesis, we performed a detailed investigation that include inhibi- strictly transferrable between heterogeneous protein environments. The calcu- tion kinetics, pH-dependent studies, steady-state and time-resolved tryptophan lated lineshapes were compared to experiments and across different mutant fluorescence studies, anisotropy-decay kinetics and MD simulations. In the sites in the different binding states of CaM. The EFP breakdown of the different þ absence of the crystal structure of Co2 -protein, we generated model structures contributions to the vibrational frequency shifts yields further information on of these two holoproteins and performed extensive MD simulations at micro- the structural origins of the lineshape variation. Since the microenvironment second time-scale which provided a structural insight into the difference of around the probe is sensitive to global conformational changes in relatively conformational changes induced by the metal-ions in the active-site of the pro- disordered proteins, the overlap between computed lineshapes for a certain tein. These observations along with the time-resolved fluorescence studies us- simulated sub-population and experimental lineshapes across the different ing intrinsic tryptophan probe explain the basis for higher catalytic activity in mutant sites can offer a likelihood estimate for that subpopulation to occur, þ the Co2 -protein. Both holoproteins exhibit different kinetic mechanisms of in- thus facilitating structural modeling of relatively flexible proteins. hibition with same inhibitors suggesting that metal induced active-site confor- mations of these proteins were different. Further, MD simulation studies with 352-Pos Board B122 inhibitors revealed differences in their binding. These observations are well Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism correlated with our experimental data. Additionally, the active-site architecture of the Robustness in the Cyanobacterial Circadian Oscillator of these two holoproteins was found to be different compared with that of the Shogo Sugiyama1, Tetsya Mori2, Mark Byrne3, Takayuki Uchihashi4, human homologue which was validated by the inhibition studies, thereby high- Carl H. Johnson2, Toshio Ando1,5. 1Department of Physics, Kanazawa University, Kanazawa, Japan, lighting a possibility for the development of a new class of inhibitors which is 2 specific to this pathogen. Department of Biological Science, Vanderbilt University, Nashville, TN, USA, 3Department of Chemistry, Physics, and Engineering, Spring Hill 350-Pos Board B120 College, Mobile, AL, USA, 4Department of Physics, Nagoya University, Dynamic of APOBEC3G in Complex with ssDNA Revealed by High-Speed Nagoya, Japan, 5Bio-AFM Frontier Research Center, Kanazawa University, AFM Kanazawa, Japan. Yangang Pan1, Zhiqiang Sun2, Luda S. Shlyakhtenko2, Reuben S. Harris3, The circadian rhythm in cyanobacteria is essentially generated by an oscillator Yuri L. Lyubchenko2. composed of three Kai proteins (KaiA, KaiB and KaiC). The Kai system is very 1University of Nebraska Medical Center, Omaha, NE, USA, 2University of unique because the self-sustainable oscillation of KaiC phosphorylation can be Nebraska Medical Center, Omaha, NE, USA, 3University of Minnesota, reconstructed in vitro only by incubating KaiC with KaiA, KaiB and ATP. Minneapolis, MN, USA. KaiC is the main component of Kai system and forms a homohexameric double APOBEC3G (A3G) is a cytosine deaminase that belongs to APOBEC3 family of ring comprised of the CI (N-terminal) and CII (C-terminal) rings. The circadian proteins. In the absence of virus infectivity factor (Vif), APOBEC3G is able to rhythm is generated by the oscillation of KaiC phosphorylation state with a restrict HIV-1 infection by deamination of viral ssDNA or creating a roadblock 24-hours period. It is known that KaiA stimulates KaiC phosphorylation, during cDNA synthesis. A3G has two domains: its N-terminal domain (NTD) is whereas KaiB promotes KaiC auto-dephosphorylation by sequestering KaiA. responsible for interacting with nucleic acids and Vif, and its C-terminal cata- Therefore, to reveal how the Kai system expresses the robust circadian cycle, lytic domain (CTD) carries out the deamination activity. Up to date, there is it is essential to understand the interaction between Kai proteins. However, no high-resolution structure of full-length A3G, yet, recently, computational the molecular and kinetic details of this interaction are still elusive. Here, we models for the full-size monomeric A3G were developed and their dynamics apply high-speed atomic microscopy (HS-AFM) to observe dynamic interac- were assessed. To validate the computational models, we used time-lapse tions between Kai proteins. The HS-AFM images clearly show that KaiA binds high-speed AFM (HS-AFM), which confirmed the dynamic transition between to the CII ring of KaiC, while KaiB binds to the CI ring of KaiC. Also, we found two A3G structures: globular and dumbbell. Here, using HS-AFM, we presented that the affinity of KaiA for KaiC strongly depends on the phosphorylation state data on the dynamic behavior of A3G in the complex with ssDNA. Similar to free of KaiC. KaiA repeatedly binds to and dissociate from hyper-phosphorylated A3G, in the complex we observed both, globular and dumbbell forms of A3G, KaiC with a bound state lifetime of 0.4 s, whereas in the case of hypo- however, we found the higher yield of dumbbell structures for A3G in complexes phosphorylated KaiC, the lifetime of KaiAKaiC bound state is 1.3 s. with ssDNA than for A3G alone. These data show the effect of ssDNA on the From a Monte Carlo simulation, we confirm a role of the phosphoform- dynamic behavior of A3G. Moreover, we found that the population of dumbbell dependent differential affinity (PDDA) of KaiA for KaiC in the circadian structures increases with the length of ssDNA, which correlates with the depen- rhythm. The simulation results suggest that PDDA broadens the concentration dence of the deaminase activity of A3G on the length of ssDNA template range of Kai proteins that enables stable rhythmicity and can explain how the observed in (1). In addition to a higher yield of the dumbbell conformations of oscillation is resilient to intrinsic and extrinsic noises. A3G in the complex, we observed a transient dissociation of one of the A3G domain from the ssDNA template. Overall, these findings demonstrate not 353-Pos Board B123 only a very dynamic structure of A3G in the complex with ssDNA, but show Using a Fluorescent Unnatural Amino Acid to Characterize the Contribu- an interplay between A3G domains, which may shed new light on the functional tion of Pre and Post-chemistry Enzyme Structural Dynamics to High Fidel- activities of A3G in the complex with ssDNA. ity DNA Replication Tyler Dangerfield. 351-Pos Board B121 University of Texas at Austin, Austin, TX, USA. Relating the Vibrational Lineshapes of the Thiocyanate (SCN) Probe to The DNA Polymerase of Bacteriophage T7 is one of the most extensively char- Structures and Dynamics in Calmodulin Ensembles via Computational acterized model enzymes for understanding high fidelity DNA replication. Approaches Crystal structures of the open, binary (E-DNA) complex and the closed, nucle- Rosalind J. Xu, Shannon R. Dalton, Kristen L. Kelly, Casey H. Londergan. otide bound ternary (E-DNA-dNTP) complex reveal large movements of the Chemistry, Haverford College, Haverford, PA, USA. fingers domain as nucleotide binds and the fingers ‘‘clamp down’’ on the cor- The structure and dynamics of calmodulin (CaM), with and without Ca2þ and rect base pair. However, the contribution of this structural transition to DNA a 26-residue synthetic peptide (M13) comprising the binding domain of skeletal replication fidelity has been greatly disputed and is further complicated by

BPJ 8559_8561 Sunday, February 18, 2018 69a the difficulty in site specific labeling of the T7 DNA polymerase with a fluoro- targeting sequence from the end of ribosome exit tunnel. Such discrimination phore using traditional cysteine – maleimide coupling chemistry. The work pre- is comparable to the difference between substrate and non-substrate. Using sented here aims to determine the contribution of the motions of the fingers e. coli as a model system, our results show that SRP targets its substrate exclu- domain of the T7 DNA Polymerase to its high DNA replication fidelity, sively after the emergence of a transmembrane domain or signal sequence from through the use of a fluorescent unnatural amino acid that is sensitive to its local ribosome exit tunnel. chemical environment and incorporated site-specifically in the fingers domain of the enzyme. The rates of the individual steps in the pathway were measured 357-Pos Board B127 using transient kinetic techniques, including stopped flow and quench flow Protein:Protein Interactions Control Sensitivity of a Transcription methods, and data from all experiments was globally fit in Kintek Explorer Response to Input Signal 1 1 1 2 to derive rate constants for the individual steps in the pathway. Our data sup- Dorothy Beckett , Chenlu He , Jingheng Wang , Gregory Custer , 2 ports the previously proposed model where the conformational change preced- Silvina Matysiak . 1Chemistry & Biochemistry, University of Maryland, College Park, MD, ing chemistry is not rate limiting but still contributes to fidelity, and provides 2 new information on post-chemistry conformational dynamics of the enzyme USA, Fischell Department of Bioengineering, University of Maryland, and their contribution towards DNA replication fidelity. College Park, MD, USA. Transcription regulation frequently occurs through multi-step assembly pro- 354-Pos Board B124 cesses that can include small ligand binding, protein-protein and protein- Probing Substrate Sequestration in Carrier Proteins using Vibrational nucleic acid interactions. However, for the majority of these assembly Spectroscopy Labels and Molecular Dynamic Simulations processes the relationship between these coupled equilibria and the sensitivity Caroline A. McKeon, Louise K. Charkoudian, Casey H. Londergan. of the transcription response to the regulatory input signal is not known. The Haverford College, Haverford, PA, USA. E. coli biotin operon repression complex assembly, which responds to input Carrier proteins are considered ‘‘lynchpin’’ enzymes of biosynthetic pathways. biotin concentration, occurs via three coupled equilibria including corepressor The E. coli acyl carrier protein (ACP) has multiple alpha helices that form a binding, holorepressor dimerization and dimer binding to DNA. A genetic hydrophobic, solvent-protected pocket and can provide a hiding spot to protect screen has yielded superrepressor mutants that repress biotin operon transcrip- growing substrates bound to the ACP’s phosphopantetheine (Ppant) arm. The tion in vivo at biotin concentrations much lower than that required by the wild action of ‘‘chain sequestration’’ is thought to be important for driving the type repressor. In this work, isothermal titration calorimetry and sedimentation acyl chain biosynthetic process. Site specific vibrational spectroscopy tech- equilibrium measurements were used to determine the superrepressor biotin niques are being used to investigate Ppant arm dynamics via nitrile probes binding and homodimerization properties. The results indicate that, although on unnatural amino acids incorporated into the sequestration cavity. Molecular all variants exhibit biotin binding affinities similar to that measured for BirAwt, dynamics simulations in aim to characterize the Ppant arm’s shifting conforma- five of the six superrepressors show altered homodimerization energetics. Mo- tional distribution and substrate-loading selectivity by analyzing geometric lecular dynamics simulations predict complex structural origins of the altered quantities and the solvent exposed surface area (SASA) of specific pieces of dimerization. Modeling of the multi-step repression complex assembly process the arm. Ultimately, incorporating a nitrile probe group into the simulation for these proteins reveals that the altered sensitivity of the transcription will provide a direct connection to experimental data through the CN infrared response to biotin concentration is readily explained solely by the altered super- spectroscopic lineshape. A thorough understanding of Ppant arm dynamics will repressor homodimerization energetics. These results highlight how a tran- inform further characterization of experimental data from carrier proteins and scription regulatory response to input signal can be fine-tuned via linked efforts to manipulate ACP’s interactions with partner enzymes. equilibria. 355-Pos Board B125 358-Pos Board B128 Development and Characterization of Two Phenylalanine Derivatives for Conformation-Independent Dynamics of O2 Rebinding to Myoglobin use as Raman Probes in Proteins Seongchul Park, Jaeheung Park, Joohyang Shin, Manho Lim. Eliana V. von Krusenstiern, Joie Ling, Casey H. Londergan. Chemistry, Pusan National University, Busan, Republic of Korea. Chemistry, Haverford College, Haverford, PA, USA. Time-resolved vibrational spectroscopy was used to probe rebinding dynamics Two promising Raman probe functional groups - terminal alkynes and aromatic of O2 to myoglobin (Mb) in the time range from femtosecond to microsecond nitro groups - were chosen to study the possibility of their ability to report on after excitation of oxymyoglobin (MbO2)inD2O with a 575-nm pulse at 293K. their molecular surroundings. These probes each have strong Raman signals The OO stretch mode in MbO2 is known to exhibit two conformational bands 16 and the potential to report on different physical aspects of their microscopic but one conformation band in Mb O2 is split due to a perturbing vibration, re- 16 1 surroundings. To insert the probes into proteins, phenylalanine derivatives sulting in three absorption bands for Mb O2 (1149, 1125, and 1104 cm ) and 18 1 were synthesized with the probes added at the para-position. Extensive Raman two absorption bands for Mb O2 (1084 and 1067 cm ). Therefore, after the solvatochromic experiments on model compounds containing the aromatic Q-band excitation the difference transient spectra exhibit three photo-bleaching 16 18 alkyne and nitro probes were used to determine the sensitivity of key vibra- bands for Mb O2 and two transient absorptions for Mb O2. All the transient tional mode frequencies to their environment. The two p-substituted phenylal- bleach signals decay with the same kinetics and were described by two expo- anine derivatives were incorporated into interior positions of the e. coli fatty nential functions plus an equation describing bimolecular reaction. The recov- acid pathway’s acyl carrier protein via unnatural amino acid incorporation tech- ered time constants for the exponential function were 3 ps (75%) and 36 ns niques to better understand sequestration of substrates inside the pocket of the (14%) and the bimolecular rate constant was 1.6 107 M1s1. The 3-ps protein. decay were attributed to thermal relaxation of six-coordinate heme subsequent to fast electronic relaxation without photodeligation as well as fast geminate 356-Pos Board B126 recombination (GR) of O2 after photodeligation. The 36-ns decay were attrib- Co-translational Targeting by Signal Recognition Particle Activates Only uted to GR of photodeligated O2. When photoexcited in the Q-band, about 46% after Cytosolic Exposure of Signal Sequence of MbO2 undergoes photodeligation, about 76% of the deligated O2 geminately Hao Hsuan Hsieh1, Shu-ou Shan2. rebinds, and the remaining O2 escapes into solvent and bimolecularly rebinds. 1Chemistry, Caltech, Pasadena, CA, USA, 2Chemistry, Caltech, Pasadena, Fast and efficient GR of O2 indicates that the heme pocket is efficient in trap- CA, USA. ping the deligated O2. Conformation-independent dynamics of photoexcited Signal recognition particle (SRP) and its receptor (SR) co-translationally target MbO2 suggests that the O2 binding characteristics is independent of conforma- membrane and secretory proteins to membrane specifically and efficiently tional substates of MbO2 or transition between conformational substates is through multiple layers of regulation. Differential SRP binding to ribosome, faster than 36 ns. SRP-SR association rate, and GTP hydrolysis ensure specificity for substrate over non-substrate. However, there is a debate on whether SRP interacts 359-Pos Board B129 with its substrate when a target sequence is still buried inside ribosome exit tun- Mechanisms of Output Signaling from a Circadian Oscillator nel during the early stage of translation. Previous studies, which only focus on Jeffrey A. Swan1, Joel Heisler2, Courtney Dailley1, Andy LiWang2, SRP binding to ribosome, produce contradictory results. In this study, we Carrie L. Partch1. examine the full picture of SRP’s multiple layers of regulation at each transla- 1Biochemistry, UC Santa Cruz, Santa Cruz, CA, USA, 2Biochemistry, UC tion intermediate carrying increasing length of nascent chain. FRET-based Merced, Merced, CA, USA. SRP-ribosome binding affinity assay and fluorescence detected SRP-SR asso- Cyanobacteria possess a biological clock that presents unique opportunities to ciation rate measurement give the formation of key intermediates in SRP tar- understand the biophysical and thermodynamic processes that underlie circa- geting cycle as a function of nascent chain length. Both parameters differ for dian oscillation. This is due, in large part, to the relative simplicity of its core more than two orders of magnitude between N-terminal and full exposure of post-translational oscillator, KaiABC. The central hub of this timekeeping

BPJ 8559_8561 70a Sunday, February 18, 2018 apparatus, KaiC, belongs to the AAAþ family of hexameric ATPases, with embedded hairpin motif, re-localizes to LDs from the ER due to a change in ATP hydrolysis correlated with the period of oscillation both in vitro and conformation of the hairpin motif. The ARG residue in middle of the hairpin in vivo. Recent work has elucidated the structural basis for the day to night maintains a strong interaction with the phospholipid head groups, limiting transition, where KaiB binds to the KaiC hexamer the post-ATP hydrolysis the range of conformations available in bilayer membranes, which does not state, thus evicting the output protein SasA and setting into motion a series occur in LD membranes. Class II proteins are translated in the cytoplasm of phosphorylation events that result in the expression of night-specific genes. and directly bind to the LD surface, and usually contain amphipathic helices. To better understand the mechanism of the displacement of SasA at dusk, Our results also show that CTP:phosphocholine cytidylyltransferase (CCT), a we solved the crystal structure of the thioredoxin-like domain of SasA in class II protein with an amphipathic helix, senses large, persistent membrane complex with a monomeric form of the N-terminal domain of KaiC. Based packing defects unique to the LD surface. The large, hydrophobic residues of on this novel structure, we hypothesize that KaiB evicts SasA by binding to the amphipathic helix read out the specific packing defects in LD membranes, the hexamer in between SasA subunits, and then cooperatively recruiting in contrast to other proteins targeting to organelles, which rely upon recognition additional KaiB monomers to displace SasA. To test this model, binding of specific lipids or proteins. studies are actively being pursued to address stoichiometry, kinetics and ATP dependence in the competition between SasA and KaiB for the N-termi- 362-Pos Board B132 nal domain of KaiC. Additional structural studies are also underway targeting Dynamic Characterization of Photosynthetic Proteins on Thylakoid Mem- the full length KaiC-SasA complex as well as the autoinhibited state of branes by High-Speed AFM SasA. Together, we aim to use these experiments to provide a detailed and Bibiana Onoa, Shingo Fukuda, Masakazu Iwai, Krishna K. Niyogi, coherent picture of the kinetic and thermodynamic principles that govern to Carlos Bustamante. rhythmicity in the post-translational oscillator of the cyanobacterial circadian University of California, Berkeley, CA, USA. clock. Structural characterization of photosynthetic proteins within their native mem- brane environment is possible at high spatial resolution. However, the macro- 360-Pos Board B130 molecular organization and dynamics of these densely-packed membranes are Characterizing the Structural and Functional Role of W215 in Thrombin poorly understood. Using high-speed atomic force microscopy (HS-AFM), we Riley Peacock, Jessie Davis, Elizabeth Komives. present both inter and intra-molecular dynamics of Spinacia oleracea grana University of California San Diego, San Diego, CA, USA. thylakoids complexes. Regarding complex mobility, we found two types of The serine protease, thrombin, regulates the balance between the anticoagula- membranes. Although all the membranes display a macroorganization resem- tion and coagulation in the blood clotting pathway. Binding of thrombomodulin bling that of a photosynthetically-active-state, and have a large immobile frac- to thrombin switches its catalytic activity from fibrinogen to protein C, trig- tion (70-80%); the difference emerges from the small mobile fraction. In the gering the switch from the coagulation to the anticoagulation pathway. majority of the grana discs (90%) the mobile fraction is quasi-static with mo- Recently, the mutant W215A was reported to lose activity towards fibrinogen lecular displacements smaller than 10 nm2. In the remaining number of grana without significant loss of activity towards protein C, even in the absence of discs, the molecular displacements are in a much broader range indicating that thromobomodulin, and that W215A demonstrates a lower binding affinity for the supraorganization may be transitioning towards a photosynthetic or photo- sodium. Our lab has obtained experimental and computational results suggest- protective state. Single particle tracking clearly reveals two distinctive diffu- ing that enzyme dynamics are important for connecting the main allosteric site sion regimes. A large fraction of thylakoid complexes (85%) are very confined on thrombin, where thrombomodulin binds, to the active site. To understand (6 nm) and almost immobile. The remaining fraction behaves like ‘‘random- how mutation of Trp215 may alter thrombin specificity, hydrogen-deuterium walkers’’ with potential for long distance displacement. Nevertheless, the exchange experiments (HDXMS) were carried out to compare the dynamics average diffusion coefficient of both populations is close 1 nm2 s1. Moreover, of W215A with those of WT under high and low sodium conditions, HDXMS we have also captured complex to complex rotation of up to 80 degrees, which experiments were performed on the mutants W215I, F227A, and F227V, and might have significance for efficient electron transport and stabilization of high activity assays were utilized to map out the catalytic effect of each mutant. Re- order metastable crystalline arrangements. Individual complex lateral and ver- sults indicate that amide exchange in the 170’s loop dramatically increased for tical molecular fluctuations (size and height) are evidences to support that all mutants, though exchange in the sodium binding loop and N-terminus of the complex assembly, and supramolecular organization is a result of additive heavy chain increased notably only in W215A. Lowering the concentration of forces: protein:protein plus protein:lipid and lipid:lipid interactions. Further- sodium only increased the deuterium exchange for a few sections across the more, the AFM temporal resolution allowed small-lipid-embedded-proteins enzyme, which mostly appear near surface electrostatic sites that may weakly (SLEPs) detection with a probability of 30%. SLEPs detection has been interact with sodium ions. Activity assays suggested that only F227V matches elusive by conventional static scanning microscopy. This dynamic character- the catalytic ability of WT despite destabilizing the 170’s loop. We hypothesize ization of individual thylakoid complexes and SLEPs opens possibilities to that these changes result mostly from the alteration of the substrate binding address questions regarding photochemical responses and photosynthesis sites, and the occupation of the active site by the side chain of position 215 regulation. in all mutants except F227V, rather than a decrease in the ability to bind sodium. 363-Pos Board B133 High-Speed AFM Correlation Spectroscopy (HS-AMF-CS): mS Protein Dynamics without Labels Posters: Membrane Protein Dynamics I George R. Heath1, Beatrice Ramm2, Petra Schwille2, Simon Scheuring1. 1Anesthesiology, Weill Cornell Medicine, New York, NY, USA, 2Max 361-Pos Board B131 Planck Institute of Biochemistry, Martinsried, Germany. Mechanism of Protein Targeting to Lipid Droplets Interactions in crowded conditions underpin many processes at biological Morris E. Sharp1, Coline Prevost2,3, Maria-Jesus Olarte2, membranes, however we can only experimentally observe these dynamics Robert V. Farese Jr.2,3, Tobias C. Walther2,3, Gregory A. Voth4. across limited length and time scales. Developing a detailed understanding 1Department of Chemistry, University of Chicago, Chicago, IL, USA, of the mechanisms at play can require data from multiple techniques and 2Department of Genetics and Complex Diseases, Harvard T. H. Chan School modification of the molecules of interest through labeling, which can influ- of Public Health, Boston, MA, USA, 3Department of Cell Biology, Harvard ence the inherent properties of interest. Here we develop and apply high-speed Medical School, Boston, MA, USA, 4Department of Chemistry, James AFM correlation spectroscopy (HS-AFM-CS), a technique whereby we Franck Institute, and Institute of Biophysical Dynamics, University of oscillate the AFM tip at a single point and directly detect the motions of mol- Chicago, Chicago, IL, USA. ecules under the tip. This gives sub-nanometer spatial resolution combined Lipid droplets (LDs) are organelles that store neutral lipids in cells. LDs have a with microseconds temporal resolution, and can be used in conjunction with core of neutral lipids, including triacylglycerols and sterol esters, surrounded by HS-AFM imaging modes. Using this method, we investigate the oscillatory a phospholipid monolayer. LDs can grow dramatically to store neutral lipids in behavior of the Min protein system used by E. Coli to define the cell division its core, which requires a commensurate increase in the size of the LD mem- midpoint. Previous studies have shown two proteins, MinD and MinE, are brane. The growth and shrinkage of LDs is regulated by proteins, which target required for a binding-assembly-disassembly-unbinding oscillation to occur. LDs during various stages of the LD lifecycle. Here we show that proteins Yet the precise oscillation mechanism remains unknown. Using HS-AFM- target to LDs through two distinct pathways. Class I proteins are initially in- CS we simultaneously measure mobility and surface concentration at model serted in the ER membrane and re-localizes as membrane embedded proteins membranes to decipher effects of protein crowding versus aggregation on to the LD surface through membrane bridges. Using molecular dynamics sim- the assembly and disassembly mechanism of MinD and MinE. This work dis- ulations, we show that GPAT4, a Class I protein that contains a membrane plays how HS-AFM-CS can assess the dynamics of unlabeled bio-molecules

BPJ 8559_8561 Sunday, February 18, 2018 71a over several orders of magnitude and separate the various contributions spa- and common receptor, which are the Interleukin-2 receptor (IL-2R). Although, tio-temporally. both ligands show almost the same pharmaceutical function, they secret, indeed, different cell signals and possess different functions between IL-2 364-Pos Board B134 and IL-15 including side effect such as autoimmunity. Through 1000 times Investigation of Thermodynamic Dissociation Kinetics to Determine the irradiation (The time interval was 1 second) of the X-ray in each in-vivo X- Binding Strengths within a Membrane Protein Complex: Photosystem II ray monitoring, both time constants of moving diffraction from individual Supercomplex gold nanocrystals on the IL-2 and the IL-15 bonded with IL-2R on live NK Eunchul Kim, Ryutaro Tokutsu, Akimasa Watanabe, Jun Minagawa. cell membrane were 1.8102 s and 7.1102 s, respectively. We supposed National Institute for Basic Biology, Okazaki, Japan. these drastically different dynamics of broadly similar IL-2 / IL-2R and IL- Protein-protein interactions as well as the formations of protein complexes 15 / IL-2R commence dissimilar intracellular signals. Furthermore, these mea- have been intensively studied by various methods in both in vivo and in vitro. surements are the first verifying intracellular dynamics through observing the However, although membrane proteins have important roles in cells, interac- extracellular dynamics of membrane receptor at the functional state. This is an tions within membrane protein complexes have been hardly studied than that interesting ascension, considering traditional speculations based on different within soluble proteins due to the lack of suitable methods, though one tertiary structures between IL-2 / IL-2R and IL-15 / IL-2R. We will reports approach using mass spectrometry has been recently developed to elucidate individual dynamics of these ligands inducing NK cells’ proliferation and membrane protein complexes. Here, we present an approach to determine bind- maturation. ing strengths within a membrane protein complex by investigating the thermo- dynamic dissociation kinetics of an isolated membrane protein complex using 367-Pos Board B137 time-resolved fluorescence spectroscopy. We demonstrated different bindings Neuronal KV2.1 Clusters Influence the Diffusion Landscape of the Adja- of light-harvesting complex II (LHCII) trimers in a photosystem II (PSII) cent Astrocyte Membrane supercomplex isolated from a green alga Chlamydomonas reinhardtii, where Ashley N. Leek1, Diego Krapf2, Michael Tamkun1. the different binding types of the LHCII trimers have been indirectly predicted 1Biomedical Sciences, Colorado State University, Fort Collins, CO, USA, by the presence or absence of LHCII trimers in negatively stained PSII super- 2Electrical and Computer Engineering, Colorado State University, Fort complex particles observed by electron microscopy but have not been deter- Collins, CO, USA. mined. As a result of analysis on thermodynamic dissociation kinetics, we The Kv2.1 voltage-gated potassium channel, together with its cell adhesion identified three different activation energies for the dissociations of LHCII tri- molecule auxiliary subunit, AMIGO, form micron-sized clusters in the mers, which imply the presence of multiple hydrogen bonds and/or ion-dipole plasma membrane of neuronal somas. These clusters are the result of a stable bonds between LHCII trimers and core complexes. interaction between the Kv2.1 C-terminus and the cortical ER membrane, 365-Pos Board B135 which in turn induces endoplasmic reticulum-plasma membrane (ER-PM) þ Cytochrome P450 Prefers to be in Liquid-Ordered Domains in the Endo- junctions that influence ER Ca2 store homeostasis, endo- and exocytosis, plasmic Reticulum and the neuronal diffusion landscape. Within intact brain tissue these Carlo Barnaba, Bikash R. Sahoo, Ayyalusamy Ramamoorthy. neuronal Kv2.1-induced ER-PM junctions are closely associated with astro- Biophysics, University of Michigan, Ann Arbor, MI, USA. cytic membranes. However, little is known about whether this Kv2.1-contain- High-resolution mapping of protein/lipid interactions is essential to fully ing organelle influences the astrocyte membrane. Such neuron to astrocyte understand the biological function of a membrane protein, but poses tremen- communication seems probable given that the AMIGO beta subunit may dous challenges to most biophysical and biochemical approaches. These chal- also function as an adhesion molecule. This study addresses the effect of lenges are more severe for membrane proteins like cytochrome P450 that the neuronal Kv2.1/AMIGO clusters on the diffusion landscape of the adja- contain a very large soluble domain and exhibit a completely different time cent astrocyte plasma membrane. Employing a hippocampal neuron/astrocyte scale of dynamics as compared to its transmembrane domain [Durr et al., co-culture system and cell-type specific promoters, in combination with sin- BBA Biomemb, 1768(12), 2007; Yamamoto et al., Sci Rep, 3, 2013; Yama- gle-particle tracking, we examined the diffusion of extracellular epitope- moto et al., Sci Rep, 7, 2017]. In this study, we use peptide-based lipid nano- tagged glutamate transporters (GLT1a) on the astrocyte surface. Analysis discs to ‘‘trap’’ the microsomal cytochrome P450 2B4 alone or also in of protein distribution on the cell surface indicated a higher concentration complex with its redox partners [Zhang et al., Angew Chem, 55(14), 2016]. of GLT1a in the region of astrocytic membrane directly adjacent to the We effectively utilized the unique nature of peptide-based nanodiscs, that al- neuronal Kv2.1/AMIGO clusters. Individual GLT1a transporter trajectories lows for the lipid exchange among nanodiscs, in order to characterize the im- exhibited anomalous diffusion over the entire astrocyte surface. Comparison mediate membrane environment preferred/stabilized by cytochrome P450. We of trajectories from the astrocyte membrane adjacent to the neuronal Kv2.1/ report the first evidence that CYP2B4 is able to induce the formation of AMIGO clusters to those obtained distant from the clusters suggests the ‘‘raft’’-like domains in a biomimetic of the endoplasmic reticulum (ER). Kv2.1/AMIGO clusters influence diffusion on the adjacent astrocyte mem- NMR experiments were used to identify and quantitatively determine the brane. Thus, these data indicate that neuronal Kv2.1/AMIGO clusters alter lipids present in the nanodiscs. In addition, biophysical experiments were sup- the diffusion landscape of the adjacent astrocyte membrane. To our knowl- ported by molecular dynamics simulations to identify a sphingomyelin bind- edge, this study is the first to 1) examine membrane dynamics at a putative ing motif in P450, which is largely conserved among microsomal P450s. The cell-cell junction and 2) demonstrate a novel form of neuron to astrocyte protein-induced raft formation increased the thermal stability of P450 and also communication. dramatically altered the ligand binding kinetics. These results unveil lipid/pro- tein dynamics which can possibly contribute to the delicate mechanism of 368-Pos Board B138 redox catalysis in lipid membrane systems [Barnaba et al., F1000Research, The Tyrosine Kinase Lck Translational Dynamics and its Interrelation 6, 2017]. with its Conformational State Geva Hilzenrat1, Elvis Pandzic2, Katharina Gaus1. 366-Pos Board B136 1EMBL Australia Node in Single Molecule Science, University of New South In Vivo X-Ray Monitoring of Dynamics between Interleukin 2 and Inter- Wales, Sydney, Australia, 2Biomedical Imaging Facility, Mark Wainwright leukin 15 on NK Cells Analytical Centre, University of New South Wales, Sydney, Australia. Jaewon Chang1,2, Masahiro Kuramochi1,2, Youngsuk Beak3, Following T Cell receptor (TCR) engagement, a cell-signalling chain of events Kouhei Ichiyanagi4, Hiroshi Sekiguchi5, Yuji C. Sasaki1,2. begins as a part of T cell activation, including protein phosphorylation events. 1The University of Tokyo, Kashiwa, Japan, 2AIST-UTokyo Operando-Oil, The lymphocite-specific protein tyrosine kinase (Lck) is a key player in T cell Kashiwa, Japan, 3Chabiotech, Seongnam, Republic of Korea, 4Materials signalling. Lck is highly conserved a protein which is anchored to the plasma Structure Science, High Energy Accelerator Research Organization, KEK, membrane via post-translational modifications. It is recruited in the earliest Kashiwa, Japan, 5SPring8, Sayo, Japan. stages of the downstream signalling cascade and serves as a crucial link be- There are many proteins on cell membranes co-acting with other molecules. tween various participating signalling proteins. Lck demonstrates two main Our previous studies using in-vivo X-ray monitoring revealed dynamics of conformational states: an open, active state and a closed, inactive state. Past these membrane proteins related to their functional state, that is, membrane studies on fixed T cells had shown that activation of T cell results in conforma- protein is one of the most important active site of cells. Recently, we suc- tional changes of Lck, as well as a change in its 2D spatial distribution over the ceeded in measuring different dynamics of Interleukin-2 (IL-2) and Inter- membrane, such that active Lck proteins are more likely to cluster. Localising leukin-15 (IL-15) bounded to same receptors on Natural Killer (NK) cells Lck in live cell conditions can improve our knowledge on Lck translational dy- membranes using in-vivo X-ray monitoring with Cu-Ka light (1.54A, Rigaku namic properties under more physiological conditions. Single molecule local- FR-D) at the sub-second level. The IL-2 and the IL-15 have high homology isation microscopy serves as an exciting avenue in order to study Lck location

BPJ 8559_8561 72a Sunday, February 18, 2018 in space and in time. Post-imaging analysis of Lck trajectories made it possible D(t)=Gta1. However, currently straightforward approaches to quantify a gen- for us to detect key characteristics of Lck dynamics over the T cell membrane, eral form of D(t) are lacking to our knowledge. In this study, we develop a such as changes in its confinement states. We show how different Lck mutants, novel mathematical and computational framework to compute the mean corresponding to different conformations, at the two T cell activation states square displacement of diffusing molecules and a diffusion coefficient D(t) (resting vs. activated) result in differences in Lck confinement. from each individual time point of confocal FRAP data without the single power law assumption. Additionally, we developed an auxiliary equation 369-Pos Board B139 for D(t) which can distinguish normal diffusion and single power law anom- Domain Interfaces Facilitate Protein Association and Aggregation in alous diffusion from other types of anomalous diffusion directly from FRAP Multicomponent Lipid Bilayers data points. Importantly, by applying this approach to FRAP data of a varity Asanga Bandara Ekanayaka Mudiyanselage, Afra Panahi, of mambrane proteins and a lipid probe, we demonstrate that the signle power George A. Pantelopulos, John E. Straub. assuption is not enough to describe various types of D(t) of membrane Chemistry, Boston University, Boston, MA, USA. proteins. Model lipid bilayers that mimic the lateral heterogeneity observed in biological membranes are increasingly used to probe protein assembly, cellular signaling, 372-Pos Board B142 vesicle fusion, and membrane protein activity. Consensus is yet to emerge on In Vivo Measurements of Tetraspanin 8 Interactions in Living Cells the driving forces of protein co-localization in domain separated membranes. Daniel Wirth. Difficulty in locating domain boundaries and accurate predictions of their Johns Hopkins University, Baltimore, MD, USA. contribution to free energy presents further challenges. Employing coarse- Tetraspanins are small membrane proteins (200 – 350 amino acids), that span grained molecular dynamics simulations of multicomponent lipid membranes the plasma membrane four times. They are involved in key biological processes with multiple copies of transmembrane proteins, we investigate protein migra- such as cell migration, fusion and signaling events by functioning as organizers tion and local membrane properties such as membrane curvature and thickness of the cell membrane and are widely and abundantly expressed in different cell that drives these processes. We report that proteins predominantly migrate to types. To date, there are 34 known proteins of the Tetraspanin superfamily in the interface between ordered and disordered stripe domains and stabilizes mammals, referred to as Tspan 1 – 34. The most characteristic feature of the the interface by reducing domain interface free energies by 40%-50% via a members of the Tetraspanin family is their ability to interact laterally with mul- new interface detection algorithm. tiple partner proteins and with each other to form the so called TEMs (Tetraspa- nin enriched microdomains). However, little is known about the assembly of 370-Pos Board B140 TEMs and the molecular interactions between Tetraspanins themselves and TLR4 Recruitment into Lipid Rafts Studied by Coarse Grained MD Simu- with their partner proteins. In this study, we utilize the Fully Quantified Spec- lation tral Imaging method (FSI) to investigate molecular interactions of fluorescent 1 1 2 Paulo C.T. Souza , Tsjerk A. Wassenaar , Munir S. Skaf , protein-tagged Tspan8 in live cells. Siewert J. Marrink1. 1University of Groningen, Groningen, Netherlands, 2University of Campinas, 373-Pos Board B143 Campinas, Brazil. Molecular Basis of EPHA2 Pre-oligomers and the Inhibitory Function of Toll-like receptors 4 (TLR4) are transmembrane proteins of the innate im- Its SAM Domain mune system responsible for recognizing lipopolysaccharides (LPS), essential Xiaojun Shi1, Ryan Lingerak1, Chuan Yu2, Jeannine Muller-Greven2, components of the Gram-negative bacteria outer membrane. The association SoonJeung Kim2, Paloma Gill-Rodriguez2, Fatima Raeselle Javier2, of LPS to TLR4 promotes its migration to cell membrane rafts and its homo- Deanna Bowman1, Yixuan Hou3, Yifan Ge4, Matthias Buck2, dimerization, which triggers an inflammatory response. Given its role in the Bingcheng Wang2, Adam W. Smith1. innate immune system, TLR4 is an important target in drug development, 1University of Akron, Akron, OH, USA, 2Case Western Reserve University, especially for the treatment of infections and acute inflammations such as Cleveland, OH, USA, 3Ohio State University, Wooster, OH, USA, 4Harvard sepsis. In the last years, in vitro and in vivo experiments demonstrated that Medical School, Boston, MA, USA. dietary saturated fatty acids (SFAs) can also activate TLR4, linking obesity Eph receptors are the largest family of receptor tyrosine kinases (RTKs). The and inflammation. Conversely, polyunsaturated fatty acids (PUFAs) have activation of Eph receptors are involved in neural development, tissue showed anti-inflammatory properties and can inhibit TLR4 activation by patterning and vascular growth. Earlier structural studies have revealed the de- LPS. The molecular reasons underlying TLR4 trafficking between the mem- tails of multiple dimerization/clustering interfaces in the extra cellular domain brane microdomains and also the agonist/antagonist action of SFAs and (ECD). These findings lead to the prediction of pre-clustering of Eph receptors PUFAs are largely unknown. In this study, we applied MARTINI coarse prior to ligand-binding. Recently, we developed a new membrane protein con- grained molecular dynamics (MD) simulations to study the influence of trol system and a mathematical model for live cell PIE-FCCS experiment, SFAs, PUFAs and LPS on the TLR4 partitioning between liquid-order (Lo) which allows more quantitative measurements of the oligomerization states and liquid-disorder (Ld) phases of a raft membrane model. In agreement of membrane proteins. Using this PIE-FCCS method, we measure the oligo- with existing experimental data, our simulations indicate that LPS and fatty merization degree of EphA2 receptors in live cells. Our data support the pre- acids influence the partitioning of the TLR4 transmembrane domain: SFAs dicted pre-clustering of EphA2. The contribution of the reported ECD and LPS induce the recruitment into the Lo phase while PUFAs keep the pro- interfaces in the oligomerization of EphA2 is also investigated both prior and tein in the Ld phase. Our results provide insights into the driving forces un- after ligand-binding. Comparing to the detailed structural knowledge of the derlying this sorting mechanism. Furthermore, our simulations reveal that ECD, there is only limited knowledge on the intracellular domain (ICD). In TLR4 phase location affects its orientation in the membrane with a direct our previous study, we reported that the SAM domain of EphA2 inhibits the impact on the possible dimerization binding modes. oligomerization and activity of the receptor. In this study, we investigated the oligomerization state and activity of isolated ICD constructs in live cell 371-Pos Board B141 membranes. By deciding the oligomerization states of different ICD constructs, A Novel Computational Framework for D(t) from FRAP Data Reveals we discovered a functional kinase-kinase interaction. In addition, we also iden- Various Anomalous Diffusion Types tified two residues in the SAM domain which are closely related to its inhibitory Minchul Kang. function. The mutation of these two residues led to the attenuation of the inhi- Texas A&M University-Commerce, Commerce, TX, USA. bition of oligomerization and activity in both ICD constructs and full length re- Diffusion of membrane proteins and other biomolecules in lipid membranes ceptors. Together, our work demonstrates the different spatial arrangement of play a pivotal role in almost all the aspects of cellular signaling. For this EphA2 compared to other RTKs and provides new molecular details that reason, various tools have been developed for diffusion measurement decode the working mechanism of EphA2. including Fluorescence Recovery After Photobleaching (FRAP). Although FRAP was invented in the 1970s, it has remained strong and evolved into 374-Pos Board B144 an indispensable biophysical tool tracking cellular organelles, proteins, and Interactions between the Transmembrane Domains of Plexin, Semaphorin, molecules not only in the cell membranes but also in the cyto-/nucleo-plasms and Neuropilin in a spatio-temporal manner. Due to complex cellular environments, biolog- Shaun M. Christie1, Soon-Jeung Kim2, Paul D. Toth1, ical diffusion is often correlated over time and described by a time dependent Jeannine Muller-Greven2, Matthias Buck2, Adam W. Smith1. diffusion coefficient, D(t) althogh the underlying mechanisms have not 1Chemistry, University of Akron, Akron, OH, USA, 2Physiology and been fully understood. Since D(t) potentially provided important information Biophysics, Case Western Reserve University, Cleveland, OH, USA. of cellular structures and proteins’ transporting mechanisms, many efforts Plexins and semaphorins are a large family of secreted and membrane bound have been made to quantify D(t) by FRAP assuming the single power law signaling proteins, all with a conserved Sema domain. Broad expression across

BPJ 8559_8561 Sunday, February 18, 2018 73a tissue types is thought to aid in organization of tissues, and neuoral and vascu- tions affecting conserved regions resulted in loss of function. Second, we char- lature guidance. The plexins are the receptor moiety and act through intracel- acterized the expression and sub-cellular localization of FisB mutants that were lular domains that contain a region with homology to GTPase activating fused to YFP. While some of the mutants found to be defective in the heat-kill proteins. Canonical signaling occurs through trans interactions via the Sema assay failed to locate to the mother cell membrane, some were correctly tar- domains of semaphorin ligand and plexin receptor or in some cases by the geted but their dynamics were altered compared to wild-type. Further charac- use of neuropilin co-receptors. Recent developments show cis interactions terization of the mutants will reveal factors that are important for FisB’s may exist between certain family members, where the ligand semaphorin ex- function. pressed in the same cell as the receptor causes changes in trans signaling. Some of these associations have been attributed to the transmembrane (TM) do- 377-Pos Board B147 mains. Due to the sequence characteristics of the TM domains of these families, Organization of I-BAR Proteins on Tubular and Vesicular Membranes our study aims to experimentally test the homo- and hetero-cis interactions of Zack Jarin1, Feng-Ching Tsai2,3, Patricia Bassereau2,3, Gregory A. Voth4. 1 these TM domains in cell membranes. Here we use pulsed interleaved fluores- Institute for Molecular Engineering, University of Chicago, Chicago, IL, 2 cence cross correlation spectroscopy to resolve the contribution of the TM USA, Laboratoire Physico Chimie Curie, Institut Curie, PSL Research 3 domain to plexin-semaphorin-neuropilin oligomerization in live cells. We de- University, CNRS UMR 168, Paris, France, Multiscale Physics-Biology- signed constructs consisting of the TM domain and short regions of the extra- Chemistry and Cancer, Sorbonne Universites, UPMC Univ Paris 06, Paris, 4 cellular and intracellular domains attached to a fluorescent protein. Using these France, Department of Chemistry, Institute for Biophysical Dynamics and we found a diverse set of correlations between TM domains of the protein fam- James Franck Institute, University of Chicago, Chicago, IL, USA. ily, with most interactions in agreement with the computational models. We Inverse Bin/Amphiphysin/Rvs (I-BAR) proteins are a family of proteins also began to determine how the TM correlates and oligomerizes in context responsible for initiating and driving membrane deformation orders of magni- with the full length proteins. These results suggest that the TM domain can tude of larger than a single protein. While protein-mediated membrane remod- used as a potential therapeutic target and could aid in the creation of TM-spe- eling is key to many cellular processes, the mechanism used by I-BAR cific inhibitor peptides for plexin-semaphorin-neuropilin signaling related proteins is thus far unknown. Simulations of I-BAR domains can improve diseases. our understanding of membrane remodeling by adding near atomistic detail to protein aggregation, the precursor to large-scale deformation. We employ 375-Pos Board B145 a multiscale approach that generates a bottom-up coarse-grained (CG) model Computationally Determined Free Energy Profiles of the Mucin-1 Trans- that bridges the gap between atomistic simulations and our recent Mesoscopic membrane Homodimer Membrane Simulations with Explicit Proteins (MesM-P) model. Using our Christina M. Freeman, Alexander J. Sodt. unique, hierarchical approach to CG model development, we simulate the ag- NICHD, National Institutes of Health, Bethesda, MD, USA. gregation behavior of I-BAR proteins at micron length scale while remaining Determining the principles governing lipid-protein interactions, specifically consistent with atomistic simulations. We find at low surface coverage, i.e. low those in single-pass transmembrane protein dimerization, are fundamental bound density, I-BAR domains form transient, tip-to-tip strings. As surface problems in the field of membrane biophysics. Mucin-1 (MUC1), a mucus con- coverage increases, a disordered mesh begins to form. We demonstrate this stituent found on the apical side of epithelial cells, typically acts as a barrier complex aggregation behavior on lipid bilayers in the geometry of interest against pathogens but has also been identified as an oncogene. When overex- to in vitro experiments: the surface of giant unilamellar vesicles (e.g. periodic pressed, the C-terminal subunit of MUC1 is known to homodimerize and un- flat sheets), inside of lipid bilayer tubules, and on the surface of small lipo- dergo nuclear translocation as well as interact with growth factors and other somes. By studying various geometries, we add fundamental insight into kinases in signaling cascades to promote metastasis, inflammation, and drug how I-BAR proteins sense curvature and the effect of membrane curvature resistance. on aggregation behavior. MUC1 homodimerization in the membrane occurs due to a pair of disulfide 378-Pos Board B148 bonds formed between two adjacent monomers. Strong dimerization affinity Interaction of KRas4B Protein with C6-Ceramide Containing Lipid Model in bacterial membranes comparable to glycophorin A has been observed exper- Membranes imentally, and weak affinity is seen even without the cytosolic membrane-prox- Lei Li, Roland Winter. imal cysteine residues. Several mutations in the TMD, most notably Ala-1180- Department of Chemistry and Chemical Biology, Technical University of Leu, also affect dimerization. Dortmund, Dortmund, Germany. All-atom molecular dynamics simulations of the transmembrane and juxta- Ras proteins are oncoproteins and play a pivotal role in cellular signalling path- membrane domains of MUC1 have been used to better quantify the dimer struc- ways. Over 30% of cancers are driven by mutant Ras proteins and KRas4B pro- ture and binding energetics. Umbrella sampling and free energy perturbation tein is the mostly mutated Ras isoform. All Ras proteins’ signaling strongly methods were used to generate potential of mean force (PMF) curves, which depends on their correct localization in the cell membrane. Natural ceramides were then compared to experimental assay activity. are important cellular lipids, which are secondary messengers and play an important role in the regulation of programmed cell-death. Unlike the C16, 376-Pos Board B146 C18, and C20 long fatty-acid chain ceramides, the shorter C6-ceramide is Deciphering the Role of FisB during Sporulation of Bacillus Subtilis known to be a cell membrane permeable lipid. Recent studies show that C6-cer- through Mutagenesis amide could induce KRas4B dependent cancer cell death by acting synergisti- Anna Georgieva1, Ane Landajuela2, Erdem Karatekin2. 1 2 cally with chemotherapy. Further analysis indicated that C6-ceramide could University of California, Riverside, Riverside, CA, USA, Yale, New directly inhibit the growth activity of KRas4B mutated cells. However, the Haven, CT, USA. mechanism of C6-ceramide inhibiting KRas4B mutated cells’ activity is still Membrane fission is an essential biological process for all forms of life. While unknown. We established a C6-ceramide containing heterogeneous model two dedicated machineries, dynamin and ESCRT-III have been described for membrane and studied the effect of KRas4B on the lateral organization of eukaryotic organisms, little is known about this process in prokaryotic organ- the lipid bilayer. The incorporation of the ceramide leads to significant differ- isms. Previous studies have described the involvement of fission protein B ences in the partitioning behavior compared to other raft-like membranes, and (FisB) in membrane fission during the sporulation of Bacillus subtilis. Sporu- induces formation protein nanoclusters dispersed in the fluid-like domains of lating cells divide asymmetrically, generating a large mother cell and smaller the membrane, probably via some kind of lipid sorting mechanism. No direct forespore. After division, the mother cell membrane engulfs the forespore. interaction between the C6-ceramide and KRas4B is observed in FCCS Membrane fission releases the forespore into the mother cell cytoplasm. Cells experiments. lacking FisB are severely impaired in the fission reaction, but the molecular mechanism is not known. FisB is a single-pass transmembrane protein that is 379-Pos Board B149 expressed in the mother cell after asymmetric division. It forms small, mobile Lipid-Anchored Ras Proteins Sense/Modulate Plasma Membrane Curva- foci in the mother cell membrane surrounding the forespore 2 hours into spor- ture in an Isoform- Specific Manner ulation that accumulate at the pole where fission occurs at 3 hours. FisB also Hong Liang, Alemayehu Gorfe, John F. Hancock, Yong Zhou. binds cardiolipin, a lipid that localizes to the poles and around the forespore and Dept of Intgrative Biology and Pharmacology, University of Texas Medical whose level is upregulated during sporulation. Here, we generated a battery of School, Houston, TX, USA. FisB mutants, guided by a predicted structural model of FisB and conservation Ras proteins, including isoforms H-, N- and K-Ras, are lipid-anchored small of residues, and characterized their phenotypes. First, we employed a heat-kill GTPases and play key roles in cell growth, division, proliferation, and migra- assay to evaluate the sporulation efficiency of the mutants. Most of the muta- tion. Constitutively active Ras mutants are oncogenic and found in 20% of all

BPJ 8559_8561 74a Sunday, February 18, 2018 human tumors. Ras signaling is highly compartmentalized to the plasma mem- pass transmembrane proteins. The most investigated substrate of g-secretase brane (PM), making their trafficking critical to their biological function. Ras is the C-terminal fragment of the amyloid precursor protein (APP) C99, which trafficking is still poorly understood. Ras C-termini are lipid-modified, which is cleaved at different positions by PS1. Among other peptides, C99 process- potentially allows direct sensing and modulation of PM curvature, a key ultra- ing results in ‘‘amyloid beta’’ (Ab) fragments of different lengths with the 40 structural event in membrane trafficking. (Ab40) and 42 (Ab42) amino acid long variants being the primary products. We measured Ras lateral oligomerization, an essential step in Ras signaling, on Since they are known to aggregate and form fibrillar structures (the main com- giant plasma membrane vesicles (GPMVs) using fluorescence lifetime imag- ponents of amyloid plaques, found in the brains of FAD patients) the Ab42 ing-fluorescence resonance energy transfer (FLIM-FRET). Nanometer-scaled peptides have been heavily implicated in Alzheimer’s disease. In healthy pa- membrane curvature changes in the form of thermal undulations were manip- tients, the production of Ab40 is favored but this balance can be shifted to- ulated via altering osmotic pressure. Hypotonic conditions disrupted local wards Ab42 by a large number of known PS1 mutations. The exact clustering of H-Ras anchor but enhanced K-Ras oligomerization, suggesting mechanism responsible for the formation of the different Ab fragments is still that H-Ras favors curved membrane and conversely for K-Ras. Super resolu- unknown. To shed some light on the matter we conducted atomistic micro- tion electron microscopy-spatial mapping was used to quantify the lateral second time-scale molecular dynamics simulations of three different C99- oligomerization of Ras on intact cell PM. Expression of positive curvature- PS1 complexes, studying three different sites at which C99 is believed to inducing amphiphysin 2 BAR domain (BARamph2) enhanced H-Ras lateral bind to PS1 and enter its catalytically active center. We uncovered that the oligomerization, but disrupted K-Ras oligomerization. On the other hand, ex- binding modes differ mainly in number of key protein ligand interactions pressing negative curvature-inducing IRS53p BAR domain (BARIRS53p) almost (with respect to known mutation sites), active site accessibility and PS1 completely abolished H-Ras oligomerization, while having little effect on conformation. Based on the available data we conclude that prior to cleavage, K-Ras. EM-bivariate co-localization analysis shows that H- or K-Ras co-local- C99 most likely binds to the cavity formed by the PS1 transmembrane do- ized extensively with both BAR domains. This was correlated with elevated mains 2 3 and 5. mitogenic signaling (preferentially regulated by K-Ras) but diminished PI3K signaling (preferentially regulated by H-Ras) when exposed to hypotonic me- 382-Pos Board B152 dium. Atomic force microscopy shows that expressing H-Ras anchor induced Modeling of Claudin-15 Paracellular Channels via Molecular Dynamics more PM protrusions and decreased the elastic Young’s modulus of apical Simulations 1,2 1,2 1 PM of live kidney cells, suggesting that H-Ras anchor induces PM curvature. Giulio Alberini , Fabio Benfenati , Luca Maragliano . 1Center for Synaptic Neuroscience and Technology, Istituto Italiano di Our data suggest that Ras senses and modulates membrane curvature in an iso- 2 form-specific manner. Tecnologia, Genova, Italy, Department of Experimental Medicine, Universita` degli Studi di Genova, Genova, Italy. 380-Pos Board B150 Tight-junctions between epithelial cells are tissue-specific structures that regu- Membrane Allostery Recruits Unique Hydrophobic Binding Sites Promot- late the flux of solutes across the barrier, parallel to cell walls. While it is known ing Substrate Specificity of Lipolytic Enzymes that the tight-junction backbone is made of strands of proteins from the claudin Varnavas D. Mouchlis, J. Andrew McCammon, Edward A. Dennis. family, the molecular mechanism of its function is still unclear. Recently, the Pharmacology, and Chemistry and Biochemistry, University of California, crystal structure of a mammalian claudin-15 was reported, and a structural San Diego, La Jolla, San Diego, CA, USA. model of claudin-15-based paracellular channels has been proposed. The model Addressing selectivity of Phospholipases A2 (PLA2) towards phospholipids is suggests association of claudins in the same cell via cis interactions, and across crucial for defining the structural features contributing to molecular recogni- adjacent cells via trans interactions. Although very promising, the structure tion between enzyme and substrate. This is fundamental to understanding represents only a static conformation, with missing residues in the extracellular enzyme-substrate binding and interactions. PLA2 enzymes are the upstream regions that might potentially cause steric clashes. Here, we present detailed regulators of the eicosanoid pathway liberating arachidonic acid (AA). The atomic models of paracellular architectures that we have generated starting liberation of AA sets off a cascade of molecular events that involves down- from the putative assembly and refined via structural modeling and molecular stream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX), dynamics simulations in double membrane bilayer and water environment. Our leading to inflammation (Nat. Rev. Immunol. 2015, 15, 511-523). Aspirin results show that the configuration of the channel is overall stable. The mono- and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting mer-monomer cis interfaces are preserved via stable hydrogen bonds that were COX and LOX which rely on PLA2 enzymes to provide them with AA. predicted by cysteine crosslinking experiments. Extracellular residue loops in Therefore, PLA2 enzymes could potentially also be targeted to diminish trans interaction regulate the size of the pore, which displays a stationary radius inflammation at an earlier stage in the process. This study focuses on three of 2.5-3.0 A˚ at the narrowest region. Finally, we employ the refined model to human PLA2 enzymes: the cytosolic (cPLA2), calcium-independent (iPLA2) perform Free Energy calculations of ions and small molecules diffusion and lipoprotein-associated (Lp-PLA2). A lipidomics high-throughput assay through the channel, in order to elucidate the mechanism of selective was developed allowing us to define unique specificity of PLA2s towards a permeability. wide variety of phospholipids. Hydrogen/deuterium (H/D) exchange mass spectrometry was used to experimentally identify distinct peptide regions 383-Pos Board B153 that interact with phospholipid vesicles. Molecular dynamics simulations, Effect of Mild and Harsh Detergents on the Stability of the Model Mem- guided by H/D exchange data, showed that the active sites of these enzymes brane Protein Proteorhodopsin are allosterically regulated by membranes. Membrane phospholipids bind to Sadegh Faramarzi, Blake Mertz. West Virginia University, Morgantown, WV, USA. allosteric sites located on the interfacial surface of PLA2s shifting their conformation from the ‘‘closed’’ to the ‘‘open’’ state (PNAS 2015, 112, Despite the widespread use of surfactants in the study and manipulation of E516-E525). This process facilitates extraction and binding of a phospholipid membrane proteins, an atomic-level perspective of the physical properties molecule into the active site. Similar methods were employed to understand that lead to membrane protein solvation remains relatively unknown. In addi- the association mechanism of the interfacial surface with the membrane tion, standard experimental biophysical approaches such as X-ray scattering bilayer. This constitutes the first detailed study revealing that the formation and NMR are inadequate for these studies because protein-surfactant systems of amphipathic helices into the membrane play a significant role in the recruit- tend to possess a polydisperse size and shape profile. In this work, we have car- ment of unique hydrophobic binding pockets promoting substrate specificity ried out molecular dynamics (MD) simulations on proteomicelle complexes of a model bacterial membrane protein, proteorhodopsin (PR), solvated with of PLA2s. either a mild detergent (n-dodecyl b-D-maltoside (DDM)) or a harsh detergent 381-Pos Board B151 (n-dodecylphosphocholine (DPC)). We observed that the helicity of PR and the Investigating Complex Formation of C99 with Gamma-Secretase, Using thickness of the micelles around PR do not differ significantly between two de- Atomistic MM-MD Simulations and Free Energy Calculations tergents. However, DPC interacts with aqueous solvent more than DDM, leav- Manuel Hitzenberger, Martin Zacharias. ing PR solvent-exposed. As a result, PR undergoes significant structural Physics Department, Technical University of Munich, Garching Bei fluctuations in DPC, leading to an asymmetric proteomicelle shape. Increased Munchen,€ Germany. interactions of DPC with water molecules is confirmed by higher total head- The intramembrane aspartyl protease g-secretase is mostly known for its group-water interaction energies (including electrostatic, Van der Waals and assumed role in the development of familial Alzheimer’s disease (FAD). Be- nonbonded). We conclude that the stabilization or destabilization effect of a ing a hetero-tetramer, it consists of four different proteins: presenilin-1 (PS1), surfactant depends on detergent-solvent interactions more than detergent-pro- nicastrin, APH-1 and PEN-2. The catalytic center of this complex is located tein interactions. Our findings help explain experimental observations indi- deep within the hydrophobic region of presenilin where it cleaves single- cating that PR is more stable and can form ordered oligomers in DDM,

BPJ 8559_8561 Sunday, February 18, 2018 75a while it predominantly exists as an unstable monomer in DPC [1]. [1] Hussain expedite the MD simulations and allow for slower and more realistic extraction et al. (2015) J. Mol. Biol. 427:1278. velocities and lower pulling forces for simulating SMFS experiments. These factors increase the likelihood of observing transient intermediates. We simu- 384-Pos Board B154 late the forced unfolding of membrane proteins using our CG model, Upside Interrogating Membrane Protein Conformational Dynamics within Native (1), incorporating new membrane potentials. Upside can reversibly fold some Lipid Bilayers with Hydrogen-Deuterium Exchange Mass Spectrometry soluble proteins up to 97 AA in CPU-hours without the use of fragments or ho- Eamonn Reading. mology. Our membrane potentials are derived from statistics of known struc- Department of Chemistry, King’s College London, London, United tures, accounting for burial depth in the membrane and side chain exposure Kingdom. levels. In the simulations of the forced unfolding of bacteriorhodopsin, we Membrane protein structure and function is influenced by interactions with are able to reproduce the characteristic features displayed in experiments, lipids and their membrane environment. However, the influence of the actual including the unfolding of individual and pairs of helices, worm-like chain native lipid composition encountered in the membrane has largely eluded behavior of the elastic unfolded segments and the back-and-forth transitions be- investigation. As membrane proteins represent important drug targets it is tween states with a comparable resolution as the experiments. The difference in imperative, therefore, to develop methods capable of gaining structural insights the unfolding pathway are compared for the isolated monomer and in trimeric within their native lipid environment. To address this need we establish a form. Our method is ready to be applied to other transmembrane protein sys- generic method that combines hydrogen-deuterium exchange mass spectrom- tems including GlpG, halorhodopsin. etry (HDX-MS) with the styrene-maleic acid lipid particle (SMALP) technol- (1) Jumper et. al, Trajectory-Based Parameterization of a Coarse-Grained ogy for the investigation of membrane protein conformational dynamics within Forcefield for High-Throughput Protein Simulation, http://www.biorxiv.org/ native lipid bilayers. We validate how our approach can be used to probe native content/early/2017/07/27/169326. membrane protein conformational dynamics using the rhomboid protease, GlpG, captured within three different native lipid compositions. We demon- 387-Pos Board B157 strate exquisite sensitivity of protein regions important for function and stabil- Continuum Theory of HIV-Budding ity to alterations in their native lipid environment. The potential for these Sanjay Dharmavaram1, Baochen She1, Ioulia Rouzina2, Robijn Bruinsma1. 1 emerging techniques in examining ligand, drug, and lipid interactions with Phys/Astro, University of California, Los Angeles, Los Angeles, CA, USA, 2 membrane proteins, in well-defined native lipid environments, is an exciting The Ohio State University, Columbus, OH, USA. prospect which we anticipate will impact on the membrane protein structural The emergence of release of immature HIV virions from the surface of in- biology field, as well as on drug discovery pipelines. fected cells presents multiple biophysical puzzles. For human cells, virion release requires the ESCRT machinery but this is not the case for yeast 385-Pos Board B155 cells. Next, the development of the bud appears to be characterized by inter- Assessing the Structure of Transmembrane Oligomeric Intermediates of mittent pausing. Finally, the classical Lipowsky mechanism - based on an a-Helical Toxin using Molecular Dynamics Simulations Helfrich theory - for exocytosis from multi-component membrane, relies on Rajat Desikan1, Ganapathy Ayappa2, Prabal K. Maiti3. line tension. Line tension can be ruled out for the case of HIV-1 by the 1Chemical Engineering, Indian Institute of Science Bangalore, Bangalore, large Gag-free sectors of the surface of HIV virions after release. The talk India, 2Chemical Engineering, Biosystems Science and Engineering, Indian will propose a new budding mechanism for assembling protein shells that Institute of Science Bangalore, Bangalore, India, 3Physics, Center for is based not on line tension but on the role of Gauss membrane curvature. Condensed Matter Theory, Indian Institute of Science Bangalore, Bangalore, It accounts naturally for the pausing and presence/absence of ESCRT India. machinery. Pore forming toxins (PFTs) are the primary vehicle for infection by several 388-Pos Board B158 strains of bacteria. These proteins which are expressed in a water soluble Towards Piecing Together the RAS-RAF Puzzle: Dynamics of Membrane- form (monomers), bind to the target membrane and conformationally transform (protomers), and self-assemble to form a multimer transmembrane pore com- Associated KRAS4b-RAF RBD/CRD Ternary Complex plex through a process of oligomerization. PFTs are broadly classified into or Timothy Travers, Cesar A. Lo´pez, S. Gnanakaran. alpha or beta toxins. In contrast to beta-PFTs, the paucity of available crystal Los Alamos National laboratory, Los Alamos, NM, USA. structures coupled with the amphipathic nature of the transmembrane domains The RAS family of small guanine nucleotide-binding proteins are critical for various cellular signaling pathways and are among the most common culprits have hindered our understanding of alpha-PFT pore formation. We use molec- in human cancer, with KRAS4b mutations occurring at high rates in pancre- ular dynamics (MD) simulations to examine the process of pore formation of the bacterial toxin Cytolysin A from Escherichia coli (ClyA) in lipid bilayer atic, lung, and colorectal cancer. Membrane-anchored RAS binds to effectors membranes. Using atomistic MD simulations ranging from 50- 500 ns, we from the RAF kinase family at two domains: (i) the RAS-binding domain show that transmembrane oligomeric intermediates or ‘arcs’ form stable pro- (RBD) whose complex with RAS has been solved experimentally, and (ii) teo-lipidic complexes consisting of protein arcs with toroidal lipids lining the the cysteine-rich domain (CRD) that mutational assays show can bind RAS free edges. By creating initial conditions where the lipids are contained within independently. However, the structure of the RAS/RBD/CRD ternary complex still remains unknown, including its membrane orientation and interactions. the arcs, we study the dynamics of spontaneous lipid evacuation and toroidal We have used a combination of all-atom and coarse-grained molecular dy- edge formation. This process occurs on the timescale of tens of nanoseconds, suggesting that once protomers oligomerize, transmembrane arcs are rapidly namics (MD) simulations to investigate these questions, focusing on the stabilized to form functional water channels capable of leakage. Using umbrella complex between KRAS4b and the RBD/CRD domains of BRAF that sampling with a coarse grained molecular model, we obtain the free energy of experiments show is the initially-activated RAF isoform. First, single-domain insertion of a single protomer into the membrane. A single inserted protomer simulations of CRD or RBD alone indicate their associations with the membrane. Next, binary complex simulations based on the RAS/RBD has a stabilization free energy of 53 kJ/mol and forms a stable transmembrane crystal structure show multiple dominant membrane orientations, where water channel capable of leakage. Our simulations reveal that arcs are stable and viable intermediates that can occur during the pore formation pathway RBD could either be away from the membrane or making direct membrane for ClyA. interactions. Finally, extensive simulations based on computational docking of CRD to the RAS/RBD crystal structure suggest a viable ternary complex 386-Pos Board B156 model where CRD embeds into the membrane via both of its hydrophobic Including H-Bonding and Lipid Exposure in Near-Atomic Level Folding loops, RAS interacts with the membrane at the surface covering helices 4 Simulations of Helical Membrane Proteins: II. Applications to Single- and 5 of the G domain, and RBD away from the membrane. We are currently Molecule Force Spectroscopy investigating if interactions of KRAS4b with the RBD/CRD domains of the Zongan Wang. CRAF isoform lead to changes in the ternary complex structure and to differ- Department of Chemistry, University of Chicago, Chicago, IL, USA. ential membrane interactions. These studies should provide insights that will Single-molecule force spectroscopy (SMFS) is a powerful technique to study contribute to our understanding of both normal and cancer-activated signaling the unfolding of soluble and membrane proteins. Coarse-grained (CG) models pathways.

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Posters: Intrinsically Disordered Proteins (IDP) reactive oxygen species on A-syn aggregation has been established. In addition, cytochrome c along with A-syn, has been found to be a crucial component of and Aggregates I Lewy bodies. Aggregation of A-syn induced by cytochrome c/H2O2 complexes with or without the presence of lipid membranes have been investigated 389-Pos Board B159 extensively. a-Synuclein is a Neuron Specific Lectin In this study, we used biophysical experiments along with fluorescence cor- Melissa Birol, Siobhan Toal, Elizabeth Rhoades. relation spectroscopy (FCS) to study the role of cytochrome c in attenuating University of Pennsylvania, Philadelphia, PA, USA. A-syn folding/aggregation. FCS is a convenient technique to study the diffu- Monomeric a-Synuclein (aS) is a small intrinsically disordered, neuronal sional and conformational dynamics of labeled biomolecules under single protein which garners significant interest in the biomedical field because molecule resolution. Using FCS, we observed that A-syn fluctuated between fibrillar deposits of aS are the hallmark of Parkinson’s disease (PD). While different forms of varying diameter, an event which was directly affected by significant efforts have focused on deciphering pathological mechanisms of cytochrome c binding. The data emphasized the role of cytochrome c as a aS, comparatively little is understood of its native function. Our work peroxidase, while A-syn protected the cells at the initial stages of acute identifies that aS is a lectin that binds specifically to complex, N-linked reactive oxygen induced apoptosis. The presented data showed that the con- glycans. Using cell-derived proteoliposomes, we find that aS associates tributions of different components in the A-syn-cytochrome c binding vary selectively with neuronal-lineage cell membranes and induces vesicle clus- systematically depending on different factors. These factors included exper- tering. Cleavage of surface exposed N-linked glycans decreases aS binding imental conditions, the nature of A-syn aggregation states (native vs oligo- to the membrane, disrupts vesicle crowding and alters the conformation mers vs fibrils) and the availability of the reactive oxygen species (ROS). of bound protein. An array-based screen identified a small number of These factors are implicated in the oxidative alterations of both A-syn and pentasaccharide-core glycans, underscoring the specificity of the interac- cytc, radical formation and di-tyrosine adduct formation. We found direct tion. Intriguingly, only the N-terminally acetylated, physiological form of relevance of these factors to the nucleation and multi-merisation of A-syn the protein is impacted by deglycosylation and returns hits in the glycan- in vitro. array screen. Recent findings indicate that aS pathology spreads into the brain and monomeric, oligomeric and fibrillar aS can progress from cell 392-Pos Board B162 to cell and trigger the aggregation of the endogenous protein in recipient Illuminating the Self-Assembly of Alpha-Synuclein Amyloid Fibrils neurons. Cellular internalization of aS is specific to neuronal-lineage cells Jervis V. Thevathasan, Jonas Ries. and we identify that this is dependent upon cell-surface N-linked glycans. Cell Biology and Biophysics, European Molecular Biology Laboratory, PD-relevant aS mutants also show reduced cellular uptake and deficient Heidelberg, Germany. glycan binding. The classification of aS as an intrinsically disordered lectin Amyloid formation of alpha-synuclein is a major pathological hallmark of provides new considerations for understanding its long-sought native func- Parkinson’s disease (PD). It is therefore imperative to have a detailed mech- tion and role in disease, as well as for aS-targeted therapeutics for treatment anistic understanding into amyloid self-assembly process and structure. To of PD. this end, we use a variant of single molecule localization microscopy called ‘points accumulation for imaging in nanoscale topography’ (PAINT) in 390-Pos Board B160 combination with fluorogenic amyloid binding dyes. Sequential localization Exploring the Role of O2 on the Metal Ion Specific Modulation of Alpha- of each fluorogenic binding event affords visualization of amyloid fibrils Synuclein Structure with an unprecedented spatial resolution of approx. 15 nm. Here we demon- Heather R. Lucas. strate the versatility of PAINT in visualizing amyloid fibrils with dyes Department of Chemistry, Virginia Commonwealth University, Richmond, spanning the visual light spectrum. Using polarization detection for single VA, USA. molecules to infer the precise orientation of each dye molecule we are The role of transition metals in neuronal health has become increasingly able to deduce information of dye binding characteristics with respect to more appreciated in recent years. A tight regulatory system exists to maintain amyloid structure. We then extend this imaging strategy to visualize the normal function; however, the abnormal accumulation of metals in specific self-assembly process of single amyloid fibrils in real-time. We observe regions of the brain is associated with both aging and neurodegeneration. fibril elongation to proceed from both ends of a seed fragment. Interestingly, Metal imbalance has long been linked to Parkinson’s disease (PD) due to the elongations from either end of a seed are different, with one end having the accumulation of iron in the substantia nigra pars compacta, while copper a faster rate compared to the other. Fibril elongation was found to have pe- levels in this same region are diminished. Despite the redox characteristics riods of stagnation followed by burst of elongation resulting in a step-wise and ability of these transition metal ions to produce damaging free radicals, growth. their potential for promoting normal brain function under homeostatic condi- tions must be considered. Both copper and iron trigger conformational 393-Pos Board B163 changes within the intrinsically disordered protein aS, the principal compo- Alpha-Synuclein Modulation of Vesicle Exocytosis in Secretory Cells nent of Lewy body inclusions, which are the proteinaceous deposits within Meraj Ramezani1, Marcus Wilkes1, Tapojyoti Das2, David Eliezer2, PD brains. This presentation will focus on new insights into potential protag- David Holowka1, Barbara Baird1. 1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, onistic and antagonistic effects of metal-protein interactions. The conforma- 2 tional dynamics associated with iron, copper, and manganese binding to aS NY, USA, Department of Biochemistry, Weil Cornell Medicine, New York, NY, USA. will be discussed as well as their ability to activate O2 and generate radicals. Unique changes are observed in the protein secondary and quaternary struc- Alpha-synuclein (a-syn) is the most prominent factor in the etiology of Par- ture upon the introduction of stoichiometric quantities of individual metal kinson’s disease (PD), a common and debilitating neurodegenerative disorder ions based on circular dichroism and dynamic light scattering analyses. that impairs motor function. Duplication or triplication of the a-syn gene, as Immunoblotting techniques have further revealed that metal-bound aS can well as a series of point mutations such as A53T, E46Q and A30P lead to follow distinct pathways towards toxic oligomers or b-sheet fibrils depending early onset hereditary PD. Elevated levels of a-syn are observed in many on the metal. Furthermore, a clear involvement of ambient oxygen in the patients who develop sporadic forms of PD, further pointing to the critical production of post-translational modifications has been observed. Such need to understand the pivotal role of a-syn. A-syn is a 140-amino acid, pre- metal-induced changes can result in altered protein-protein and protein- synaptic protein found predominantly in neurons, but also present in other membrane interactions, which could have physiological implications. Taken cells. A prominent role for a-syn in regulating synaptic vesicle trafficking together, these results contribute to the ongoing conversation within the field has been implicated in several studies, but the precise functions and func- about the role of metals and aS in PD progression. tional mechanisms of this important protein remain unclear. We are exam- ining the influence of the human a-syn protein on the stimulated exocytic 391-Pos Board B161 trafficking of recycling endosomal vesicles in mammalian RBL-2H3 and Investigations on the Factors Responsible for the Cytochrome C-alpha PC-12 cells utilizing VAMP8-pHluorin. This assay reveals that a-syn ex- Synuclein Binding-Aggregation Landscape pressed ectopically at low levels inhibits stimulated vesicle exocytosis, Sumanta Ghosh. whereas ectopically expressing a-syn at high levels does not inhibit and Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical may enhance exocytosis. We have evaluated several mutants of a-syn and Biology, Kolkata, India. observed interesting similarities and differences compared to wild type. The aggregation of a-synuclein (A-syn) has been implicated strongly in the pa- These comparisons can be related to the binding of these a-syn variants to thology of Parkinson’s disease (PD). A-syn is an intrinsically disordered liposomes and micelles, as measured by NMR spectroscopy, enabling an protein with a heterogeneous folding/aggregation landscape. The influence of analysis of structure-function relationships.

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394-Pos Board B164 conformational changes in aSyn upon binding. Together these measurements Developing Novel FRET Based Biosensors that Monitor a-Synuclein provide insight into how aSyn achieves glycan selectivity through N-terminal Assembly for Use in High Throughput Screening acetylation. Malaney Young, Anthony R. Braun, Jonathan N. Sachs, Chi Hung Lo. University of Minnesota, Minneapolis, MN, USA. 397-Pos Board B167 Aggregation of endogenous a-synuclein is the hallmark of Parkinson’s disease Structural Dynamics of Monomeric a-Synuclein on the PS-mS Time Scale pathology. Under normal conditions, a-synuclein plays an important role in Derived from MD Simulations neurological function. Under pathologic conditions, misfolded a-synuclein Reinhard Klement1, Timo Graen1, Asaf Grupi2, Elisha Haas2, forms cytotoxic oligomers and fibrils. There has been significant effort to iden- Helmut Grubmueller1. 1 2 tify small molecule inhibitors of a-synuclein fibrillization. However, recent MPI for biophysical Chemistry, Goettingen, Germany, Bar-Ilan University, evidence suggests the kinetically unstable oligomeric species, and not fibrils, Ramat Gan, Israel. are the source of a-synuclein cytotoxicity. We interrogated the self- alpha-synuclein[aS] is an intrinsically disordered protein[IDP], which can association of the oligomeric species under aggregation prone conditions using form pathogenic beta-sheet aggregates. A number of dynamically reordering FRET. To achieve this, we developed a set of cell-free FRET based biosensors regions exist, in which secondary structure elements form and melt on ns-ms that monitor oligomer-oligomer interactions and fibril formation through fluo- timescale. Here we used unrestrained molecular dynamics simulations to rescence lifetime. Using fluorescence lifetime provides a 30-fold increase in quantify the timescales at which the aS monomer reorders. We find that sec- sensitivity over more traditional FRET measurements. We have achieved an ondary structure forms and dissolves on time scales of a few ms, very similar optimal FRET of 14.99% for oligomeric interactions and a seeded induced to those of tertiary structure rearrangements. Remarkably, both largest change in FRET of 15.34%. We are currently optimizing these biosensors beta-sheet formation rates and propensities were seen for the NAC and flank- for scale up to facilitate high throughput screening. Hits will be validated for ing regions, involved in fibril formation. Since the on-rate for beta-sheet ag- FRET dose response and biophysical changes in aggregation kinetics via thio- gregation is much slower, a conformational selection mechanism seems flavin T assays and AFM characterization. plausible for aggregation. We also investigated the spontaneous alpha-helix formation in the structurally variable region between residues 1-100. The 395-Pos Board B165 on-rates for alpha-helix formation are very low or not observed at all. This Interaction of the Copper Chaperone Atox1 with Alpha-Synuclein suggests an inducted fit or much slower conformational selection mechanism Istvan Horvath, Tony Werner, Pernilla Wittung-Stafshede. for alpha-helix formation upon membrane binding compared to beta-sheet Biology and Bioengineering, Chalmers University of Technology, formation. Both structure and dynamics of IDPs are very sensitive to force Gothenburg, Sweden. field inaccuracies. We therefore performed extensive test simulations using Transition metal binding appears as a common feature of many amyloidogenic four force field/water model combinations and compared the results to proteins. The bound metals may affect the function and also aggregation prop- NMR, SAXS and FRET data. erties of the polypeptides. Copper (Cu) binding to a-synuclein has been widely studied and a number of binding sites have been identified in the protein. Due to 398-Pos Board B168 its high affinity for copper, a-synuclein might be a player in the cellular homeo- Systematic Development of Small Molecules to Inhibit Specific Steps of stasis of this metal ion. Most previous in vitro studies were focused on Cu(II) a-Synuclein Aggregation in Parkinson’s Disease interaction with a-synuclein. There are only a few reports on Cu(I) binding to Roxine Staats, Patrick Flagmeier, Michele Vendruscolo. a-synuclein, although this is the main intracellular redox state of copper. Also, Chemistry, University of Cambridge, Cambridge, United Kingdom. the Cu(I) ions are not water soluble and therefore bound to various Cu a-Synuclein (aS) is an abundant neuronal protein closely associated with Par- chaperone proteins in the cell. For this reason we aimed to identify if the Cu kinson’s Disease (PD). Its aggregation, which is a defining hallmark of this con- chaperone Atox1 is capable of removing bound Cu or delivering Cu(I) to dition, is widely held responsible for progressive damage to neuronal cells, and a-synuclein.Aggregation studies using Thioflavin-T fluorescence detection for an exacerbation of the complex network of interplaying factors which showed that in the presence of Cu(I)-loaded Atox1, a-synuclein amyloid for- constitute the overall PD pathology. Currently, no disease-modifying treatment mation is greatly reduced while in the presence of free copper ions a-synuclein is available for PD, at least in part because the disordered nature of aS compli- aggregation is promoted. Interaction studies between Atox1 and a-synuclein cates efforts to design therapeutic agents aimed at abrogating its aggregation. In using surface plasmon resonance showed Cu-dependent binding between this study, we aim to establish a strategy for the systematic development of the two proteins with an affinity in the micromolar range. Gel filtration studies small-molecule inhibitors of aS aggregation. This aim will be achieved by ush- on mixtures of Atox1 and a-synuclein in the presence and absence of ering a recently developed, highly quantitative, three-pronged kinetic analysis Cu(I) showed that the complex is not stable, but metal analysis revealed that of aS aggregation into drug discovery efforts. We will use this approach to Cu transfer from Atox1 to a-synuclein took place. Based on these results we establish a structure-activitiy relationship between a library of compounds hypothesize that a-synuclein is a new target protein for Atox1-mediated and their efficacy in inhibiting specific microscopic processes of aS aggrega- Cu(I) delivery. tion. By elucidating the chemical features that cause the inhibiton of the respec- tive processes of aS lipid-induced aggregation, fibril elongation, and secondary 396-Pos Board B166 nucleation, we aim to demonstrate that this kinetic approach can be employed Characterizing Alpha-Synuclein Binding to Glycans in the rational development of aS anti-aggregation compounds targeting Karen Acosta1, Elizabeth Rhoades2. specific processes. By establishing the three-pronged kinetic analyis as a 1 Biochemistry and Molecular Biophysics, University of Pennsylvania, drug development tool, we will foster a systematic approach to aS anti- 2 Philadelphia, PA, USA, Chemistry, University of Pennsylvania, aggregation drug discovery, which may accelerate the rate of compound devel- Philadelphia, PA, USA. opment and address the high rate of attrition of PD clinical trials by virtue of Alpha-synuclein (aSyn) is a small, neuronal protein which forms aggregates more precise mechanistic elucidation. and insoluble plaques in Parkinson’s disease. Our understanding of the role of aSyn in disease etiology is impaired, in part, by a lack of knowledge sur- 399-Pos Board B169 rounding the native function of aSyn. Due to the localization of aSyn to the syn- Identification of a Specific Residue Side Chain Controlling the Self- aptic termini of neurons, it has been suggested that its native function may Assembly and Cytotoxicity of Islet Amyloid Polypeptide involve synaptic vesicle fusion and trafficking. aSyn binds to synthetic lipid Phuong Trang Nguyen, Elizabeth Godin, Ximena Zottig, Steve Bourgault. bilayers and can sense and alter physical membrane properties, including the Chemistry, UQAM, Montreal, QC, Canada. induction of lipid bilayer curvature and thinning. Thus, while interactions be- Polypeptide assembly into amyloid fibrils plays key roles in over 50 amyloid- tween aSyn and lipids in cellular membranes have been heavily studied, less related diseases including Alzheimer’s diseases and diabetes mellitus type 2 attention has been given to other components of these membranes. Recent (DM-2). The deposition of the islet amyloid polypeptide (IAPP) as insoluble work from our lab has suggested that aSyn preferentially binds to complex, amyloid fibrils in the pancreatic islets correlates proportionally to the severity N-linked glycans. Interestingly, only N-terminally acetylated aSyn shows of DM-2. The propensity of IAPP to form amyloid fibrils is strongly dependent this selectivity. While N-terminal acetylation has been shown to enhance on its primary sequence. We and others have recently reported the key contri- alpha-helicity in the N-terminus of aSyn, how it mediates glycan-binding is butions of asparagine at residue 21 in the amyloidogenesis and the toxicity of not known. Here we used isothermal titration calorimetry to quantify the inter- IAPP. Furthermore, Asn21 is located within the amyloidogenic region. To gain action between aSyn and a variety of N-linked glycans. Single molecule Fo¨rster more insight into the importance of this position, single mutations at this res- resonance energy transfer (smFRET) was used to characterize structural and idue were performed ([N21A] IAPP, [N21Q] IAPP, [N21F] IAPP, [N21Dab]

BPJ 8562_8565 78a Sunday, February 18, 2018

IAPP and [N21P] IAPP). The kinetics of IAPP amyloid formation and its ana- the oligomers in physiological solutions (at a sub-nM concentration) as well as logs were evaluated by a combination of thioflavin T (ThT) fluorescence assay on supported anionic lipid bilayers (POPC:POPG:Cholesterol = 1:1:1) exposed and transmission electron microscopy (TEM). We observed that mutation of to such solutions. Single molecule imaging results indicate that solution state Asn21 modulates amyloid formation. The process of fibrillogenesis was also and membrane-bound oligomers mostly consist of monomers to trimers monitored by circular dichroism (CD) spectroscopy and we observed that the (neglecting any pre-measurement photobleaching). Dimers and trimers have conformational transition disordered-to-beta-sheet is associated with IAPP/ higher membrane affinity than the monomers. This higher membrane affinity IAPPs N21 modified. Among these IAPP analogs, atomic force microscopy is possibly linked to the conformational changes which occur during aggrega- (AFM) revealed two distinct fibrils morphologies with varied diameters, thick- tion. We explore aggregation-associated conformational transitions using nesses, and lengths of [N21F] IAPP, [N21Q] IAPP and IAPP. Using rat pancre- fluorescence, vibrational and Solid State NMR (ssNMR) based methods that atic beta-cells (INS-1E), we observed that [N21F] IAPP and [N21Q] IAPP we have established earlier for amyloid beta. We find that the high membrane fibrils are toxic, whereas IAPP amyloid fibrils are not. This study highlights affinity species contains a mixture of both alpha-helices and beta-sheets. Our that the self-assembly and the toxicity of IAPP may be controlled by a precise study, therefore, quantifies the structure and membrane interaction of small intermolecular interaction involving a specific residue in the amyloidogenic n-mers of amylin as a function of n, paving the way for size-specific drug- region. targeting of amylin aggregates.

400-Pos Board B170 403-Pos Board B173 Exosomes from Human Pancreatic Islets Suppress IAPP Amyloid Structural Analyses of a Linker Region of the Amyloid Precursor Protein Formation Mizuho Imamura1, Shingo Kanemura2, Masaki Okumura2, Pernilla Wittung-Stafshede. Shigeru Shimamoto1, Yuji Hidaka1. Biology and Biological Engineering, Chalmers University, Gothenburg, 1Kindai University, Higashi-osaka, Japan, 2Tohoku University, Sendai, Sweden. Japan. Human islet amyloid polypeptide (IAPP) is the major component of amyloid The amyloid precursor protein (APP) is a receptor like membrane bound deposits found in pancreatic islets of patients with type-2 diabetes (T2D). protein that has an N-terminal extracellular, a trans-membrane, and a short Because T2D cases are increasing in the world and there is no cure, it is of C-terminal intracellular domain. The N-terminal domain consists of several uttermost importance to identify molecular mechanisms and involved pro- domains, such as heparin binding, copper binding, acidic, and linker domains. cesses. IAPP is normally secreted in conjunction with insulin from pancreatic It is known that the processing of APP by b- and g-secretase in the linker re- b-cells to regulate glucose metabolism. Extracellular vesicles (EVs), such as gion triggers the production of the amyloid b peptide (Ab, the pathogenic pep- exosomes, are small vesicles released by virtually all (including pancreatic) tide) and aggregates derived from it (amyloid). However, little is known cells to aid in cell-cell communication and tissue homeostasis. Since IAPP concerning the processing mechanism and the tertiary structure of the b-sec- amyloid formation is affected by synthetic lipid vesicles in vitro, we predicted retase recognition sites that are included in a linker region, although various that also EVs may have effects. To address this question, we isolated and APP mutants in which the b-secretase processing site are mutated have been characterized EVs from pancreatic islets of healthy and T2D patients, reported to be associated with the pathogenesis of Alzheimer’s disease. In or- followed by probing their effects on IAPP amyloid formation using a combi- der to obtain structural information on the processing site in the linker region, nation of analytical and biophysical methods. We discovered that pancreatic the linker and related mutant proteins were over-expressed, purified, and EVs from healthy patients inhibit IAPP amyloid formation by peptide scav- measured. The proteins were expressed using a T7-promoter system enging (direct binding); in contrast, T2D pancreatic and human serum EVs (pET17b vector, Novagen) in E. coli cells. Then, the proteins purified by had no effect on IAPP amyloid formation. In accord with differential effects Ni-affinity, ion exchange, and size exclusion chromatography, and analyzed on IAPP amyloid formation, the protein and lipid contents differed between by Circular Dichroism (CD) and small angle X-ray scattering (SAXS) in healthy pancreatic EVs and T2D/serum EVs. Taken together, we propose various concentrations of trifluoroethanol (TFE). The results indicated that an EV-mediated process that tempers IAPP amyloid formation in the pancreas the linker proteins can be classified into an intrinsically disordered protein at normal conditions, which breaks down in T2D due to alterations in secreted and that a-helical structures in the proteins are induced in the presence of vesicles. TFE. To further obtain structural information related to its pathogenesis, we 401-Pos Board B171 determined the apparent radii of gyration, the molecular masses, and the Human Islet Amyloid Polypeptide: Identifying Early-Stage Aggregation molecular shapes of these mutants by SAXS analyses. Comparisons of the Mechanisms through Molecular Simulation calculated structures of a wild type and mutant proteins will be discussed in Ashley Z. Guo, Juan J. de Pablo. this paper. Institute for Molecular Engineering, The University of Chicago, Chicago, IL, USA. 404-Pos Board B174 Human islet amyloid polypeptide (hIAPP) is implicated in the onset of type II Hsp70 Delays Amyloid Aggregation of Amylin by Inhibiting Primary diabetes and is known to aggregate into amyloid fibrils. However, it is prefibril- Nucleation lar species, not mature fibrils, that are proposed to be cytotoxic. In order to Neeraja Chilukoti, Bankanidhi Sahoo, Mithun Maddheshiya, better understand the role of hIAPP aggregation in the onset of disease, as Kanchan Garai. well as to design effective therapeutics, it is crucial to understand the mecha- Interdiciplinary Sciences, Tata Institute of Fundamental Research, nism of early-stage hIAPP aggregation. In this work, we use atomistic molec- Hyderabad, India. ular dynamics simulations combined with multiple advanced sampling Amyloid aggregation of amylin is implicated in the pathology of Type-2 dia- techniques to examine the formation of the hIAPP dimer and trimer. Metady- betes (T2DM). In vitro and in vivo studies have shown that small heat shock namics calculations reveal a free energy landscape for hIAPP dimer formation, protein Hsp70 can inhibit amyloid aggregation and provide protection against which suggests multiple possible transition pathways. We employ the finite cytotoxicity in amyloid aggregation diseases. However, molecular mechanisms temperature string method to identify favorable pathways for dimer and trimer of the interactions of Hsp70 with the amyloid proteins is still poorly under- formation, along with relevant free energy barriers and intermediate structures. stood. Here we use Hsp70 family of proteins such as the Hsp70 from e.coli, Results provide valuable insights into mechanisms and energetics of hIAPP Mycobacterium Tuberculosis (MTB) and human Hsc70 to investigate the aggregation. effects of these chaperones on aggregation of amylin. Our results suggest that kinetics of fibrillization of amylin, as measured by thioflavinT fluores- 402-Pos Board B172 cence, is delayed dramatically in presence of even substoichiometric concentra- Amylin (hIAPP) Aggregates on the Membrane tions of Hsp70. However effects are reduced significantly if Hsp70 is added in Simli Dey, Anoop Rawat, Bappaditya Chandra, Barun Kumar Maity, the growth phase of aggregation indicating that Hsp70 possibly inhibits the Perunthiruthy K. Madhu, Sudipta Maiti. early phase of aggregation. Kinetics of interactions measured by FRET be- Chemical Science, Tata Institute of Fundamental Research, Mumbai, India. tween alexa488-labeled Hsp70 and Tetramethylrhodamine-labeled amylin Amylin is a 37 residue intrinsically disordered peptide whose aggregation is (TMR-amylin) is found to be slow typically taking several hours indicating associated with Type II diabetes. Small oligomers (n-mers, with n<20) bind that Hsp70 interacts with one or more species of amylin that form in a time strongly to small unilamellar vesicles and are likely to be the key to its toxicity. dependent manner. These could be oligomers or some rare forms of monomers However, we do not know which n-mer is the key. Here we use Single Mole- of amylin. We then use field flow fractionation (FFF) coupled to Multi-angle cule Photo-bleaching (smPB) techniques to study the size (‘n’) distribution of light scattering (MALS) and fluorescence correlation spectroscopy (FCS) to

BPJ 8562_8565 Sunday, February 18, 2018 79a characterize the complexes of Hsp70 and TMR-amylin. Radius of gyration of in the binding process. Upon binding, Segment 1 of ArkA sampled a wider the complexes were below the detection limit of MALS. Molecular weight range of contacts, with the domain, compared to simulations started from the measured by MALS, and Hydrodynamic size and fluorescence brightness of experimental bound structure. The presence of many nonnative contacts shows the complexes measured by FCS indicate interactions of Hsp70 with mono- there is more than one bound ArkA conformation and therefore we may be meric or small oligomeric forms of amylin. Taken together our results indicate simulating an encounter complex. Most nonnative contacts are still to SI of that Hsp70 inhibits aggregation of amylin possibly by preventing primary Abp1SH3 indicating that it is not favorable for ArkA Segment 1 to bind to other nucleation by binding to the ‘misfolded’ monomers and/or small oligomers surfaces of the SH3 domain. Further investigation will consider how electro- of amylin. static interactions steer ArkA to the binding site. 405-Pos Board B175 408-Pos Board B178 Conformational Changes and Flexibility for ArkA Binding to Abp1-SH3 Intrinsically Disordered Proteins Link Alternative Splicing and Post- Kristina Foley, Robyn Stix, Gabriella Gerlach, K. Aurelia Ball. Translational Modifications to Complex Cell Signaling and Reulation Chemistry, Skidmore College, Saratoga Springs, NY, USA. Jinhong Zhou1, Suwen Zhao1, A.K. Dunker2. SH3 domains are the most common protein interaction domains and are found 1iHuman Institute, Shanghai Tech, Shanghai, China, 2Biochemistry and across all forms of life. However, little is known about how flexible peptides Molecular Biology, Indiana University, Indianapolis, IN, USA. bind to these domains. These flexible peptides are often intrinsically disordered Intrinsically disordered proteins and regions (IDPs and IDRs) lack well-defined proteins (IDPs) which are difficult to model using only experimental methods. tertiary structures, yet carry out various important cellular functions, especially One SH3 domain found in yeast, Abp1SH3, has a binding site for the ArkA those associated with cell signaling and regulation. In eukaryotes, IDPs and IDP. Molecular dynamics simulations were used to model the Abp1SH3 IDRs contain the preferred loci for both alternative splicing (AS) and many domain and the Abp1SH3-ArkA complex. SH3 domains are believed to have post-translational modifications (PTMs). Furthermore, AS and/or PTMs at a two-step binding process in which part of the peptide binds to Surface I these loci generally alter the signaling outcomes associated with these IDPs (SI) and afterwards, the rest of the protein binds to Surface II (SII). It was found or IDRs. However, the prevalence of such functional modulations remains un- that there is a greater amount of flexibility in the residues located in SI and SII known. Also, the signal-altering mechanisms by which AS, and PTMs modu- compared to other residues in the SH3 domain. Also, several side chain late function and the extent to which AS and PTMs collaborate in their dihedral angles in SI and SII have different angle preferences between the signaling modulations have not been well defined for particular protein exam- free and bound states. Mutations to the ArkA peptide were made including ples. Here we focus on three important signaling and regulatory IDR- several residues mutated to alanine and a K-3R mutation. It was found that containing protein families in humans, namely G-protein coupled receptors although lysine and arginine are similar in structure, this mutation resulted in (GPCRs), which are transmembrane proteins, the nuclear factors of activated fewer contacts between ArkA and Abp1.The mutations to alanine gave insight T-cells (NFATs), which are transcription factors (TFs), and the Src family to the specificity SII has for binding the ArkA peptide, shown by a change in kinases (SFKs), which are signaling enzymes. The goal here is to determine binding pattern between the proteins. Simulations were also run with a linker how AS and PTMs individually alter the outcomes of the signaling carried between Abp1 and ArkA, to mimic experimental procedures. It was found out by the various IDRs and to determine whether AS and PTMs work together that there are similar contacts between Abp1 and ArkA with and without the to bring about differential cellular responses. We also present data indicating linker, however, there is an increased flexibility in Abp1 in the presence of that a wide range of other signaling IDPs or IDRs undergo both AS- and the linker compared to the normal bound structure. This increased flexibility PTM-based modifications, suggesting that they, too, likely take advantage of may be compensating for the reduction of entropy caused by the linker. signal outcome modulations that result from collaboration between these two post-translational events. Hence, we propose that the widespread cooperation 406-Pos Board B176 of IDPs, AS and/or PTMs substantially contributes to the vast complexity of Conformational Changes of ArkA12 eukaryotic cell signaling systems. Robyn Stix, Kristina Foley, Gabriella Gerlach, K. Aurelia Ball. Skidmore College, Saratoga Springs, NY, USA. 409-Pos Board B179 SH3 domains are common protein interaction domains that are found across all Large-Scale Analysis of the Evolution of Functions Mediated by Intrinsi- forms of life and bind flexible intrinsically disordered proteins (IDPs). IDPs are cally Disordered Regions difficult to model using only experimental methods. Molecular dynamics (MD) Mary O.G. Richardson1, Alex S. Holehouse1, Iris Langstein2, simulations mimicking experimental conditions were used to model the ArkA Philipp Korber2, Rohit V. Pappu1. IDP which binds to the SH3 domain found in yeast, Abp1SH3. ArkA IDPs are 1Department of Biomedical Engineering, Washington University in St. Louis, believed to bind to SH3 domains in a multi-step binding process. Conforma- St. Louis, MO, USA, 2Biomedical Center, Molecular Biology, LMU Munich, tional analysis was applied to the ArkA IDP to gain a better understanding of Munich, Germany. conformations that may promote this binding. ArkA was found to sample a Intrinsically disordered regions (IDRs) make up ca. 30% of the proteome of higher population of polyproline II helices compared to circular dichroism most eukaryotes, and are involved in a diverse set of functions. Conventional data, which is likely due to restriction of the omega bond of proline to the trans sequence alignment tools suggest that the sequences of disordered regions conformation in the MD simulations. Accelerated molecular dynamic simula- are poorly conserved, implying that they are under weak evolutionary selection. tions that allow isomerization from trans to cis of the omega bond of proline are However, biophysical studies have established the presence of quantifiable currently being run to generate a more accurate conformational ensemble for sequence-to-conformation relationships for IDRs that are essential for their the ArkA peptide. functions. These relationships are satisfied by a broader swath of sequences with similar physico-chemical properties when compared to the narrow range 407-Pos Board B177 of sequences that satisfy sequence-to-structure relationships in intrinsically Initial Binding Interaction between ArkA and Abp1SH3 foldable proteins. In this study, we combine sequence analysis with extensive Gabriella Gerlach, Kristina Foley, Robyn Stix, Lia Ball. extant functional data of near-saturating mutagenesis of S. cerevisiae to Chemistry, Skidmore College, Saratoga Springs, NY, USA. examine the relationships between protein truncation and cell viability. We SH3 domains are the most common protein interaction domains and are found identify sequence features that most strongly correlate with dispensable regions across all forms of life with at least 400 in humans alone. Theses domains often and contrast these with sequence features associated with those of disordered bind to flexible proteins known as intrinsically disordered proteins (IDPs). regions that are crucial for cell viability. These results provide the necessary However, little is known about the binding mechanism between IDPs and foundations for furthering our understanding of the interplay amongst SH3 domains. One SH3 domain found in yeast, Abp1SH3, has a binding site sequence, function, and conservation of IDRs. The results highlight the diver- for the IDP ArkA. Molecular dynamics simulations were used to model the sity of sequences with similar physico-chemical features that give rise to con- binding mechanism of Abp1SH3 with ArkA, consisting of Segments 1 and 2. servation of functions. Abp1SH3 is believed to have a three-step binding process beginning with the formation of an encounter complex where an ensemble of ArkA conformations, 410-Pos Board B180 each forming different contacts with the domain, are populated in an equilib- Controlled Liquid-Liquid Phase Seperation of Recombinant Oleosin rium exchange. For the peptide to fully bind, it locks into a specific conforma- Ellen H. Reed, Daniel A. Hammer. tion. From the encounter complex, Segment 1 of ArkA would bind to Surface I Chemical and Biomolecular Engineering, University of Pennsylvania, of the domain (SI), and afterwards, Segment 2 of ArkA would bind to Surface II Philadelphia, PA, USA. of the domain (SII). We preformed simulations in explicit water starting with Recombinant proteins enable the design of materials with tailored functionality Segment 1 of ArkA and the SH3 domain placed farther apart than the maximum and responsiveness. We designed a protein that, upon oxidation of a cysteine cut-off distance for non-bonded interactions to observe the initial interactions residue, phase separates into micron sized liquid droplets. These droplets are

BPJ 8562_8565 80a Sunday, February 18, 2018 similar to membrane-less organelles found in cells. This work is based on the (Matsushita et al., 2017). Next, we examined whether the diffusion motion in natural plant protein, oleosin. Oleosin is an amphiphile with distinct hydrophil- the lysozyme solution dependent on the temperature. The mean squared curves ic and hydrophobic domains: N and C-terminal hydrophilic segments and a in the diffusion motion of gold nanoparticles were increased when the temper- hydrophobic central core. From wild type oleosin, a truncated version was en- ature in the lysozyme solution is decreased. One possibility for this result is the gineered that reduced the hydrophobic core and added glycines. An amino acid fast diffusion in the lysozyme solution at lower temperature is triggered by the was then mutated to add a cysteine in the N-terminal hydrophilic segment. invisible solute cluster. This idea has that the solute cluster which generates the Above an upper critical solution temperature, the protein formed micelles, protein aggregation might be controlled by thermal regulation. So, we are now and below this temperature, the protein condensed to form liquid droplets. examining the aggregation process in the lysozyme solution by using a thermal The phase transition was thermoreversible. The cysteine containing protein regulation plate under the microscopy. This phenomenological analysis will had a higher propensity to form droplets than the variant with no cysteines. help to understand how lysozyme solutions do trigger to the protein aggregation We predict that this is because disulfide bonding of the cysteine residues though a solute cluster. created dimers. In other systems, dimers have been shown to have a higher pro- pensity to form droplets. A family of oleosins was synthesized with a single 413-Pos Board B183 cysteine at various locations in the protein backbone. Placing the cysteine Protein Interactions Control Dynamics of Liquid Compartments 1,2 € 1 2 closer to the N-terminus resulted in a higher transition temperature. We predict Tyler S. Harmon , Frank Julicher , Anthony A. Hyman . 1Max Planck Institute for the Physics of Complex Systems, Dresden, that this is because cysteine residues closer to the N-terminus are exposed on 2 the protein surface and therefore oxidizes more readily. This protein construct Germany, Max Planck Institute of Molecular Cell Biology and Genetics, provides a novel way to control protein liquid droplet formation and dissolu- Dresden, Germany. tion. We envision this work having applications as a membrane-less organelle Micron-sized, non-membrane bound cellular organelle can form as the result mimic in synthetic protocells and as a targeted drug delivery system. of liquid-liquid phase separation. These liquid compartments have been impli- cated in many functions ranging from cellular signaling to macromolecular 411-Pos Board B181 assembly. The physical properties of these compartments are important for Controllable Protein Phase Separation and Modular Recruitment to Inves- their function and thus should be tuned to match their intended purpose. tigate Biochemical Compartmentalization in Membraneless Organelles For many of these liquid compartments the optimal functionality appears to Benjamin S. Schuster, Matthew C. Good, Daniel A. Hammer. be in a relatively well hydrated, dynamic, and easily reversible state. Multiple University of Pennsylvania, Philadelphia, PA, USA. diseases are associated with a hardening transition where these liquid com- Many intrinsically disordered proteins (IDPs) self-assemble via liquid-liquid partments transition from a functional liquid-like state to an aberrant solid- phase separation into spherical droplets, which function as membraneless like state over a long period of time. For other compartments, they appear organelles with critical cellular functions. Despite significant interest, funda- to be designed to mature naturally from a liquid compartment into a more mental questions remain unanswered about the functional capabilities of mem- solid compartment. Therefore, the physical mechanism controlling the liquid braneless organelles. It is well established that membrane-bound organelles to solid transition is at the heart of how cells regulate and control these provide aqueous compartments adapted for specific biochemical reactions, compartments. but it is unclear what roles membraneless organelles play in regulating the ki- We designed and developed a three-dimensional polymer lattice model to build netics of intracellular reactions. The field has been hindered because few tools a framework for investigating mechanisms for hardening in liquid compart- exist to systematically investigate the consequences of localizing enzymes and ments. We designed lattice polymers to explore several mechanisms affecting substrates to membraneless organelles. Therefore, we manipulated the intrinsi- the time dependence of protein dynamics. This allows us too test which protein cally disordered, arginine/glycine-rich RGG domain from the P granule protein properties are important for controlling the slowing of the dynamics of the LAF-1 to demonstrate controllable phase separation and cargo recruitment to a liquid compartments. We analyzed the transition to solid-like through quanti- synthetic membraneless compartment. First, we demonstrated methods to con- fying the time dependence of diffusion rates, density, and reversibility of disso- trol phase behavior by externally triggering droplet assembly and disassembly. lution. These results are a promising first step to reach a molecular picture of This was accomplished by using specific proteases to manipulate the valency of the hardening process. IDP domains and presence of solubility-enhancing domains. Second, we char- acterized permeability of these compartments to soluble macromolecules and 414-Pos Board B184 devised strategies to target and colocalize cargo molecules into the droplets. Quantitative Measurement of Stability and Heterogeneity of Protein Am- Soluble cargos were recruited using either RGG domains or coiled-coiled inter- yloids using Disaggregation by Chemical Denaturants action domains as recruitment modules, and cargo release was triggered by pro- Timir Baran Sil, Bankanidhi Sahoo, Subhas Chandra Bera, Kanchan Garai. teolytic removal of the recruitment domains. Droplet assembly and cargo Tifr Centre for Interdisciplinary Sciences, Tata Institute of Fundamental recruitment were robust and occurred in cytoplasm. Our results using this plat- Research, Hyderabad, India. form suggest it is now possible to controllably recruit multiple enzymes and Amyloids are protein aggregates known to be involved in several neurode- substrates to stimulus-responsive membraneless organelles. This system pro- generative diseases e.g., Alzheimer’s and Parkinson’s disease. However, vides a much-needed experimental framework to answer unsolved problems biological activities of the amyloids still remain unclear due to highly hetero- in the biophysics of membraneless organelles and to harness IDP compartments geneous and polymorphic nature of the amyloid aggregates. We hypothesize for bioengineering applications. that kinetics of dissolution of the amyloids can be dependent on the molec- ular structure of the amyloids. Here, we use amyloids of tetramethylrhod- 412-Pos Board B182 amine labelled Amyloid-b(1-42) (TMR-Ab42) which are fluorescently dark Microscopic Observations of Protein Brownian Motions in Supersaturated as shown by Garai and Frieden previously. Our results using fluorescence Solutions correlation spectroscopy reveals that fluorescence of TMR increases upon Kazuki Yoshimura1,2, Msahiro Kuramochi1,2, Yuji C. Sasaki1,2. dissolution of TMR-Ab amyloids in 4M GdnCl. Increase of fluorescence is 1University of Tokyo, Kashiwa, Japan, 2AIST-UTokyo Operando-Oil, correlated with the increase in the concentration of monomeric TMR- Kashiwa, Japan. Ab42. We find that TMR-Ab42 amyloid dissolved minimally in native buffer Protein aggregation (crystallization) process is a microscale phenomenon of but the rate of dissolution increases exponentially with increase in the con- typical protein solution, and cause a wide variety of disease such as ALS, Alz- centrations of Urea or GdnCl. However, dissolution of the amyloids doesn’t heimer’s and Parkinson’s. Protein aggregates are generated by mis-folded pro- seem to follow thermodynamic solubility. In case of the amyloids we find teins, and localize to specific areas in the cells. However, its phenomenological that soluble concentration increases with the increase in total concentration mechanism for aggregation process is not well understood. Our previous study of the fibrils. Furthermore, fibrils incubated at a particular concentration was performed for the understanding of this aggregation process using the of denaturant do not dissolve any further in the same solvent even upon Diffracted X-Ray Tracking method, and suggested that the local aggregations 100-fold dilution. Taken together, our data indicate that amyloids are a are generated by solute clusters in the supersaturated protein solutions (Mat- mixture of different species and each type of species requires a certain min- sushita et al., 2017). In this study, we monitored the local aggregation dynamics imum concentration of denaturant for dissolution. Our data of apparent sol- in the lysozyme solution containing the gold nanoparticles (approximately ubility as a function of concentration of denaturants can be fit assuming 100nm), which is a crystal precursor metastable state, under the Bright-field Gaussian distribution where the peak of the distribution indicates mean sta- microscopy. This method can monitor indirectly the local dynamics in the lyso- bility and width of the distribution indicates heterogeneity in a particular zyme solution by detecting translational motion in the gold nanoparticles. preparation of the amyloids. Methodologies used here are general; hence, We found that the diffusion motion in the lysozyme solution could be detected they can be employed to characterize stability and heterogeneity of amyloids in the order of milliseconds. Our result was consistent with our previous work of other proteins as well.

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415-Pos Board B185 Nuclear localized human uracil DNA glycosylase 2 (hUNG2) initiates base Thermodynamic and Hydrodynamic Properties of a Doxorubicin Labeled excision repair (BER) of uracil lesions in genomic DNA. Similar to many ELP-Drug Carrier DNA glycosylases, hUNG2 possesses a 90 amino acid unstructured Valeria Zai-Rose1, Wolfgang Kramer2, Reid Bishop3, John J. Correia1. N-terminal domain (NTD) that encodes the nuclear localization signal as 1Biochemistry, University of Mississippi Medical Center, Jackson, MS, USA, well as several sites for post translational modification and interaction with 2Millsaps College, Jackson, MS, USA, 3Belhaven University, Jackson, MS, replication proteins. Here, we present evidence that within crowded environ- USA. ments such as the cell nucleus, the hUNG2 NTD endows enhanced nanoscale The Low Critical Solution Temperature (LCST) of the thermoresponsive en- translocation properties to the enzyme. Based on in vitro measurements gineered ELP biopolymer is being exploited for the thermal targeted delivery comparing hUNG2 to a catalytic hUNG2 construct lacking the NTD under of Doxorubicin (Dox) to solid tumors. Here we show that ELP labeled with crowded conditions, these enhancements are manifested in a near 10-fold Dox undergoes an enhanced cooperative liquid-liquid phase separation DNA length dependent increase in kcat,a3-fold increase in the overall cat- (LLPS) from a soluble to insoluble coacervation state with increasing concen- alytic efficiency (kcat/KM), and an improved ability for the enzyme to trans- tration and fraction of labeled Dox. The phase change is entropically driven as locate between damaged sites separated greater than 20 bp. These indicated by the positive entropy of the reaction. We show by turbidity and translocation studies were further implemented in human cells to develop DLS that ELP phase change is monophasic, while mixtures of ELP and the first in vivo assay of its kind, able to detect nanoscale translocation events Dox-ELP are biphasic, with Dox-ELP exhibiting LCST first and ELP parti- of hUNG2 between uracil lesion sites 10 to 80 bp apart. hUNG2 translocation tioning into droplets of increasing Rh. Circular Dichroism (CD) shows that between target sites in human cells increased over that seen under similar below LCST ELPs are soluble and consist of both random coils and tempera- conditions in vitro. These findings together reveal a novel role for both the ture dependent b-turn structures (Lyons et al., 2013; 2014), and Dox-labeling hUNG2 NTD as well as the crowded nuclear environment in facilitating further enhances b-turns formation. DLS measurements at 50-300 uM ELP DNA damage detection. reveal a significant increase in Rh below LCST from 5.96 þ/- .03 nm at 5Cto7.70þ/- .73 nm at 28-31C consistent with weak self-association. 418-Pos Board B188 The Rh values for Dox-ELP at 50-200 mM increased from 8.46 þ/- .37 nm DNA Synthesis Determines the Binding Mode of the Human Mitochondrial SSB Protein at 5 C to 13.51 þ/- .72 nm at 24-26 C. A plot of D20,W vs. concentration Fernando Cerron1, Jose Morin2, Javier Jarillo3, Elena Beltra´n-Heredia3, obtained from the diffusion measurements gives a negative Kd value o Grzegorz Ciesielski4, Francisco Cao3, Laurie S. Kaguni4, Borja Ibarra1. (D = D (1þKdC)) that correlates with self-association. Dox labeling further 1IMDEA Nanociencia, Madrid, Spain, 2Biotechnology Center (BIOTEC). decreases the Kd, which is evidence of stronger attractive intermolecular inter- Technical University Dresden., Dresden, Germany, 3Universidad actions and enhanced self-association. We propose that the phase change initi- 4 ated below LCST is a result of ELP self-association that is stabilized by Dox Complutense Madrid, Madrid, Spain, Michigan State University, Lansing, labeling. The correlation of b-turn formation and self-association suggests a MI, USA. mechanism where modular interactions initiate and stabilize LLPS. The Single-stranded DNA-binding proteins (SSBs) play a key role in genome decrease in the soluble state of the Dox-ELP further stabilizes the thermal tar- maintenance, binding and organizing single-stranded DNA (ssDNA) interme- geting to solid tumors. diates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentrations. It has long been suggested Posters: DNA Replication, Recombination, and that different binding modes might be used selectively for different Repair functions. To study this possibility, we used optical tweezers to determine and compare the structural and energetic properties of long, individual 416-Pos Board B186 HmtSSB-DNA complexes assembled on preformed ssDNA, and on ssDNA Quaternary Interactions and DNA Twist Modulate the Cooperative generated gradually during ‘in situ’ DNA synthesis. We show that HmtSSB Binding of AGT binds to preformed ssDNA in two major modes, depending on salt and pro- Michael G. Fried, Manana Melikishvili. tein concentrations. However, when protein binding was coupled to strand- Molecular and Cell Biochemistry, University of Kentucky, Lexington, KY, displacement DNA synthesis, only one of the two binding modes was USA. observed under all experimental conditions. Our results reveal a key role Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic for the gradual generation of ssDNA in modulating the binding mode of a O6-alkylguanine and O4-alkylthymine adducts in single-stranded and multimeric SSB protein and consequently, in generating the appropriate duplex DNAs. The search for these lesions, through a vast excess of nucleoprotein structure for DNA synthetic reactions required for genome competing, unmodified genomic DNA, is a mechanistic challenge that maintenance. may limit the repair rate in vivo. Here, we examine influences of DNA sec- ondary structure and twist on protein-protein interactions in cooperative 419-Pos Board B189 AGT complexes formed on lesion-free DNAs that model the unmodified A Novel DNA Repair Mechanism for the Processing of Low-Level UV- parts of the genome. We used a new approach to resolve nearest neighbor Induced Damage in Bacteria 1 2 3 3 (nn) and long-range (lr) components from the ensemble-average coopera- Luke Springall , Craig Hughes , Michelle Simons , Stavros Azinas , 4 1 tivity, u . We found that while nearest-neighbor contacts were signifi- Bennett Van Houten , Neil Kad . ave 1School of Bioscience, University of Kent, Canterbury, Kent, United cant, long-range interactions dominated cooperativity, and this pattern 2 held true whether the DNA was single-stranded or duplex. Experiments Kingdom, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom, 3School of Biological Sciences, University of with single plasmid topoisomers showed that the average cooperativity 4 was sensitive to DNA twist, and was strongest when the DNA was slightly Essex, Colchester, Essex, United Kingdom, UPMC Hillman Cancer Center, underwound. This suggests that AGT proteins are optimally juxtaposed University of Pittsburgh, Pittsburgh, PA, USA. when the DNA is near its torsionally-relaxed state. Most striking was the Nucleotide excision repair (NER) is the primary mechanism for removal of decline of binding stoichiometry with linking number. As stoichiometry ultraviolet light (UV)-induced DNA photoproducts and is mechanistically and affinity differences were not correlated, we interpret this as evidence conserved across all kingdoms of life. The first stages of bacterial that supercoiling occludes AGT binding sites. These features suggest NER involve damage recognition by UvrA2 and UvrB, followed by that AGT’s lesion-search distributes preferentially to sites containing UvrC-mediated incision either side of the lesion. Subsequently, the torsionally-relaxed DNA, in vivo. damaged oligonucleotide is displaced and the correct DNA resynthesized in its place. Here, using a combination of in vitro single molecule, and in 417-Pos Board B187 vivo imaging and growth studies we show that a UvrBC complex is capable Characterizing the Enhanced Nanoscale Translocation Properties of of lesion identification and processing in the absence of UvrA. Using single hUNG2 Facilitated by its Disordered N-terminal Domain In Vitro and in molecule fluorescence colocalization of protein complexes with fluores- Human Cells cently tagged damage on DNA tightropes, we observe that 46% (56%) Gaddiel Rodriguez, Alexandre Esadze, Brian P. Weiser, of UvrAB complexes preferentially bind to damage, and surprisingly Joseph D. Schonhoft, Philip A. Cole, James T. Stivers. UvrBC binds at statistically similar levels (52%55%). Removal of the Pharmacology and Molecular Sciences, Johns Hopkins University, damage sensing UvrB ß-hairpin reduces this preference in both complexes. Baltimore, MD, USA. Both UvrAB and UvrBC complexes have greater localization to damage

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than UvrA (29%53%) and UvrC (18%56%) alone. To confirm the rele- us to begin building a model of how UvrA2 ATPase activity is coupled to vance of this response in vivo we used fluorescence imaging in cells. lesion search, recognition and response. uvrA- complemented with UvrCþeGFP and UvrBþeGFP moved from diffusing in the cytoplasm to binding DNA after UV treatment. This reveals 422-Pos Board B192 UvrB and UvrC can be brought to damaged DNA in the absence of UvrA. Function of the Intrinsically Disordered N-Terminus of Uracil DNA Surprisingly, ectopic expression of UvrC in the uvrA- strain increased sur- Glycosylase vival at low UV levels. These data provide evidence for a previously unre- Brian P. Weiser, Gaddiel Rodriguez, Alexandre Esadze, Philip A. Cole, alized mechanism of DNA repair that involves direct lesion recognition and James T. Stivers. processing by a UvrBC complex. Pharmacology & Molecular Sciences, Johns Hopkins University, Baltimore, MD, USA. 420-Pos Board B190 Uracil DNA Glycosylase (UNG2) is the primary enzyme in humans that re- IPMK and PTEN Regulate Nuclear Phosphoinositide-Dependent ATR moves uracil bases from the genome. UNG2 contains a structured catalytic Signaling upon DNA Damage domain that is preceded by a 91 residue N-terminus that is intrinsically disor- Yu-Hsiu Wang1, Anushya Hariharan1, Giulia Bastianello2, dered. We continue our work here characterizing the function of the disor- Yusuke Toyama1, G.V. Shivashankar1, Marco Foiani2, Michael P. Sheetz1. dered N-terminus of UNG2 by comparing the properties of the full-length 1Mechanobiology Institute, National University of Singapore, Singapore, protein to a truncated catalytic domain construct that lacks the N-terminus. Singapore, 2IFOM, the FIRC Institute of Molecular Oncology, Milan, Italy. We show that, compared to the catalytic domain, the full-length protein has Phosphoinositide lipids (PPIs) are enriched in the nucleus and the role of an enhanced ability to translocate between uracil base substrates on the PPI signaling in nucleus has remained enigmatic for many years. Existing same DNA strand in vitro. We hypothesized that the enhanced translocation evidence suggest that the overall lipid profiles of nuclear PPIs changes of the full-length protein might be due to more extensive interactions of the following ionizing radiation. We further demonstrate that nuclear PPIs accu- protein with DNA, possibly mediated through its N-terminus. To test this, mulate at DNA damage sites shortly following global DNA damage induc- we developed a protocol to express and purify the disordered N-terminus of tion by UV or chemically. However, whether nuclear PPIs play a role in UNG2 (UNG2 residues 1-91) for binding studies. The isolated N-terminal mediating DNA damage repair is still an open question. Here, we investi- polypeptide UNG2(a.a.1-91) binds DNA with micromolar dissociation con- gate the potential roles of nuclear PPIs in mediating repair protein recruit- stants that are dependent on solution ionic strength. Finally, we present exper- ment by sequestering specific PPIs with the expression of nuclear-targeted iments that examine the activity of the full-length protein compared to the PPI-binding domains. The expression of PIP2- and PIP3-sequestering pleck- catalytic domain when uracil bases are incorporated into DNA substrates strin homology (PH) domains significantly suppresses recruitment of Ataxia that model the replication fork. We conclude from our studies that the long telangiectasia and Rad3-related protein (ATR) and ATR-interacting protein N-terminus of UNG2 affects the intrinsic ability of its catalytic domain to (ATRIP) to DNA damage sites, and thereby reduces Chk1 activation. Local identify and remove uracil bases from DNA, and that this is mediated through DNA damage by laser microirradiation shows that PPI-binding domains the direct interaction of the N-terminus with DNA. < rapidly ( 1s) accumulate at damage sites with local enrichment of PPIs 423-Pos Board B193 as confirmed by anti-PIP2 immunostaining. Accumulation of PIP2 in com- The 50 Nuclease Domain of DNA Polymerase I Mediates a Novel DNA plex with the nuclear receptor protein, SF1, at damage sites is further phos- Transfer Pathway during Proofreading phorylated by inositol polyphosphate multikinase (IPMK). The resulting Raymond Pauszek, Rajan Lamichhane, Arishma Rajkarnikar Singh, SF1-PIP3 complex is required for ATR-ATRIP recruitment and is down Edwin van der Schans, David Millar. regulated at the overexpression of phosphatase and tensin homologue The Scripps Research Institute, La Jolla, CA, USA. (PTEN). Suppressed recruitment and activation of ATR is confirmed with The high fidelity of many DNA polymerases is due, in part, to their ability to Latrunculin A and wortmannin treatment as well as IPMK or SF1 depletion. remove erroneously incorporated nucleotides at the terminus of the growing Other DNA repair pathways involving different PI3K-like kinases, ATM primer strand. This ‘‘proofreading’’ activity is catalyzed by the 30-50 exonu- and DNA-PKcs, are unaffected by PPI sequestration, suggesting specificity clease (exo) domain in E. coli DNA polymerase I (Pol I). Structural studies in nuclear PPI-mediated signaling. Together, these findings reveal that nu- have shown that this domain is spatially separated from the 50-30 polymerase clear PPI metabolism mediates an early damage response through the domain (pol), which is responsible for incorporation of dNTPs by template- IPMK-dependent pathway to specifically regulate ATR-ATRIP-pChk1 directed polymerization. Pol I also contains another spatially distinct domain, signaling axis. the 50 nuclease (nuc) domain, which is connected to the enzyme core by a flexible amino acid linker. This domain is involved in cleavage of down- 421-Pos Board B191 stream flaps formed during Okazaki fragment maturation and base excision Coordinated Actions of Four ATPase Sites on UvrA2 during Initiation of repair. Using a single-molecule FRET system, we resolved three distinct Nucleotide Excision Repair conformations of Pol I-DNA complexes. Experiments with Pol I mutants es- 1 2 2 1 Brandon C. Case , Silas Hartley , David Jeruzalmi , Manju M. Hingorani . tablished that these states correspond to DNA bound at the pol, exo, or nuc 1Molecular Biology and Biochemistry, Wesleyan University, Middletown, 2 domains. Notably, the DNA substrate can transfer among all three domains CT, USA, Chemistry and Biochemistry, City College of New York, New without dissociation from the enzyme. We determined rate constants for site York, NY, USA. switching of model DNA substrates containing a correctly paired primer ter- Nucleotide excision repair (NER) protects genomic DNA from a wide variety minus, a terminal mismatch or an internal mismatch. The presence of mis- of lesions, including nucleotide adducts and pyrimidine dimers. In bacteria, matches accelerated transfer of DNA from the pol domain to the exo NER is initiated by the UvrA2(B2) scanning complex when UvrA2 locates a domain, as expected during proofreading. Surprisingly, the mismatches lesion and UvrB binding confirms lesion recognition. Each monomer in the also accelerated transfer of DNA along the pol to nuc to exo domain UvrA2 dimer has two ATPase sites (proximal and distal), and previous studies pathway. These observations reveal the existence of a second intramolecular indicate that each site plays a distinct role in lesion detection. These studies, proofreading pathway, in which mismatched DNA first transfers from the pol however, have lacked the resolution needed to determine the temporal order domain to the nuc domain and then to the exo domain. Our results suggest and stoichiometry of ATP binding, hydrolysis and product release at all sites. an unexpected role for the 50 nuclease domain in the proofreading activity of Hence, there is uncertainty about how the four ATPase sites work together to Pol I. drive UvrA2 actions during lesion detection and initiation of NER. Our goal is to address this question by first determining the UvrA2 ATPase mechanism on 424-Pos Board B194 and off a lesion. To this end, we have initiated kinetic analysis of wild type B. Direct Observation of MUTYH and the Cancer-Associated Y150C Variant stearothermophilius UvrA2 and Walker A and Walker B ATPase mutants. Binding to OG: A Mismatches at the Single Molecule Level 1 2 2 2 Thus far, our results show that when UvrA2 is not bound to DNA, ATP bind- Shane R. Nelson , Andrea J. Lee , Scott D. Kathe , Thomas S. Hilzinger , ing to the proximal sites permits ATP hydrolysis at the distal sites; however, April M. Averill2, Susan S. Wallace2, David M. Warshaw1. ADP is released slowly leaving the latter predominantly ADP-bound. Contact 1Molecular Physiology & Biophysics, University of Vermont, Larner College with undamaged DNA accelerates the ATPase cycle at both the proximal and of Medicine, Burlington, VT, USA, 2Microbiology and Molecular Genetics, distal sites, but the latter still appear predominantly ADP-bound. Contact with University of Vermont, Larner College of Medicine, Burlington, VT, damaged DNA also accelerates the ATPase cycle, but ADP release from the USA. distal sites is stimulated such that ATP hydrolysis becomes the slowest step in Of the four DNA bases, guanine is the most likely to be damaged by reactive the reaction. Thus, when UvrA2 binds a lesion it switches from a predomi- oxygen species. Oxidized guanine (specifically, 8-oxoguanine) mispairs with nantly distal ADP-bound to ATP-bound state. These distinct responses allow adenine (instead of cytosine) during DNA replication (an OG:A mismatch),

BPJ 8562_8565 Sunday, February 18, 2018 83a ultimately resulting in genomic G -> T transversion mutations. MUTYH is a of the linear genome has profound consequences for the regulatory and cod- mammalian DNA repair glycosylase that locates and removes the adenine in ing capabilities for these regions. We report here comprehensive eccDNA these OG:A mismatches. Y150C is the mouse homolog of a human MUTYH profiles for C. elegans and in three human cell types, defining loci of variant that is associated with colorectal adenomas and carcinomas. Struc- eccDNA formation associated with both unique and repetitive regions. In- tural information indicates that tyrosine 150 is analogous to the critical vitro synthesis of eccDNA analogs, facilitated by biophysical models of ‘‘wedge residue’’ present in many other DNA glycosylases, which is essential DNA cyclization, are a key methodology for characterizing pathways of for lesion recognition. Here, we directly observe, at the single molecule level eccDNA dynamics. Such eccDNA mimics will help elucidate the mecha- in vitro, fluorescently tagged wildtype (WT) MUTYH and the Y150C variant nisms by which cells differentiate between endogenous and exogenous cir- while they scan suspended DNA ‘‘tightropes’’ comprised of concatenated, cular DNAs. linearized plasmids that contain specifically positioned OG:A mismatches every 2700 base pairs. In bulk assays, Y150C demonstrates reduced catalytic 427-Pos Board B197 activity. Although we find that both the WT and Y150C diffuse along undam- Circulomics: Ultrasensitive Characterization of Extrachromosomal Circu- aged DNA with the same diffusion constant (1x105nm2/s) as they scan for lar DNA (eccDNA) Distributions and Functions in Eukaryotes 1 2 1 damage, Y150C remains bound to and scans the undamaged DNA for a Massa Shoura , Stephen Levene , Andrew Fire . 1Pathology and Genetics, Stanford University School of Medicine, Stanford, much shorter period of time than WT (3.9s and 32.7s, respectively). Both 2 the WT and Y150C recognize and pause at OG:A mismatches, suggesting CA, USA, Bioengineering and Physics, The University of Texas at Dallas, that Y150C lesion recognition is not impaired. However, once recognized Richardson, TX, USA. Y150C pauses at a mismatch for only a short time (median 1.9s), while The eukaryotic genome is classically depicted as being organized into linear the WT enzyme remains bound at these mismatches for at least the duration chromosomes. However, a significant and an important fraction of the of our 5-minute recordings. Taken together, our data show that mutation of genome also exists in the form of circular molecules, known as. extrachro- the tyrosine ‘‘wedge residue’’ does not impair MUTYH’s ability recognize mosomal circular DNA (eccDNA). This molecular species confers a high and pause at OG:A mismatches, although the variant is unable to form a degree of plasticity to our genetic information and is also a manifestation long-lived complex. of genomic instability and alteration. The presence of eccDNA is a charac- teristic of almost all cancers and a variety of human diseases that are 425-Pos Board B195 associated with developmental defects and premature aging. Therefore, a Fluorescence Lifetime of NADH Reveals PARP-Dependent Increase of precise knowledge of the biogenesis and function of our circular genome Oxidative Phosphorylation Critical for Cell Survival is helpful in understanding genome dynamics and instability. Using rigorous Michael M. Murata1, Xiangduo Kong2, Kyoko Yokomori2, methods and analyses including a novel method for characterizing Michelle A. Digman1. eccDNAs, Circulome-seq, we have obtained human eccDNA profiles pre- 1Biomedical Engineering, University of California, Irvine, Irvine, CA, sent in both healthy and malignant cells. Our method reveals several new USA, 2Biological Chemistry, University of California, Irvine, Irvine, CA, classes of cell-specific eccDNAs originating from coding regions. Of USA. considerable interest is a small number of affected proteins with substantial The DNA damage response (DDR) pathway triggers a chain of reactions that roles in disease processes. Given the strong association between proteins are critical for the maintenance of cell survival and genome integrity. with a requirement for isoform diversity and eccDNA formation, we are Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that currently evaluating the roles for both eccDNAs and the resulting catabolizes nicotinamide adenine dinucleotide (NADþ) to form poly(ADP- somatically-modified chromosomes in both normal physiology and diseased ribose) on target proteins for initial chromatin organization and DNA repair states. This work presents an emerging understanding of how genome orga- pathway choice at damage sites. It has been reported that this depletion of nization, as a whole, interacts dynamically with cell fate and function. Our NADþ leads to a loss of a ATP and subsequent metabolic collapse ultimately study also highlights a novel juxtaposition of rigorous biophysical tools with leading to cellular death in DNA damaged cells. However, it is not clear Next Generation Sequencing (NGS) and provides a foundation for develop- whether or not regulating metabolism can overcome this PARP-dependent ment of a genome-wide understanding of eccDNA distribution, dynamics, cell death pathway as a cell survival mechanism. We are investigating if in- and mechanistic processes. hibition of oxidative phosphorylation increases cellular damage sensitivity and if that correlates with cell survival. This may be dependent on PARP ac- 428-Pos Board B198 tivity and the effect can last longer than the transient depletion of the total Massively Parallel Measurement of DNA Mismatch Repair Efficiency NADH level. Here we use the phasor approach to FLIM to measure the ratio In Vivo of free to bound reduced nicotinamide adenine dinucleotide (NADH) over Tunc Kayikcioglu, Chang-Ting Lin, Taekjip Ha. time reflecting the unique relationship between energy metabolism and Johns Hopkins School of Medicine, Baltimore, MD, USA. DNA repair. We used micro-irradiation to induce complex DNA damage Due to mutagens or errors in DNA synthesis, single base pair mismatches occur within a defined submicron region in the HeLa cell nucleus. Changes in frequently in vivo. To ensure accurate propagation of genetic information, cells NADþ were monitored using cells stably expressing a NADþ biosensor monitor and repair such DNA defects. The efficiency of this cellular response is localized to different subcellular regions. Our results show that NADþ/ known to depend on the type and the sequence context of the mismatch in bac- NADH depletion is transient and demonstrate a significant shift from free teria as well as in single- and multi-cellular eukaryotes. Although methods to to bound NADH in a damage dose- and PARP-dependent manner. Our results quantify repair propensities of individual mismatches exist, only a small subset show that the DNA damage signaling can enhance oxidative phosphorylation of possible mismatches have been examined due to the labor-intensive nature of energy metabolism to promote cell survival. This work was supported these assays. Here, we present a high-throughput approach that can quantify the by grants from NIH P41-GM103540, NSF MCB-1615701, and CRCC mismatch repair efficiency. CRR-17-426665. We tagged each plasmid with a random barcode sequence. As the descen- dants of each plasmid will inherit the same unique barcode, the replication 426-Pos Board B196 products of the ancestor plasmid can be traced. Into this barcoded library, Circulomics: The Structural Genomics of Endogenous and Exogenous we inserted a DNA library with 150 different mismatches and observed their Extrachromosomal Circular DNAs fate after transformation into Escherichia coli. If a mismatch gets repaired Stephen D. Levene1, Massa J. Shoura2, Andrew Z. Fire2. before the first replication, one of the strands is converted into the proper 1Dept. of Bioengineering, University of Texas at Dallas, Richardson, TX, complementary of the other strand. Subsequent replications of such ancestors USA, 2Dept. of Pathology, Stanford University School of Medicine, Stanford, yield a pure product. On the contrary, an ancestor plasmid evading repair CA, USA. gives rise to mixture of two products that differ at the position of the The mechanisms by which a cell distinguishes between endogenous and mismatch. Mismatch repair efficiency can hence be quantified by next gener- exogenous genomes has widespread implications for many biological ation sequencing of the DNA products. Repair efficiency was above 95% for processes, including gene expression. We have developed a multiphasic majority of mismatch types and contexts, which was greatly reduced in approach, combining biophysical, biochemical, and informatic methods, DmutS and DmutL strains, validating the approach. We also identified for characterizing endogenous populations of extrachromosomal circular some poorly repaired mismatches. Our assay is generally applicable to other DNA (eccDNA). Although identified in several organisms over the last thir- organisms and can reveal valuable information about the sequence- ty years, this population of chromosome-derived circular DNAs has not dependence of mismatch repair efficiency with implications in evolution of been well studied. Nevertheless, eccDNA formation from specific regions genome, codons and regulatory elements.

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429-Pos Board B199 This work provides evidence for a DNA recombination pathway where 5-hy- Physical Biology of Meiotic Chromosome Pairing in the Nematode Lineage droxymethylcytosine (5hmC) is a recombination marker that is recognized Baris Avsaroglu1,2, Kayla Baskevitch1,2, Abby Dernburg1,2. and cleaved by Endonuclease G (Endo G). We hypothesize that EndoG acts 1UC Berkeley, Berkeley, CA, USA, 2HHMI, Berkeley, CA, USA. as a resolvase to terminate Holliday junction migration by specifically cleaving Meiosis is a specialized cell division in which haploid gametes are generated 5hmC sites. EndoG is historically characterized as a non-specific DNA cutting from diploid precursors. Fundamental aspects of meiosis are widely conserved. protein that cleaves G/C rich sequences of DNA. EndoG has been implicated However, many features of the process have diversified in various species. For in a variety of cellular recombination functions, and was recently found to example, in the nematode, Caenorhabditis elegans early steps in meiotic recom- preferentially cleave 5hmC DNA in a sequence-specific context to promote bination, including the formation of programmed double-strand breaks by the recombination. 5hmC is an epigenetic marker that represents up to 1% percent enzyme Spo11, end resection, and strand invasion, are not required for homol- of cytosines in the mammalian genome, and it is found in all vertebrate organ- ogous chromosomes to pair and synapse, as they are in budding yeast, isms. In this study, we demonstrate that the Holliday junction, the 4-stranded mammals, and plants. The emergence of recombination-independent homo- DNA intermediate in homologous recombination, is a preferred substrate for log-pairing mechanisms in this organism has been accompanied by loss of EndoG. EndoG cuts 5hmC-modified Holliday junctions to produce unique the gene encoding DMC-1, the meiosis-specific paralog of the RAD-51 recom- cleavage products, suggesting 5hmC is a marker in EndoG mediated recombina- binase protein. Whole-genome sequence information has recently revealed that tion. Furthermore, we present the single-crystal structure of mouse EndoG and loss of DMC-1, as well as its cofactors HOP-2 and MND-1, has occurred inde- propose a mechanism for vertebrate EndoG recognition of 5hmC. An a-helix pendently in several roundworm lineages. In Pristionchus pacificus, a nematode seen at the DNA binding site of the homologous enzymes from Drosophila species that diverged from C. elegans around 200 MYA, genes encoding all and C. elegans has unraveled into a long structured loop, allowing the side three proteins are present. We have developed tools to investigate how meiotic chains of Ala109 and Cys110 to enter the binding site and potentially recognize mechanisms have changed during nematode evolution. Using immunofluores- the 5hmC modification. The unraveling of this helix is attributed to a two amino cence we have examined the dynamics of the recombinase RAD-51, the acid deletion near the binding site, which is conserved for all vertebrate EndoG meiotic chromosome axis protein HOP-1 and the synaptonemal complex sequences. Although EndoG is found in all eukaryotic species, we suggest component SYP-4 during meiotic progression. We find that HOP-1 staining EndoG has evolved to recognize 5hmC in vertebrate species. initially localizes along the full lengths of the chromosomes. Shortly after syn- apsis, both SYP-4 and HOP-1 become restricted to a limited segment along 432-Pos Board B202 each pair of chromosomes. Additionally, analysis of targeted deletion alleles Computational Analysis of DNA Homologous Recombination Pathway in a of Ppa-spo-11 and Ppa-dmc-1 has revealed that SPO-11, as well as DMC-1 Foldback Intercoil Structure are required for pairing and synapsis. In addition, we apply simple polymer Byung Ho Lee1, Soojin Jo1, Byung-Dong Kim2, Sung Ha Park1, Moon Ki Kim1. models with confinement and tethering to quantitatively address the physical 1 2 principles of chromosome pairing in P. pacificus. Our results indicate P. pacif- Sungkyunkwan University, Suwon, Republic of Korea, Seoul National icus shares some core meiotic mechanisms with other eukaryotes. University, Seoul, Republic of Korea. Four-stranded DNA structures including foldback intercoil (FBI) have been 430-Pos Board B200 studied not only to understand various life phenomena in vivo, but also to A Genome Edited Pig with the Hypertrophic Cardiomyopathy-Mutation design new DNA nanostructures. A stem-loop FBI structure is constructed R723G in the MYH7-gene by intertwining two DNA duplexes in the major groove. Each strand shares Judith Montag1, Bjo¨rn Petersen2, Anna Katharina Flo¨gel1, Edgar Becker1, the same helical axis and its diameter almost coincides with a conventional Andrea Lucas-Hahn2, Gregory J. Cost3, Christian Muhlfeld€ 4, Theresia Kraft1, B-DNA. Although DNA homologous recombination as well as deletion can Heiner Niemann2, Bernhard Brenner1. be successfully explained by the FBI structure, only few studies have been re- 1Molecular and Cell Physiology, Hannover Medical School, Hannover, ported unlike other four-stranded DNA structures such as DNA G-quadruplex Germany, 2FLI of Farm Animal Genetics, Neustadt, Germany, 3Sangamo structure and DNA supercoil structure. Therapeutics, Richmond, CA, USA, 4Functional and Applied Anatomy, In this work, we first constructed a 3D computer model of FBI structure and Hannover Medical School, Hannover, Germany. then carried out several computational analyses including MD and NMA in Familial Hypertrophic Cardiomyopathy (HCM) is the most common inherited order to investigate the DNA homologous recombination process. First of all, cardiac disease. About 30% of the patients are heterozygous for mutations in a 3D computer model of FBI structure was generated with homologous and the MYH7 gene. This gene encodes the ß-myosin heavy chain (MyHC), the mo- palindromic DNA base sequences. Its diameter and helicity are 22 A and tor protein of the sarcomeres in human heart. Hallmarks of HCM are cardio- 10.5 bp/turn, respectively. Validity of this 3D FBI model was confirmed by myocyte disarray and hypertrophy of the left ventricle, the symptoms range comparison of a variety of distances such as adjacent P-P distance, hydrogen from slight arrhythmias to sudden cardiac death or heart failure. To gain insight bond length, and adjacent base-base distance with those of B-DNA. When into the underlying mechanisms of the diseases’ etiology we aimed to generate homologous recombination occurs in the FBI structure, it is also required to genome edited pigs with an HCM-mutation.Designer nucleases have been secure enough space for base flipping of the intercoil structure. For this, the un- successfully employed for editing of the porcine genome. Here, we used winding FBI structure was also constructed with 26 A diameter and 13 bp/turn TALEN-mediated genome editing and successfully introduced the orthologous helicity. Consequently, this computational approach enables us to better under- HCM-point mutation R723G into the MYH7 gene of porcine fibroblasts. These stand that homologous recombination occurs through base flipping of the FBI cells were successfully used for somatic cell nuclear transfer based cloning of structure. domestic pigs that were heterozygous for the HCM-mutation R723G. No off- target effects were determined in the R723G-pigs. Surprisingly, the animals 433-Pos Board B203 died within 24 h post partem, probably due to heart failure as indicated by a Molecular Dynamics Simulation Study of DNA Mismatch Recognition by shift in the alpha/beta-MyHC ratio in the left ventricle and lung edema. Most Complementary Strand Interactions in Thymine DNA Glycosylase interestingly, the neonatal pigs displayed features of HCM, including mild Ozge Yoluk1, Alexander C. Drohat2, Alexander D. MacKerell Jr1. 1Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, myocyte disarray, malformed nuclei, and MYH7-overexpression. Mutated 2 mRNA and protein were expressed, albeit at very low levels. Force generation MD, USA, Biochemistry and Molecular Biology, University of Maryland, of cardiomyocytes was essentially unaffected. The finding of HCM-specific pa- Baltimore, Baltimore, MD, USA. thology in neonatal R723G-piglets suggests a very early onset of the disease Base excision repair (BER) is a conserved repair mechanism of the cell that re- and highlights the importance of novel large animal models for studying caus- stores the genetic material back to its original state. Defects in BER compo- ative mechanisms and long-term progression of human cardiac diseases. nents lead to elevated mutation rates, eventually pushing the cell to a cancerous state. Thymine DNA glycosylase is an essential component of 431-Pos Board B201 BER that corrects G$T mutagenic mismatches, while A$T pairs are not subject Vertebrate Endonuclease G Preferentially Cleaves Holliday Junctions and to base excision by TDG. How TDG achieves this specificity is not well under- Specifically Recognizes 5-Hydroxymethylcytosine stood. One hypothesis is that rejection of A$T pairs is due to unfavorable con- Crystal M. Vander Zanden1, Adam B. Robertson2, Shing P. Ho3. tacts with the complementary strand. In this study, we use molecular dynamics 1Chemical and Biological Engineering, University of New Mexico, (MD) simulations to elucidate the substrate recognition of TDG in the presence Albuquerque, NM, USA, 2Microbiology, Oslo University Hospital, Oslo, of several different complementary bases including guanine, adenine and ino- Norway, 3Biochemistry and Molecular Biology, Colorado State University, sine. MD simulations are performed on DNA alone and the enzyme-DNA com- Fort Collins, CO, USA. plex using the CHARMM36 additive and Drude polarizable force field, with

BPJ 8562_8565 Sunday, February 18, 2018 85a utilization of the latter motivated by recent studies indicating that the Drude probed conformational changes as arising from unwinding at the lesion site model captures the base flipping equilibria close to those obtained from exper- accompanied with nucleotide flipping. Taken together, these strongly point to imental studies. a conformational capture mechanism for Rad4, whereby lesion-containing DNA has the propensity to undergo spontaneous unwinding fluctuations to 434-Pos Board B204 adopt pre-distorted conformations that Rad4 recognizes. Homology Modeling and Structural Analysis of S. Cerevisiae Msh4 and Msh5 Provide Insight into DNA Binding and Specificity 436-Pos Board B206 Sudipta Lahiri, Ishita Mukerji. Single Molecule Experiments Reveal Molecular Level Details of Molecular Biology & Biochemistry, Wesleyan University, MIddletown, CT, MutS-MutL Interactions in DNA Mismatch Activated Sliding Clamp USA. Pengyu Hao1, Sharonda LeBlanc2, Dorothy Erie2, Keith Weninger1. The MutSg homologs, Msh4 and Msh5, play a significant role in meiotic 1Physics, North Carolina State University, Raleigh, NC, USA, 2Chemistry, recombination by assisting in the proper segregation of chromosomes through University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. binding to DNA Holliday Junctions (HJ). We have employed fluorescent Maintaining the integrity of DNA is crucial for a species to preserve genomic measurements with molecular dynamic simulations to generate a homology- information. Organisms have developed several DNA repair pathways to repair modeled MD-refined structure of the protein-junction interaction. We have different types of DNA lesion. Among these pathways, the DNA mismatch used this model coupled with our experimental results to examine Msh4- repair pathway identifies and repairs erroneous insertion or deletion of nucleo- Msh5 conformational dynamics in DNA binding and identify the features of tide bases during DNA replication. The first two proteins involved in this DNA the Msh4-Msh5-junction interaction that lead to specific recognition. Our mismatch repair process, MutS and MutL, are studied in this work using single studies show that Msh4-Msh5 preferentially binds HJ DNA relative to duplex molecule techniques including smFRET and tethered particle motion tracking. DNA and our model of the Msh4-Msh5-HJ interaction illustrates how Msh4- With smFRET we have characterized complex behaviors of MutS sliding Msh5 stabilizes the stacked form of the junction. Stabilization of this junction clamps on DNA containing a mismatch. We find that inclusion of MutL in conformation is generally refractory to branch migration, which is consistent the assay greatly alters the behavior of these MutS sliding clamps. With Teth- with a potential role for Msh4-Msh5 (MutSg) in trapping HJ until they are ered Particle Motion (TPM) experiments, we characterize the effects of MutS resolved by Mlh1-Mlh3 (MutLg). Our homology modeled MD-refined struc- and MutL on the global structure of DNA. The results constrain current models ture of Msh4-Msh5 also revealed a putative DNA-binding region, in which of the DNA mismatch repair phenomena. the Msh4 subunit interacts more directly with either junction or duplex DNA relative to the Msh5 subunit. The model also indicates that the protein complex 437-Pos Board B207 contains a second cavity, which could be important for binding other DNA High-Speed Magnetic Tweezers Studies of the E. coli Replisome substrates. Consequently, we have examined Msh4-Msh5 binding to six other Samuel M. Leachman, Nynke H. Dekker. recombination intermediates including DNA overhangs, forks, D-loop with Department of Bionanoscience, Technische Universiteit Delft, Delft, single end strand invasion, pre-HJ and open junctions. Although nanomolar Netherlands. binding affinities are observed, the junction is the preferred substrate of The replisome is the nucleoprotein assembly responsible for replicating DNA Msh4-Msh5. Steady-state ATPase assays in the presence and absence of these prior to cell division. In E. coli, it comprises roughly a dozen proteins, including different recombination intermediates elucidate the role of ATP hydrolysis in DNA polymerase III, DnaB helicase, the beta processivity clamp (b2), and the binding and specificity. The ability of the Msh4-Msh5 complex to specifically clamp loader complex. Through a complex hierarchy of dynamic interactions recognize these different recombination intermediates will be presented in of varying strength, the E. coli replisome can incorporate nearly one thousand terms of binding affinity, conformational dynamics, and utilization of ATP. nucleotides per second with extremely high fidelity. As such, it represents an ideal model molecular motor, not to mention a potential and heretofore unex- 435-Pos Board B205 ploited antibiotic target. In order to study the dynamic behavior of single active Visualizing Spontaneous DNA Dynamics and its Role in Mismatch Recog- replisomes, we employ a magnetic tweezers (MT) assay incorporating a high- nition by Damage Recognition Protein Rad4 speed (several kHz) camera. DNA incorporating a hairpin is tethered between a Sagnik Chakraborty1, Debamita Paul2, Saroj Baral1, Hong Mu3, microscope coverslip and a magnetic bead under applied force. Nucleotide Peter J. Steinbach4, Suse Broyde3, Jung-Hyun Min2, Anjum Ansari5. incorporation changes the bead height, allowing us to follow replication with 1 2 Physics, University of Illinois at Chicago, Chicago, IL, USA, Chemistry, higher spatiotemporal resolution than current single-molecule fluorescence ap- 3 University of Illinois at Chicago, Chicago, IL, USA, Biology, New York proaches allow. We have previously used this approach to measure rates and 4 University, New York, NY, USA, Center for Molecular Modeling (CMM) identify transient pausing in phage polymerases and present our recent results Center for Information Technology, National Institutes of Health, Bethesda, from the E. coli replisome here. MD, USA, 5Physics and Bioengineering, University of Illinois at Chicago, Chicago, IL, USA. 438-Pos Board B208 XPC protein recognizes diverse DNA lesions including ultraviolet- ATP-Dependent Topology Discrimination by Type IIA Topoisomerases: photolesions and carcinogen-DNA adducts, initiating nucleotide excision Implications for below Equilibrium Topology Simplification repair. Structural studies showed that Rad4 (yeast ortholog) bound specifically Yeonee Seol1, Tamara Litwin1, Lauren Kim1, Laudan Nikoobakht1, to DNA flips out damaged nucleotides away from the protein, indicating that it Neil Osheroff2, Keir C. Neuman1. relies on indirect readout for damage recognition. However, characterizing 1Laboratory of Molecular Biophysics, National Institutes of Health, intrinsic DNA deformability at damaged sites and how that impacts recognition Bethesda, MD, USA, 2Biochemistry and Medicine, Vanderbilt University, has been a significant challenge. Using fluorescence lifetime measurements on Nashville, TN, USA. DNA containing model lesions sandwiched by tCo and tCnitro cytosine-analog Type IIA topoisomerases (Topo IIA) are ubiquitous enzymes that play FRET pair exquisitely sensitive to local distortions,we mapped the conforma- essential roles in maintaining topological homeostasis and in unlinking tional heterogeneities of DNA with varying Rad4-binding specificities and chromosomes prior to cell division. Topo IIA enzymes, with the exception tracked their changes upon protein binding. We reveal a direct connection be- of DNA gyrase, regulate DNA topology by removing excess DNA super- tween intrinsic DNA distortions/deformability and Rad4 recognition: high- coils, and inter- and intra-molecular links, corresponding to catenanes and specificity CCC/CCC mismatch samples a strikingly broad range of conforma- knots respectively. All Topo IIA utilize ATP for their shared catalytic activ- tions even without Rad4, from B-DNA-like to a population that resembles ity in which the enzyme introduces a transient double-strand break in one distorted conformations observed with Rad4 bound; nonspecific TAT/TAT segment of DNA and transfers a second segment of DNA through this break. mismatch is largely homogeneous and B-DNA-like. Consistent with experi- The strand passage reaction is coordinated, and made irreversible, through ments, DNA conformational populations obtained from equilibrium MD simu- the hydrolysis of ATP. The hydrolysis energy is also coupled to below equi- lations show a single conformation for DNA containing TAT/TAT but at least librium topology simplification activity of Topo IIA that results in the two distinct conformations for those with CCC/CCC. The population in dis- reduction of supercoiling, linking, and knotting to below equilibrium levels. torted conformations measured in CCC/CCC constructs increase with temper- The mechanism of this below equilibrium relaxation remains speculative. In ature. We employed laser temperature jump perturbation to examine if we this study, we investigated the correlation between topology-dependent could measure the rates of these distortional dynamics in the high-specificity DNA binding and the level of DNA supercoil reduction for two Topo IIA CCC/CCC DNA. Our studies revealed relaxation kinetics in this construct enzymes that exhibit different levels of below equilibrium relaxation: E. even in the absence of Rad4. Notably, these relaxation rates remained essen- coli Topo IV and yeast Topo II. To determine the effect of ATP on the se- tially unchanged with Rad4 bound. Previously we had identified tCo-tCnitro- lection of DNA topology, we measured the topology-dependent binding in

BPJ 8562_8565 86a Sunday, February 18, 2018 the presence of two non-hydrolysable ATP analogues: ATPgSand measured enthalpy and differential binding of ions and water terms, which AMPPNP. Topo IV, but not yeast topo II, shows a weak topology- most likely corresponds to the anti-parallel stacked-X structure. Supported dependent binding in the absence of ATP analogs. Both enzymes exhibit by Grant MCB-1122029 from NSF. significantly enhanced DNA topology-dependent binding in the presence of ATPgS and AMPPNP. Simulations of Topo IIA activity that incorporate 441-Pos Board B211 the measured DNA topology dependent binding affinities indicate that DNA Unexpected Discontinuous Supercoiling of Torsionally Buckled DNA: topology dependent binding may be a contributing factor explaining non- Evidence for a Solenoid? equilibrium simplification by type IIA topoisomerases. Andrew Dittmore, Keir C. Neuman. Laboratory of Single Molecule Biophysics, NIH, Bethesda, MD, USA. 439-Pos Board B209 Overtwisted elastic rods are known to buckle into a coiled loop (a plectoneme) Skewing the Playing Field: A Single-Molecule Study on how RSS Sequence to relieve torsional stress. This buckling event is marked by a discontinuous Influences Gene Segment Selection drop in the extension of a single supercoiled DNA molecule, as observed in ex- Soichi Hirokawa1, Nathan M. Belliveau2, Geoffrey A. Lovely3, periments. Contrary to expectations and current models, we observe a subse- Michael Anaya2, David G. Schatz4, David Baltimore2, Rob Phillips1. quent cascade of highly regular discontinuous extension changes during 1Department of Applied Physics and Materials Science, California Institute plectoneme extrusion, indicating a series of kinetic barriers. We reconcile these of Technology, Pasadena, CA, USA, 2Division of Biology and Biological data within the context of elastic rod theory and present a self-consistent model Engineering, California Institute of Technology, Pasadena, CA, USA, in which the extended DNA adopts a solenoid structure. In this scenario, kinetic 3National Institute on Aging, National Institutes of Health, Baltimore, MD, barriers to plectoneme extension arise from boundary matching conditions be- USA, 4Department of Immunobiology, Yale University School of Medicine, tween the solenoid and the plectoneme. Although evidence for a solenoid has New Haven, CT, USA. not been previously reported for DNA, extended solenoids have been imaged in V(D)J recombination, the cut-and-paste process that combines various actin filaments which bend and form superstructures on a much larger charac- antibody-encoding gene segments, provides jawed vertebrates with a combi- teristic length scale. Our data and model provide a framework for further mea- natorically diverse arsenal of unique antibodies that allows the organism to surements and theories that capture the structures and mechanics of supercoiled identify virtually any invading bacterium or infected cell. To initiate this biopolymers. process, the RAG enzymatic complex binds two recognizable recombination signal sequences (RSSs) neighboring the gene segments and subsequently 442-Pos Board B212 cleaves the DNA to expose the segments before additional enzymes join Direct Measurement of Torque Induced Telomere Strand Invasion using the two ends to create a continuous antibody-encoding gene. While the gen- Magnetic Tweezers 1 2 3 4 eral process is well understood, the root causes for the non-uniform distri- Xi Long , Terren Chang , Shankar Shastry , Joeseph W. Parks , 5 bution of gene segment selection have not been clearly parsed out. In this Michael D. Stone . 1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, project, we examine how the sequence diversity of RSSs affect RAG’s 2 3 affinity for forming paired complexes and its propensity for cleaving the USA, University of California Santa Cruz, Santa Cruz, CA, USA, Center DNA, the two key steps for passing two gene segments onward to generate for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, CA, USA, 4Department of Chemistry and Biochemistry, University of an antibody-encoding gene. Using a single-molecule method known as 5 tethered particle motion (TPM), we systemically study a range of RSS Colorado Boulder, Boulder, CO, USA, Department of Chemistry and sequences, from single mutation deviations away from the consensus Biochemistry, University of California Santa Cruz, Santa Cruz, CA, USA. sequence to endogenous RSSs, to determine that RSS sequence is a signif- Telomeres are specialized structures that protect chromosome ends from icant determinant for whether a given gene segment in any antibody gene nucleolytic processing by DNA repair machinery. The foundation of human loci will be selected to produce an antibody. We expect that this work telomere structure consists of an arrayoftandemduplexDNAsequences will set us on an initial path to better understanding how the dynamic nature (TTAGGG) and terminates with a single-stranded 3’ G-rich tail. To protect the chromosome end, telomeres are thought to adopt a lariat structure of the genome influences the likelihood that a lymphocyte will produce a 1 particular antibody. known as a telomere-loop (T-loop) . T-loops are stabilized by DNA displacement loops (D-loops) generated by the invasion of the G-rich tail into an adjacent region of duplex telomere. Recent studies suggest that Posters: DNA Structure and Dynamics I telomere-associated proteins promote strand invasion through the applica- tion of torque to the DNA2. Although the mechanism of T-loop formation 440-Pos Board B210 has been described using biochemical approaches, the torque response Folding/Unfolding Thermodynamics of Three-Way and Four-Way Junc- and internal structural equilibrium of duplex telomeric DNA are not well tions characterized. To probe the mechanical properties of telomeric DNA we Luis A. Marky, Carolyn E. Carr. developed a magnetic tweezers assay that reports in real-time on the Pharmaceutical Sciences, University of Nebraska Medical center, Omaha, response of telomeric DNA molecules to precisely applied degrees of tor- NE, USA. que. Using a single molecule DNA topology-based assay, we directly There is considerable interest in the stability and overall physical properties of monitor the torque-dependent invasion of single stranded telomere primers nucleic acid three- and four-way junctions because of their significant role in into duplex telomeric DNA. Our results demonstrate that the stability of the both genetic recombination and the folding of the secondary/tertiary structures telomeric D-loops formed during strand invasion is highly dependent on the of RNA. We used a combination of spectroscopic and calorimetric techniques sequence of the displaced strand. Specifically, we show that invasion of the to obtain a thermodynamic description of the folding/unfolding of a DNA G-rich tail induces formation of a stable telomeric D-loop. The stability of three-way (TWJ) and a four-way junction (FWJ), and their appropriate control the telomeric D-loop structure is significantly knocked down in the pres- stem-loop motifs. The transition temperature of each oligonucleotide remains ence of Liþ. These results are consistent with the formation of G-quadru- constant over a ten-fold range of total strand concentrations, indicative of their plex (GQ) structure in the displaced strand of a telomeric D-loop, which intramolecular formation. TWJ unfolds biphasically at low salt and triphasi- serves to stabilize the invaded state. Our results provide insight into the cally at high salt, with total DHs of 118/119 kcal/mol and DG s of 12.6/17.5 physiological contexts in which GQ structures may transiently form, and kcal/mol, respectively. TWJ at low salt concentration has a defined structure help to explain the mechanism by which GQ-binding ligands can disrupt and retains the base pair stacking of all three stems; actually, the thermal sta- normal telomere homeostasis. bility of its dumbbell base structure is reduced to maintain an overall folded state. While at higher salt concentration, the simultaneous unfolding of the 443-Pos Board B213 three domains/stems is lost, resulting in the sequential unfolding of these stems. Single Molecule Measurement of DNA Folding by Protamines FWJ unfolds biphasically at low and high salt concentrations, with total DHsof Luka Matej Devenica, Bishop Grimm, Ashley R. Carter. 135/149 kcal/mol and DGs of 17.5/24.9 kcal/mol, respectively. These results Physics, Amherst College, Amherst, MA, USA. show an increase in the cooperative unfolding of its stems, which significantly In sperm nuclei, DNA interacts with the small protein protamine to form highly affects their thermal stability; thus, its control molecules cannot accurately compact structures through a currently unknown process. We report prelimi- describe its melting behavior at any salt concentration. Single value decompo- nary data using an in vitro, optical trapping assay to measure the folding dy- sition analysis suggests FWJ has two folded conformations at low salt while a namics of the DNA-protamine complex. In this assay, we attach individual single folded conformation is found at higher salt, and consistent with the DNA molecules to a cover slip on one end and to an optically trapped bead

BPJ 8562_8565 Sunday, February 18, 2018 87a on the other. Movement of the optical trap applies a force on the bead, stretch- YOYO dissociation. The kinetics results indicate that for DNA-YOYO associ- ing the DNA to a particular extension that is a function of the force applied to ation the second moiety intercalation event occurs rapidly after the first event, the bead. This force-extension profile contains information about the folding which is likely due to a minimal energy difference between the first and second process, namely the kinetics and thermodynamics of the intermediate states energy transition barriers. The results also suggest that the rate-limiting steps of folding. When protamine is added, it binds to the DNA and changes its for dissociation from the mono- and bis-intercalated states both involve signif- force-extension profile, allowing us to measure the effect of protamine binding icant length changes, resulting in the observed two-exponential time depen- on the folding pathway of DNA. dence for dissociation. 444-Pos Board B214 447-Pos Board B217 Isothermal Calorimetry Investigation of DNA Compaction Under Osmotic Strong Accumulation of DNA at a Heated Air-Water Interface Stress Jonathan Liu1, Matthias Morasch2, Dieter Braun2. Kurt Andresen, Amlan Chowdhury. 1UC Berkeley, Berkeley, CA, USA, 2Ludwig Maximilian University of Gettysburg College, Gettysburg, PA, USA. Munich, Munich, Germany. DNA condensation is a complex process that allows us to test our understand- Temperature gradients provide an energy source for many nonequilibrium ing of the electrostatics of highly-charged biomolecules. Numerous work has phenomena, from fluid convection to molecular thermophoresis. We investi- looked at the ion concentrations and structure of these condensates, but fewer gate microfluidic dynamics at an air-water interface that is subjected to a tem- measurements have directly interrogated the underlying energetics. Isothermal perature gradient. We show that DNA is trapped from bulk solution and calorimetry (ITC) allows one to directly extract the endothermic and accumulated near the contact line of the interface, reaching concentrations of exothermic components of a process and therefore learn about the underlying up to a 4000-fold increase. The accumulation happens rapidly - on the order free energy of the process. Moreover, by altering the ion concentrations, va- of minutes - and exhibits a temperature dependence. We rationalize the findings lences, and osmolytes in the solution, one can explore the electrostatic and with a simulation and conclude that the accumulation is the combined result of volumetric energy contributions independently. Herein, we present data look- capillary flow, thermophoresis, and continuous evaporation-condensation cy- ing at condensed DNA under various Na, Mg, and polyethylene glycol cles. At a scale of tens of microns, the above effects counteract the prominent (PEG) concentrations to try to elucidate these various free energy components Marangoni flow. The robust accumulation mechanism has broad implications, and use these data to inform our understanding of DNA condensation and DNA especially for studies on the origins of life. The findings complement previ- electrostatics more generally. ously discovered accumulation mechanisms from thermal convection and ther- mophoresis in bulk water. 445-Pos Board B215 1-Propanol Causes Reentrant Transition on DNA whereas 2-Propanol does 448-Pos Board B218 not: Experimental Verification through Single Molecular Observation Structure-Hydration Relationships in DNA Minor Groove Binding Yue Ma, Yuko Yoshikawa, Koichiro Sadakane, Kenichi Yoshikawa. Noa Erlitzki1, Abdelbasset A. Farahat2, Arvind Kumar1, David W. Boykin1, Doshisha University, Kyotanabe, Japan. Gregory M.K. Poon1,3. Nowadays, structural characterization of genomic DNA is one of the most 1Department of Chemistry, Georgia State University, Atlanta, GA, USA, important trends in medicine, biology and agricultural sciences. The indispens- 2Department of Pharmaceutical Organic Chemistry, Mansoura University, able procedure for the analysis of DNA in living cells is to isolate DNA mol- Mansoura, Egypt, 3Center for Diagnostics and Therapeutics, Georgia State ecules as precipitates from the crude mixture in rich variety of cellular University, Atlanta, GA, USA. components. According to the standard experimental protocol in molecular Regardless of type, compounds that bind the DNA minor groove of duplex biology and medicinal chemistry, usage of 2-propanol is recommended, seem- DNA follow a structural paradigm in which one- or two-ring aromatic ingly without any reasonable physic-chemical explanation why 2-propanol is groups are connected linearly, with or without cationic termini, in register desirable. In the present study, we have measured the change of the higher- with the DNA base-steps. Since molecular insertion involves the displace- order structure of genomic DNA molecules in the presence of alcohols by ment of water from the richly hydrated minor groove and the groove-facing use of single DNA observation with fluorescence microscopy, by focusing facet of the compound, changes in solute-solvent interactions are expected our attention to unveil the different effect between 1-propanol and 2-propanol. to make characteristically large contributions to the overall thermody- We found that, with 1-propanol, the long-axis length exhibits minimum at 60% namics of binding. From a molecular design perspective, hydration changes and then tends to increase with the increase of alcohol content. On the other are important for achieving high affinity and selectivity of the targeted hand, with 2-propanol the long-axis length exhibits almost monotonous motif over nonspecific sequences. We are therefore interested in the decrease with the increase of alcohol content. These results indicate that structure-hydration relationships of designed minor-groove binders, specif- DNA undergoes reentrant transition of coil-globule-coil with 1-propanol, ically low-MW heterocyclic cations which are emerging as useful transcrip- whereas such reentrance phenomenon does not appear with 2-propanol. As a tional inhibitors in vivo. As immediate objectives, we are probing the related phenomenon, we have recently reported that ethanol causes reentrant structure-hydration relationships of substitution at the cationic termini of transition on DNA accompanied by the increase of its concentration.[1] We designed compounds harboring a defined indole-phenyl-phenyl core. will discuss the mechanism of the reentrant transition in terms of the effect Hydration changes are measured via their linkage with counter-ion release, of none-sized clusters on the ethanol and 1-propanol solutions with high the physical bases of which are well established, in DNA binding titrations concentrations. as well as volumetric measurements by high-precision densimetry. The data [1] Y. Oda, et al., ‘‘Highly Concentrated Ethanol Solution Behaves as a Good show that substitution of a terminal amidinium (Amþ)withtetrahydropyr- Solvent for DNA as Revealed by Single-Molecule Observation’’, ChemPhy- idinium (THPþ) decreases the free energy of binding to an AT-rich hairpin sChem, 17, 471(2016). by 1.5 kJ/mol per substitution through the displacement of 10 additional waters. The levels of these hydration changes agree in sign but exceed in 446-Pos Board B216 magnitude in comparison with co-crystallographic structures, implying sig- The Binding Kinetics and Mechanical Properties of DNA-YOYO-1 nificant involvement of loose, thermodynamically bound water in solution. Complexes The latter feature is consistent with the relatively strong dynamics of termi- 1 2 Ali A. Almaqwashi , Mark C. Williams . nal cations observed in molecular dynamics simulations, and suggests sta- 1Physics Department, King Abdulaziz University, Rabigh, Saudi Arabia, 2 bilization of the cationic termini as an important concept in DNA minor Department of Physics, Northeastern University, Boston, MA, USA. groove binding. The bis-intercalator YOYO-1 is widely used for probing DNA. However, its binding properties, including the modulation of DNA mechanical properties 449-Pos Board B219 upon binding, are not fully explored. We utilized optical tweezers to obtain The Effect of Cation Size on DNA Thermal Stability both equilibrium and kinetic measurements of DNA-YOYO intercalation Earle Stellwagen1, Nancy C. Stellwagen2. over a range of ligand concentration and applied stretching forces. By fitting 1Department of Biochemistry, University of Iowa, Iowa City, IA, USA, equilibrium extensions of forces 3-50 pN to the wormlike chain model, the ob- 2University of Iowa, Iowa City, IA, USA. tained parameters show that the mechanical properties of DNA when saturated Capillary electrophoresis has been used to examine the effect of cation size on by YOYO are significantly modified. The binding kinetics of DNA-YOYO can DNA thermal stability, using hairpins with 6 base pair-stems and 4 nucleotide- be fitted with a single exponential rate for association, and by a double expo- loops as the reporter system. The midpoint melting temperatures observed in nential rate for dissociation. We also observed that force inhibits DNA- solutions containing the tetrabutylammonium ion (TBAþ) are consistently

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16C lower than observed in solutions containing the same concentration of quences that melt at similar temperatures. We illustrate this point using a Naþ, even though no preferential interaction with either cation is observed. thermodynamics-based, sequence-dependent model of overstretching. The The thermal melting temperatures decrease progressively with increasing model captures the tendency of AT-rich sequences to form local melting bub- cation size, due primarily to the effect of cation size on solution viscosity. bles upon overstretching, as observed by other authors, while also predicting We postulate that the viscosity of the solution affects the hairpin 4 coil equi- that DAP-T sequences overstretch by almost exclusively forming S-DNA, as librium because of through-solvent effects on hairpin renaturation, as observed experimentally observed for G-C DNA. in other studies. The mobilities of the various hairpins are independent of sequence in Naþ but depend on sequence in TBAþ solutions. In particular, 452-Pos Board B222 the observed mobilities increase with the increasing number of thymine bases Probing Changes in Ionic Atmosphere and Hydration Accompanying in the loop, possibly because of the partial ionization of thymine in TBAþ I-Motif Formation which would increase the effective net charge of the hairpin. Lutan Liu. Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, 450-Pos Board B220 University of Toronto, Toronto, ON, Canada. Elastic Properties of DNA as the Entropic Driving Force for Dehybridiza- G-quadruplexes and i-motifs are non-canonical secondary structures of DNA tion Transitions that play a number of regulatory roles in the genome with clear connection to Sebastian Sensale1, Hsueh-Chia Chang2, Zhangli Peng1. disease. Biophysical characterization of forces stabilizing these structures 1Department of Aerospace and Mechanical Engineering, University of Notre is required for understanding the molecular origins of the subtle balance of Dame, Notre Dame, IN, USA, 2Department of Chemical and Biomolecular interactions governing the conformational preferences of guanine- and Engineering, University of Notre Dame, Notre Dame, IN, USA. cytosine-rich DNA sequences and developing an ability of controlled induction Through the use of a coarse-grained lattice model informed from all-atomic of G-quaruplex and/or i-motif structures at selected genomic loci. Here, we simulations near equilibrium, we obtain a theoretical estimate of the perform pH-dependent acoustic and densimetric measurements and UV melting sequence-dependent slow melting rates (> nanoseconds) of DNA duplexes experiments at high pressures to assess changes in hydration and ionic atmo- with activation free energies accurate within 3kT. We show that the change sphere accompanying i-motif formation by the 50-TTACCCACCCTACC- in elastic properties of short DNAs is the key entropic driving force for DNA CACCCTCA-30 DNA oligonucleotide (ODN). We also conduct pH-dependent melting and hybridization. Using a vibration and torsional continuum model, acoustic and densimetric characterizations of the 50-CTCTCACCACACCA- we relate this entropy to the bending and torsional persistence lengths CACCTCTC-30 (ODN1) and 50-CACACTCCTCACCTCTCCACAC-30 captured from the phonon spectra in all-atomic simulations, near equilibrium, (ODN2) oligonucleotides which are compositionally identical to ODN but not by capturing both the momentum and configuration degrees of freedom capable of folding into i-motif. Our acoustic and densimetric results reveal virtu- simultaneously. A key to the theory is the cooperative melting of all but ally identical pH-dependent profiles of ODN, ODN1, and ODN2 suggesting that one base pair at the transition state. This hinge transition state allows us to i-motif formation by ODN is volumetrically silent (not accompanied by changes replace the hydrogen bond vibration modes with a set of out/of-phase in volume and compressibility). This conclusion is supported by high-pressure bending modes with a much higher vibration entropy (lower phonon fre- UV melting data which reveal no change in the thermal stability of the ODN quencies). A multidimensional version of Kramers transition state theory i-motif conformation at pH 5.0 as pressure increases from 1 to 1500 atm. The allows us to assign this change to an effective vibrational entropy term volumetric similarity of the i-motif and coil states of ODN implies a fortuitous that counters the enthalpic gain required to break the hydrogen bonds, lead- compensation between changes in the intrinsic and hydration contributions to ing to an elastic theory for the melting rate of short DNAs. As the hydrogen volume and compressibility. Analysis of the pH-dependent volumetric profiles bond enthalpies are thermodynamic quantities, we estimate them from the of ODN, ODN1, and ODN2, in conjunction with the data on volumetric changes classical nearest-neighbor theory. The vibration entropy of the hinge transi- associated with protonation of an isolated cytosine, suggests that protonation of tion state is strictly kinetic but, as it relates only to the persistence lengths, the cytosines in the oligonucleotides causes release of the majority or all of the we are able to capture it from near-equilibrium simulations of the duplex. counterions from their vicinity to the bulk. Thus, at acidic pH favoring i-motif This theory allows us both to make quantitative estimates of the melting rates formation, the oligomers cease to behave as polyelectrolytes. of short DNAs as well as to differentiate between unzipping and other reac- tion pathways that may be followed in the presence of secondary structures or 453-Pos Board B223 mismatches. It represents the first theory for DNA melting with quantitative Global Structural Deformations Observed through Optimization Calcula- accuracy. tions of Smoothly Bent and Mini-Kinked Closed DNA Robert T. Young, Wilma Olson. 451-Pos Board B221 Chemistry and Chemical Biology, Rutgers University, New Brunswick, NJ, Experimentally Motivated Sequence-Dependent Models of Melting and USA. Overstretching for Diaminopurine-Substituted DNA Computational modeling of DNA has aided in understanding how the double Daniel T. Kovari1, Matteo Cristofalo2, Roberta Corti2, Domenico Salerno2, helical structure can deform with and without the assistance of proteins. Valeria Cassina2, Yoojin Lee3, Geethika Malla1, Laura Finzi1, Such deformations allow for genetic and protein regulation as well as higher- Francesco Mantegazza2, David Dunlap1. order organization within the cell. A contributing factor to such deformability 1Physics, Emory University, Atlanta, GA, USA, 2University of Milano- lies in the primary nucleotide sequence of DNA as some nucleotides have Bicocca, Milan, Italy, 3University of Pittsburgh, Pittsburgh, PA, USA. different intrinsic characteristics and helical configurations based on the local Diaminopurine (DAP) is a nucleotide analog of adenine, bearing an addi- sequence context. For example, a pyrimidine-purine (YR) dinucleotide step tional amine group on the purine moiety. Thus, it forms three hydrogen bonds has a greater degree of deformation when compared to pyrimidine- with thymidine. Despite this difference from the usual A-T base pair, incor- pyrimidine (YY) or purine-purine (RR) steps. We explore this sequence depen- poration of DAP during PCR amplification yields no loss in sequence spec- dence through minimum-energy optimization calculations with various DNA ificity, and DAP-DNA is a viable substrate for many enzymes involved in conformational rest state and initial conditions that include smoothly-bent normal (A-T, G-C) DNA biochemistry. From a bio-engineering perspective, and mini-kinked closed structures. Optimization calculations were initially DAP substitution offers a way of increasing the melting stability of a DNA conducted on various 150-nucleotide sequences consisting of adenine and gua- molecule while preserving its sequence functionality. For canonical DNA, nine with multiple initial states and a minimum of two force fields, one based sequence melting temperature depends not only on the number of hydrogen on ideal B-DNA conditions and another with sequence dependency. From these bonds formed between the two polynucleotide strands but also on sequence- initial optimization results, variant sequences were generated by replacing spe- specific effects due to base-stacking interactions. These can be accounted for cific purine nucleotides by pyrimidines to examine pyrimidine-purine and by SantaLucia’s nearest-neighbor model. Using parameters derived from purine-pyrimidine dinucleotide steps on global deformation. One of the initial melting experiments with DAP DNA, we extend the SantaLucia Model to sequence optimization calculations resulted in closed structures that contained predict sequence-specific DAP-DNA melting temperatures. Residing in the multiple kinked regions. Although bending such as this is seen in DNA contain- minor-groove of the DNA double-helix, DAP’s additional amine also affects ing poly-A tracts, this sequence contains multiple shortened adenine patches the molecules mechanical properties including its torsional and flexural rigid- that outnumber the number of kinked regions. For this sequence, variants ity. Nanomechanical assays on single molecules reveal that DAP substitution were generated with a variety of asymmetric purine to pyrimidine replacements increases the molecule’s persistence length between 25-46%. Nonetheless, to observe any possible deviations from the original optimized structure. These DAP does not alter the overstretching behavior of rotationally un- studies provide a step forward in understanding the effect of sequence on constrained molecules beyond what would be expected for poly G-C se- naturally-occurring circular DNA structures of various lengths.

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454-Pos Board B224 457-Pos Board B227 Improved Sampling in Molecular Dynamic Studies of Z[WC]-DNA and the A Coarse-Grained Simulation Study of the Effect of Salt Concentration on B to Z-DNA Transition DNA Internal Motions Sirajus Salekin, Micaela Bush, Alma Gracic, Ahmed Imamovic, Benson Ma, Edmond Chow. Ahsan A. Khoja, Jinhee Kim, Lam T. Nguyen, Sunil Pun, Ashutosh Rai, Georgia Institute of Technology, Atlanta, GA, USA. Sirajus Salekin, Alexander K. Seewald, Benjamin L. Yee, Michael G. Lerner. Earlier studies by Chow and Skolnick suggest that the internal motions of Physics and Astronomy, Earlham College, Richmond, IN, USA. bacterial DNA appear to be governed by strong forces arising from being Although DNA is most commonly found in the right-handed B-DNA struc- crowded into the small spacing of the nucleoid. However, the effect of ture, it is known that biologically active systems also contain left-handed ion concentration on the internal motions of densely packed DNA is not Z-DNA. ZII-DNA is the most common form among left-handed structures. well understood. For this study, we built and used a program called However, little is known about the transformation from right- to left- ‘BDT’ for Brownian dynamics (BD) simulations of DNA using the free- handedness. These DNA structures are too small to view physically. Hence, draining approximation. While it performs BD simulations with steric, using molecular dynamics we investigate the B to ZII transition with a pos- stretch, bend, and Debye-Huckel forces, BDT also introduces routines sibility of Z[WC]-DNA serving as an intermediate structure. Molecular sim- that have been optimized for DNA chain calculations. A fractal DNA chain ulations indicate that Z[WC] structures are stable with the current AMBER was prepared using a custom program, which arranged DNA beads along a nucleic acid force field. Along with targeted molecular dynamics we use um- Hilbert space-filling curve where straight segments were at least twice the brella sampling to produce potentials of mean force for the B to ZII transition persistence length of DNA. The simulation box was slowly reduced in vol- along both pathways, as well as a new method to increase sampling on ume until the in vivo DNA volume fraction of 13% was reached. BD sim- modestly-sized computational resources. ulations using this initial configuration were carried out at ion concentrations from 0.0001M to 0.5M, and a separate simulation was per- 455-Pos Board B225 formed in the absence of Debye-Huckel forces. Diffusion constants and Determining the Effects of Methylation on the Flexibility of CGG/CCG DNA internal motions were studied over increasing ion concentrations. Repeat DNA The results suggest that the diffusion activity of DNA increases to a limit Michaela Norbury, Catherine Volle. with increases in ion concentration. Furthermore, the diffusion values ob- Biology, Cottey College, Nevada, MO, USA. tained from a simulation of DNA near cellular conditions of 0.1M appear The human genome contains several regions composed of three repeating nu- to be close to those obtained from simulations in which Debye-Huckel in- cleotides called trinucleotide repeats (TNRs). Increasing the number of TNRs teractions were absent. This implies that the computation of Debye-Huckel in a genomic region has been linked to a number of neurodegenerative dis- interactions may actually be unnecessary in BD simulations of DNA motion eases, including Fragile X Syndrome, which is caused by the expansion of under in vivo conditions, where other forces dominate DNA motion, and CGG/CCG repeats in the Fragile X Mental Retardation 1 (FMR1) gene. Het- that BD simulations of similar conditions can benefit in computational ef- erochromatin formation silences the FMR1 gene, and the loss of the FMR1 ficiency from the removal of this calculation with minimal loss of simula- gene product leads to disease manifestation. Although these repeats create tion fidelity. rigid strands of DNA, the DNA is repeatedly bent into heterochromatin. However, if the stiffness of the repeat DNA is a barrier to heterochromatin 458-Pos Board B228 formation, what then causes the rigid strand of CGG/CCG repeat DNA to Quantifying Nucleic Acid Base Pairing Free Energy be packaged? Before the FMR1 gene is silenced, the repeat region is Rongpeng Li1, Chi H. Mak2. methylated. However, it is not known how these methyl groups affect stiff- 1University of Southern California, Los Angeles, CA, USA, 2Chemistry, ness of the repeat DNA. Thus, by performing DNA ligation experiments University of Southern California, Los Angeles, CA, USA. with unmethylated and methylated repeats, we can elucidate the effect of The free energy of base pairing and base stacking makes an important methylation on the flexibility of CGG/CCG repeats and their ability to contribution to the stability of double-stranded DNA. Different models form heterochromatin. have been proposed to attribute this free energy to their various sources. Pairing interactions are of special interest because of their importance in 456-Pos Board B226 controlling DNA complementarity. The entropy cost of forming hydrogen DNA Hybridization: Concentration-Dependent Changes in Binding Affin- bonds between base pairs is offset by the concomitant release of water ity Reveal Intrinsic Change in Hydration Energy molecules originally hydrogen-bonded to the individual bases and this Caroline Harmon, Juan Rangel, Christopher Trinh, Daryl K. Eggers. has been suggested as an important determinant of base pairing stability, Chemistry, San Jose State University, San Jose, CA, USA. but so far these entropic factors not been carefully quantified. Here we Hydration layers next to a solute have properties uniquely different from wa- study a pair of nucleobases (A:T or G:C) in an aqueous solvent with up ter in the bulk phase, remote from any surface. In the case of biopolymers, to 5,000 TIP3P water molecules using Monte Carlo simulations and the water of hydration is understood to be a major determinant of structure umbrella sampling with full electrostatics and Ewald summation. and function, yet the thermodynamic implications of hydration on binding The free energy of an A:T or a G:C pair is calculated to be stable by and conformational equilibria have been addressed poorly. Our laboratory 7 to 8 kcal/mol. Calculating the entropic contributions separately, we proposes to append the classical Gibbs-free energy equation for binding re- H2O find that the majority of this stability is due to the release of water as actions with a term for the change in hydration energy (DG ), weighted hydrogen bonds between the bases are made. Using these results, we are by the concentration of complex formed. To test our equation on DNA able to quantify for the first time how solvent entropy dominates DNA duplex formation, we studied short complementary oligonucleotides using base-pairing complementarity. two different methods, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST). Using both techniques to generate a binding curve 459-Pos Board B229 and obtain the corresponding equilibrium ratio, we found that the DNA:DNA Binding Kinetics of DNA Intercalation by Small Rhodium Complexes association ‘‘constant’’ decreased as the concentration of duplex increased. Guðfrı´ður Bjo¨rg Mo¨ller1, Liam Price1, Grace Ferris2, Micah J. McCauley1, The results were fitted to our working equation to estimate the change in hy- Ioulia Rouzina3, Megan Nu´n˜ez4, Mark C. Williams1. dration energy for each binding pair. From this analysis, we conclude that the 1Dept. of Physics, Northeastern University, Boston, MA, USA, 2Dept. of change in hydration upon duplex formation is thermodynamically unfavor- Natural Sciences and Mathematics, Lesley University, Cambridge, MA, able and large in magnitude. The large and positive value of DGH2O for USA, 3Dept. of Chemistry and Biochemistry, Center for Retroviral DNA:DNA binding may be attributed to (a) the large surface area of the Research, and Center for RNA Biology, The Ohio State University, DNA binding interface relative to other small-molecule binding models, Columbus, OH, USA, 4Dept. of Chemistry, Wellesley College, Wellesley, and (b) recent reports of a stable spine of water in the minor groove of the MA, USA. DNA double helix which should impart a large and unfavorable change in We characterize the kinetics and DNA binding properties of the small 3þ the entropy of hydration upon duplex formation. This work makes a funda- rhodium based molecules, [Rh(bpy)2(chrysi)] (chrysi) and [Rh(bpy)2 mental contribution to the application of thermodynamics in aqueous solution (phi)]3þ (phi). Both motifs bind to DNA. Phi contains an aromatic ring system chemistry and, consequently, may have broad implications for biomedical known to intercalate between adjacent bases in a fairly sequence-neutral research. Acknowledgement: This project was funded by a NIH R15 award manner. Intercalation is weaker for chrysi due to the presence of an additional to DKE, #GM110654. aromatic ring. Instead, chrysi has been shown to bind to DNA mismatches

BPJ 8562_8565 90a Sunday, February 18, 2018 preferentially, ejecting and replacing the bases along the helical stack. We demonstrate that we are able to control the reaction conditions inside the used optical tweezers on single DNA hairpin and long DNA molecules. nanofluidic channels, and thereby study complex biomolecular processes Hairpin unfolding experiments on mismatch-containing sequences show in vitro. that chrysi binds preferentially to sequences containing mismatches, stabiliz- ing the hairpin. Long double stranded DNA was stretched at a constant external force in the presence of the ligand and a change in DNA extension 461-Pos Board B231 was observed to be driven directly by ligand intercalation. Under the influ- Interactions between the Bacteriophage Protein Cox and DNA ence of increasing force, intercalation of the phi ligand into matched DNA Investigated on the Single DNA Molecule Level using Nanofluidic is observed as expected. Surprisingly this intercalation into matched DNA Channels was also observed for the chrysi ligand. Further analysis revealed the disso- Karolin Frykholm1, Ronnie P-A Berntsson2,Pa˚l Stenmark3, ciation constants (Kd) of the chrysi and phi complexes to be on the order of 1 Fredrik Westerlund1. mM, though phi showed a significantly stronger intercalating affinity than 1Chalmers University of Technology, Gothenburg, Sweden, 2Umea˚ chrysi. University, Umea˚, Sweden, 3Stockholm University, Stockholm, Sweden. The protein Cox plays several important roles in the life-cycle of P2-like 459.1-Pos Board B229.1 bacteriophages. It is involved in excision of the prophage as well as tran- Hyperstretching DNA scriptional repression and activation of different genes. The crystal struc- ture of Cox revealed that the protein forms helical oligomers with its Koen Schakenraad1, Andreas S. Biebricher2, Maarten Sebregts3, DNA-binding domains located on the outside of the filament. This sug- Brian ten Bensel2, Erwin J.G. Peterman2, Gijs J.L. Wuite2, Cornelis Storm3, gested that DNA might be bound around the helical filament, similar to Paul van der Schoot3, Iddo Heller2. 1 2 how DNA is wrapped around histones in eukaryotic cells.To investigate Instituut-Lorentz, Universiteit Leiden, Netherlands, Department of Physics and Astronomy, VU University Amsterdam, Amsterdam, Netherlands, this hypothesis, we studied the interaction of two homologs of Cox, from F 3Department of Applied Physics, Eindhoven University of Technology, phages P2 and W , with single DNA molecules, using nanofluidic chan- Eindhoven, Netherlands. nels. By measuring the extension of DNA when Cox was present in the The three-dimensional structure of DNA is highly susceptible to changes by sample we could confirm the proposed binding model. The extension of mechanical and biochemical cues in vivo and in vitro. In particular, large in- DNA decreases significantly when the protein is added, in agreement the DNA being wrapped around the protein filament. The data also suggests creases in base pair spacing compared to regular B-DNA are effected by me- chanical (over)stretching as well as by intercalation of compounds that are a large cooperativity in the binding, in agreement with the protein binding widely used in biophysical/chemical assays and drug treatments. We present to DNA as a filament. This was further confirmed by investigating a Cox single-molecule experiments and a three-state statistical mechanical model mutant with no filament formation capability. By measuring the extension that provide a quantitative understanding of the interplay between B-DNA, of the complex at two different confinements, we could confirm that the overstretched DNA and intercalated DNA. The predictions of this model protein-bound DNA is much stiffer than DNA without protein, again in agreement with the proposed formation of a protein filament around which include a hitherto unconfirmed hyperstretched state, twice the length of DNA is wrapped.In addition to revealing important insight into the DNA B-DNA. Our force-fluorescence experiments confirm this hyperstretched state and furthermore reveal that its free energy depends on DNA sequence. These binding of Cox the project also highlights the use of nanofluidic channels results pin down the physical principles that govern DNA mechanics under the for investigating DNA-protein interactions. The studies are relatively high influence of tension and biochemical reactions. A predictive understanding of throughput and the DNA used does not have to be tethered to any bead the possibilities and limitations of DNA extension can guide refined exploita- or surface for analysis. tion of DNA in, e.g., programmable soft materials and DNA origami applica- tions. 462-Pos Board B232 Interactions between DNA and HIV-1 Nucleocapsid Protein Studied using Posters: Protein-Nucleic Acid Interactions I Nanofluidic Channels Kai Jiang1, Nicolas Humbert2, Sriram Kesarimangalam 460-Pos Board B230 Kalyanavenkatramanan1, Yves Mely2, Fredrik Westerlund1. A Nanofluidic Device for Real-Time Visualization of DNA-Protein Interac- 1Biology and Biological Engineering, Chalmers University of Technology, tions on the Single DNA Molecule Level Gotheburg, Sweden, 2Faculty of Chemistry/Pharmacy, University of Robin O¨ z, Sriram Kesarimangalam Kalyanavenkatramanan, Strasbourg, Strasbourg Cedex, France. Fredrik Westerlund. HIV is a devastating disease caused by a retrovirus. A retrovirus incorpo- Biology and Biological Engineering, Chalmers University of Technology, rates its RNA into the genome of the host cell and the RNA is then reversely Gothenburg, Sweden. transcribedintoDNA.TheDNAisinturntranslatedintoproteinsthatare Single-molecule methods have become crucial biophysical tools for inves- crucial for new virus particles to form. The nucleocapsid (NC) protein of tigating DNA-protein interactions in vitro. A general drawback with most HIV-1 is a nucleic acid chaperone that plays an important role in the retro- single DNA molecule techniques is that the DNA must be tethered in at viral life cycle, in part, by facilitating numerous nucleic acid rearrangements least one of its end. This means, for example that reactions involving two throughout the reverse transcription process. However, the structural basis DNA ends are tricky to study. Nanofluidic tools have frequently been for this activity is not completely understood. NC is a small protein used in static DNA studies, where the moleculeisstretchedtoanextension composed of two zinc-finger domains and mutants can be designed and close to its contour length without any tethering. However, studying dy- tested at will. namic reactions in real-time is a large challenge since it is not possible In this study, we investigate the conformational changes of single DNA to actively control the environment within the nanofluidic channel, without molecules, confined to nanofluidic channels, caused by the NC protein holding on to the molecule. We have developed a nanofluidic device that and its mutants. Two main observations are made. Firstly, NC induces can be used to study dynamic processes on single DNA molecules in the formation of DNA concatemers when a DNA with a 12 base-pair sin- real-time. This novel design enables active control of the local environment gle-strand overhang is used. This activity seems to be related to the first in the nanofluidic channel, while keeping the DNA molecule confined. Us- 10 amino acids of the protein and correlates with the function of the protein ing this device, we are able to add analytes, such as DNA-binding proteins, in annealing RNA to its substrate in vivo. Secondly, the protein compacts on demand and simultaneously study the response of the DNA molecule by the DNA into a condensed form. The protein concentration at which fluorescence imaging. We are thus able to map the interplay between bio- compaction occurs is similar for all tested mutants. molecules in order to reveal the unknown details of different biological pro- In addition to revealing information about the interaction between NC and cesses. As a proof of concept, DNase I was successfully added to DNA, our results also demonstrate how nanofluidic channels are perfectly nanoconfined DNA stained with YOYO-1 in order to induce digestion on suited for studying interactions occurring at DNA ends. While most single demand. To further show the functionality of the nanofluidic device, we DNA molecule methods rely on tethering DNA ends to beads or surfaces, have studied real-time interactions between DNA and proteins known to DNA without tethers, and hence annealing of two ends can be readily observed change the physical properties of DNA upon binding. The results clearly in nanofluidic devices.

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463-Pos Board B233 location kinetics of the helicase Hel308 along single-stranded DNA depend Design of Novel Magnetic Tweezers and Its Use for Studying on base-specific interactions between the DNA sequence and amino acids DNA-Compacting Proteins in Hel308. Combining our single-molecule data with structural data of Roberto Fabian, Jr.1,2, Christopher Tyson3,4, Anneliese Striz5, Hel308, we propose a mechanism for sequence-specific translocation of Pamela L. Tuma6, Ian L. Pegg7,8, Abhijit Sarkar3,8. Hel308 on ssDNA. 1Catholic University of America, Washington, DC, USA, 2Vitreous State Laboratory, The Catholic University of America, Washington, DC, USA, 466-Pos Board B236 3Vitreous State Laboratory ,Catholic University of America, Washington, 4 High-Resolution Single-Molecule Analysis of UvrD Helicase using Nano- DC, USA, Biomedical Engineering Department, The Catholic University of pore Tweezers America, Washington, DC, USA, 5Biology Department, Catholic University 6 Hugh Higinbotham. of America, Washington, DC, USA, Biology Department,Catholic University of Washington, Seattle, WA, USA. University of America, Washington, DC, USA, 7Vitreous State 8 Single-molecule methods reveal enzyme dynamics that cannot be observed Laboratory,Catholic University of America, Washington, DC, USA, Physics with traditional bulk assays. We analyze UvrD helicase translocation on Department, Catholic University of America, Washington, DC, USA. ssDNA using a new technique called Single-molecule Picometer Resolution We report the development of a horizontal magnetic tweezers capable of Nanopore Tweezers (SPRNT), which has unprecedented spatiotemporal applying forces in the 0.1 - 100 piconewton (pN) range on single DNA mol- resolution. In SPRNT, a membrane porin MspA in a phospholipid bilayer forms ecules. The two ends of the DNA molecule are attached to 2.8 um paramag- an electrical connection between two salt solutions. A voltage applied across netic beads with one bead immobilized on a rigid glass surface while the the pore causes ion current to flow through and draws negatively charged other bead is suspended near a small bar magnet. The magnet is moved to- DNA bound to UvrD into the pore. UvrD is too large to fit through MspA, wards the tethered DNA at a speed between 0.25 - 10 um/s and can generate and comes to rest on the rim, arresting the DNA’s motion. Different DNA nu- forces greater than 70 pN on the DNA molecules. These forces are pro- cleotides in the pore modulate the ion current. As UvrD walks along the DNA, duced in the focal plane of the microscope objective, permitting direct < it draws the DNA out of the pore and changes the ion current. Therefore, the DNA extension measurements with a precision of 10 nm. We calibrate durations of ion-current states are kinetic measurements of UvrD. Using the tweezers using the DNA overstretching transition, and study hysteretic SPRNT, we show that UvrD moves in single nucleotide steps along DNA effects in extension-compaction cycles. We describe the tweezers in detail and characterize its ATPase activity. and present data validating its performance. We conclude with a discussion of our ongoing single experiments on the binding mechanism of the protein mIHF, a protein that may play an important role in the infection pathway of 467-Pos Board B237 tuberculosis. Analysis of Force Dependence of Translocation and Unwinding of Helicase PCRA using SPRNT Andrew H. Laszlo1, Jonathan M. Craig1, Henry Brinkerhoff1, Ian C. Nova1, 464-Pos Board B234 Matthew T. Noakes1, Jonathan W. Mount1, Jasmine O. Bowman1, Lateral Magnetic Tweezers to Study DNA: Protein Interactions Hugh Higinbotham1, Katherine Baker1, Jesse Huang1, Ramreddy Tippana2, Julene Madariaga-Marcos1, Silvia Hormen˜o1, Cesar L. Pastrana1, Momcilo Gavrilov2, Taekjip Ha2, Jens H. Gundlach1. Gemma L.M. Fisher2, Mark S. Dillingham2, Fernando Moreno-Herrero1. 1Physics, University of Washington, Seattle, WA, USA, 2Biophysics, Johns 1 Department of Macromolecular Structures, Centro Nacional Hopkins, Baltimore, MD, USA. 2 de Biotecnologı´a, Madrid, Spain, DNA:Protein Interactions Unit. Using Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), School of Biochemistry, University of Bristol, Bristol, we analyzed the kinetics of a mutant PcrA ‘superhelicase’ under varying assist- United Kingdom. ing and opposing forces as it tranlsocated along ssDNA and unwound dsDNA. The combination of single-molecule techniques with fluorescence micro- The superhelicase, dubbed ‘PcrA-X,’ was created by constraining the flexible scopy has attracted much interest because it permits correlating mechanical 2B domain into the ‘closed’ conformation by chemical crosslinking. With measurements with directly visualized DNA:protein interactions. Specif- SPRNT we are able to resolve the helicase’s stepping dynamics on millisecond ically, the combination of magnetic tweezers (MT) with total internal reflec- timescales at sub-nucleotide spatial resolution. We find that the kinetics of both tion fluorescence microscopy (TIRF) is advantageous because one can PcrA-X translocation on ssDNA and unwinding of dsDNA are force- follow many DNA molecules taking advantage of the high signal-to-noise dependent. By comparing ssDNA translocation to dsDNA unwinding, we ratio this fluorescence technique achieves. However, the stretching of long examine how DNA unwinding relates to the helicase’s stepping motion along DNA molecules across the surface of the flow cell is required to maximize its DNA track. polymer exposure to the excitation light. In the present work, we develop a laterally magnetic pulling module, which can be easily implemented in con- 468-Pos Board B238 ventional or combined magnetic tweezers (MT)-TIRF setups, to stretch Regulation of a Viral Packaging Motor’s Grips on DNA DNA molecules at a constant force. We have further characterized our pull- Mariam Ordyan1, Douglas E. Smith1, Venigalla B. Rao2, Istiaq Alam2, ing module in standard flow cells of different thicknesses and square glass Marthandan Mahalingam2. capillaries, using 1 and 2.8 mm superparamagnetic beads, and a home-built 1Physics, UCSD, San Diego, CA, USA, 2Biology, CUA, Washington, DC, device to rotate capillaries with mrad precision. We measured force- USA. extension curves in 24.5 kbp long DNA molecules, which are consistent ATP-powered viral DNA packaging motors are among the most powerful bio- with the behavior predicted by the worm-like-chain model, and allow us motors known. Here, we quantify how the nucleotide binding state regulates the to provide a range of forces achieved by the magnetic pulling module be- motor’s grip on DNA via optical tweezers measurements with rapid solution tween 0.1 and 30 pN. A formalism for estimating forces in flow-stretched exchange. In the apo state (with no nucleotide) there is almost no detectable tethered beads is also proposed and the results compared with those of grip. With low applied force the DNA usually slips at 2000 bp/s. In contrast, lateral MT, proving that lateral MT can achieve higher forces with lower with non-hydrolyzable ATP mimic bound, the motor grips the DNA strongly dispersion. Finally, an example of how lateral magnetic tweezers with (1000 less slipping). Transient slips that occur when ATP dissociates are TIRF microscopy can be used to study bacterial chromosome segregation notably slower (40 bp/s) than in the absence of nucleotide, showing that mul- will be presented. tiple ATP-bound subunits interact with the DNA and exert friction. We show that the grip in the ATP-bound state can be ruptured by application of higher 465-Pos Board B235 force, but with a 30 pN force, the estimated maximum resistance at high pro- Mechanism of Sequence Dependent Translocation of a Superfamily 2 Heli- head filling, slipping is low enough that the motor can still function. With case on ssDNA bound ADP three different motor states are observed: one that grips strongly Jonathan M. Craig. like in the ATP-bound state, one with virtually no grip like in the apo state, Physics, University of Washington, Seattle, WA, USA. and one where the DNA slips at an intermediate speed (740 bp/s). Although Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a in the apo and ADP states the motor usually has little grip, when the end of the single-molecule method for monitoring the motion of nucleic acids DNA is about to leave the capsid slipping suddenly arrests. This unique controlled by a molecular motor enzyme through a nanopore at unprece- ‘‘clamp’’ state is highly stable and packaging resumes when ATP is added. dented spatiotemporal resolution with simultaneous readout of the nucleic This mechanism prevents complete dissociation of the genome if ATP is tran- acid sequence in the enzyme. We use this resolution to show that the trans- siently unavailable.

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469-Pos Board B239 Transcription factors(TF) are proteins inside the cell regulating gene expres- Challenging a DNA Packaging Motor with a Modified Substrate sion by binding to a specific DNA sequence. Their functions are to control Juan P. Castillo1, Alexander Tong1, Sara Tafoya1, Paul Jardine2, cell division, growth, death and response to the stimuli in the environment. Carlos Bustamante1. Current research about TF are mostly by in vitro detection based on ensemble 1UC Berkeley, Berkeley, CA, USA, 2University of Minessota, Minneapolis, measurement. In this presentation, we will show a way to detect TF at single MN, USA. molecule level using fluorescent molecular beacons. Molecular beacons are The DNA packaging motor of the bacteriophage phi29 is a powerful molecular oligonucleotides fluorescently labeled by a FRET pair. They can fold to machine that couples the free energy of ATP hydrolysis with DNA transloca- two equilibrium conformations which can be distinguished by different tion in order to complete the production of new viral particles. The active part FRET efficiency and fluorophore lifetimes. Binding to the the target TF of this motor is a pentameric ring ATPase which mechanochemical cycle has will help stabilize one of the two conformations. Through single-molecule been described with exquisite detail using hi-resolution optical tweezers. It FRET efficiency and lifetime measurement, we can detect and quantify the was described that in each turn of the cycle, the motor packages DNA taking response of molecular beacons to protein binding at single molecule basis. discrete steps of 10 base pairs (bp) each, in what is called a burst of transloca- Furthermore, besides the in vitro detection, we can also detect endogenous tion. At the same time it was shown that this 10 bp burst is composed of four TF. Here we use a pore-forming protein, Streptolysin O(SLO), to reversibly 2.5 bp sub-steps, presumably reflecting the power stroke of the individual permeabilize the cell membrane and deliver molecular beacons into the living ATPases. Several models can explain what is the origin of the burst size: the cells. helical pitch of B-form DNA is 10.5 bp/turn of the double helix, suggesting that the structure of the substrate is what determines the burst size; however, 473-Pos Board B243 the non-integer nature of the sub-steps within the burst allows to hypothesize Short-Read Single-Molecule DNA Sequencing for Highly Parallel Analysis that is the ATPase’s conformational change what sets the burst size. Yet of Protein-DNA Interactions another possibility is that the DNA packaging motor switches the local confor- Rebecca Andrews1, Horst Steuer1, Arun Shivalingam2, Afaf H. El-Sagheer2, mation of the DNA substrate from B-form to A-form during packaging (DNA Tom Brown2, Achillefs N. Kapanidis1. scrunching). To test the above hypotheses we challenged the phi29 DNA pack- 1Biological Physics Research Group, Clarendon Laboratory, Department of aging motor with a double-stranded RNA substrate (that adopts the A-form of Physics, University of Oxford, Oxford, United Kingdom, 2Chemistry nucleic acids) and measured the packaging activity using optical tweezers. Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom. 470-Pos Board B240 Protein-nucleic acid interactions are central to fundamental biological pro- Construction of a Viral Helicase Nanopore for Active DNA Unwinding and cesses such as transcription, translation and DNA repair. Although the Transport sequence dependence of such processes has been studied using single- Yuejia Chen, Ke Sun, Changjian Zhao, Xialin Zhang, Jia Geng. molecule methods, there is currently no systematic, high-throughput way to State Key Laboratory of Biotherapy, Sichuan University, Chengdu, China. connect the functional properties of a single DNA molecule (i.e. its interac- The molecular nanomotors play a key role in various biological process, such as tions and processing mechanism by proteins) with its sequence. To address DNA translocation in double-stranded DNA bacteriophage. Motors with the this, we are developing a single-molecule fluorescence method that can char- ATPase and helicase activity can unwind the double-strand DNA in the pres- acterise protein-DNA interactions on libraries of DNA sequences, followed by ence of ATP in vivo. Here we report the construction of a viral helicase and reading the sequence of the DNA at the interaction site. it displays the enzyme activity in vitro with the potential in material transport Here, we present the proof-of-concept for a novel single-molecule DNA for nanomedicine. The helicase protein was purified by glutathione agarose and sequencing method which is based on the transient binding of fluorescent nu- digestion GST tag and helicase protein by PreScission Protease, and further cleic acids to individual gapped-DNA substrates. separated using AKTA purification system. The obtained viral helicase motor The analysis of the binding kinetics of the oligonucleotides to surface- can unwind dsDNA both in vitro and in lipid bilayer membrane. immobilised gapped-DNA (with gap lengths of 6-15 nucleotides) provides a way to read a single base at a time. In proof-of-principle experiments, we show that the difference in the dwell times between a fully complementary 471-Pos Board B241 oligonucleotide and an oligonucleotide with a single base-pair mismatch al- Measuring Search Times in Site Specific DNA Binding lows base identification. To increase the throughput of the method, we Allen C. Price1, Raquel Ferreira2, Sadie Piatt1, Stephen Parziale1. 1 2 achieved a tenfold increase in the working concentration of the unbound oli- Chemistry and Physics, Emmanuel College, Boston, MA, USA, Biology, gonucleotides by using fluorescence quenching. We also observed the tran- Emmanuel College, Boston, MA, USA. sient binding of oligonucleotides with weaker DNA binding sequences, Facilitated diffusion is the accepted theory of how site-specific DNA binding such as A-T exclusive regions, verifying the robustness of the sequencing proteins search for their targets. When DNA sliding is included in the search method. strategy, protein search times are predicted to exhibit a bi-model dependence We are currently linking the novel single-molecule sequencing method to on the sliding length, with a minimum occurring at an optimal sliding length. the single-molecule interactions of transcription factors with DNA. Our This peak in specific association rate as sliding length is varied has rarely method is general and can be used to study the sequence-dependence of been directly measured. We are studying facilitated diffusion using restriction many complex interactions and reaction of proteins with DNA and RNA endonucleases as a model system. Our multiplexing single molecule technique substrates. is based on DNA tethering of microbeads and can measure the time of cleavage of up to hundreds of DNAs in a single experiment. We use diffusion controlled 474-Pos Board B244 conditions to directly measure the search time of proteins which cleave dsDNA. Direct AFM Visualization of RecG Translocation after Remodeling by SSB In the work reported here, we vary the siding length by varying the salt concen- Protein tration from a few mM up to 150 mM NaCl. We have discovered a peak in spe- Zhiqiang Sun1, Mohtadin Hashemil2, Piero R. Bianco3, cific association rates of both EcoRI and NdeI, consistent with the predictions Yuri L. Lyubchenko2. of facilitated diffusion. These peaks both occur near 80 mM NaCl, significantly 1University of Nebraska Medical Center, Omaha, NE, USA, 2university of less than physiological salt concentrations. I will discuss how our technique Nebraska Medical Center, Omaha, NE, USA, 3University at Buffalo, Buffalo, works and how we are using the theory of facilitated diffusion to understand NY, USA. our results. I will further discuss our efforts to directly verify DNA sliding in The RecG DNA helicase is a guardian of the bacterial genome. It binds to a these systems using single molecule fluorescent imaging. variety of forked DNA structures thereby minimizing pathological DNA replication and facilitating stalled replication fork rescue. Although crystallo- 472-Pos Board B242 graphic data provide a model of how RecG binds to the replication fork (1), Single Molecule Detection of Transcription Factor using Fluorescent the mechanism of RecG interaction with the fork along with other proteins is Molecular Beacons not clear. According to our previous study (2), SSB promotes binding of Pin Ren1, Yuji Ishitsuka2, Paul Selvin3. RecG to stalled DNA replication fork and induces remodeling of RecG 1Center for Biophysics and Quantitative Biology, Center for Physics and enabling the protein to translocate along the DNA duplex. The process is Living Cells, University of Illinois, Urbana-Champaign, Urbana, IL, USA, spontaneous and does not require hydrolysis of ATP. In the current study, 2Singular Genomics Systems, Inc., San Diego, CA, USA, 3Department of we characterized the RecG translocation process induced by the interaction Physics, Center for Physics and Living Cells, University of Illinois, Urbana- with SSB. We utilized high-speed (HS) AFM to acquire time-lapse images Champaign, Urbana, IL, USA. of stalled DNA replication fork substrates in complex with both SSB and

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RecG and directly visualize the dynamics. The time-lapse HS-AFM experi- that all energy minima of nucleotide translocations along the center pore ments confirmed the ability of RecG to translocate on DNA. In this approximate to the R18 ring. Moreover, the strong interactions between the nu- diffusion-driven process, RecG can move over 150 bp distance from the cleotides and six R18 residues were confirmed by the trajectory analysis which fork position, suggesting that SSB alters the RecG binding mode with the is consistent with the literature2. replication fork. Computational modeling was performed to characterize 1. Perilla, J. R.; Schulten, K., Physical properties of the HIV-1 capsid from all- the interaction of RecG and the stalled fork substrate. Upon binding to the atom molecular dynamics simulations. NAT. COMMUN. 2017, 8, 15959. fork RecG undergoes structural transition, which allows the nascent and 2. Jacques, D. A.; McEwan, W. A.; Hilditch, L.; Price, A. J.; Towers, G. J.; ssDNA strands to bind to the wedge domain. 1. Singleton, M. R., S. Scaife, James, L. C., HIV-1 uses dynamic capsid pores to import nucleotides and and D. B. Wigley. 2001. Structural analysis of DNA replication fork reversal fuel encapsidated DNA synthesis. Nature 2016, 536 (7616), 349-353. by RecG. Cell 107:79-89. 2. Sun, Z., H. Y. Tan, P. R. Bianco, and Y. L. Lyubchenko. 2015. Remodeling of RecG Helicase at the DNA Replication 477-Pos Board B247 Fork by SSB Protein. Sci Rep 5:9625. Characterization of Single-Stranded DNA Binding by APOBEC3 Family Proteins using Force Spectroscopy 475-Pos Board B245 Michael Morse1, Yuqing Feng2, Robin P. Love2, Ioulia Rouzina3, Peakforce Tapping AFM Reveals that Human XPA Binds to DNA Damage 2 1 Linda Chelico , Mark C. Williams . as a Monomer Producing a 60 Bend 1Northeastern University, Boston, MA, USA, 2University of Saskatchewan, 1 2 3 4 Emily C. Beckwitt , Nina Simon , Isadora Carnaval , Caroline Kisker , Saskatoon, SK, Canada, 3Ohio State University, Columbus, OH, USA. 2 1,5 Thomas Carell , Bennett Van Houten . The APOBEC3 (A3) family of proteins are found within humans and other 1Program in Molecular Biophysics and Structural Biology, University of 2 primates and provide a degree of innate immunity to viral infection and Pittsburgh School of Medicine, Pittsburgh, PA, USA, Center for Integrated replication. A3 proteins primarily function as cytidine deaminases, enabling Protein Science at the Department of Chemistry, Ludwig Maximillian editing of viral DNA in order to inhibit replication. There is a total of seven University of Munich, Munich, Germany, 3Chemical Engineering, University 4 different A3 proteins, each with either one or two cytidine deaminase of Pittsburgh Swanson School of Engineering, Pittsburgh, PA, USA, Rudolf domains. Additionally, each of these domains can be categorized as one Virchow Center for Experimental Biomedicine, Institute for Structural 5 of three distinct zinc coordinating domains, termed Z1, Z2, and Z3. While Biology, University of Wurzburg, Wurzburg, Germany, Pharmacology and much homology exists among the various A3 proteins, these structural dif- Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, ferences greatly impact both the binding and enzymatic activity of each A3 PA, USA. protein. Using optical tweezers, we investigate the binding of A3A (single Mutations in the XPA gene are associated with extreme light sensitivity and a Z1 domain), A3H (single Z3 domain), A3G (one Z1 and one Z2 domain), 2000-fold increase in instances of skin cancer due to an inability to remove and A3F (two Z2 domains) to single-stranded DNA (ssDNA). A range of UV-induced photoproducts. We have used single molecule approaches to forces are applied to the ssDNA binding template to reveal both the gain a better understanding of XPA’s molecular role in nucleotide excision binding kinetics and energetics of the A3-ssDNA interaction. We quantify repair (NER). Atomic force microscopy (AFM) was used to obtain informa- differences between the A3 proteins ranging over an order of magnitude tion about interactions between full-length human XPA (His-tagged, in effect. These variations in binding behavior likely influence the ability 32.6 kDa) and a DNA substrate with a site-specific bulky adduct, N-(2’-de- of A3 proteins to search for potential deamination sites using both 1-dimen- oxyguanosin-8-yl)-2-acetylaminofluorene (AAF-dG), but the small size of sional sliding along the ssDNA and 3-dimensional diffusion processes to the protein presented limitations in terms of resolution using tapping AFM. access different regions of the viral DNA. Thus, these differences in We have thus adopted the use of PeakForce Tapping (Bruker) AFM in com- binding can potentially explain previously observed differences in enzy- bination with a protocol for the measurement of protein volume within a matic activity between the A3 proteins. Additionally, the binding activity protein-DNA complex. Using this technique, XPA bound to DNA has a 3 of certain A3 proteins may result in biochemical functions beyond deami- mean volume of about 31 nm at AAF-dG and non-specific sites; this nase activity, such as the stabilization of A3G binding through oligomeriza- corresponds to a molecular weight of 30 kDa, consistent with monomeric tion, which inhibits HIV-1 reverse transcription in a deaminase-independent binding. Furthermore, monomeric XPA bound to both AAF-dG and undam- manner. aged DNA induced a bend angle of approximately 60. These data differ from published EMSA studies and a recent co-crystal structure of the DNA binding domain of the yeast homolog of XPA (Rad14) showing dimer- 478-Pos Board B248 ization and a 70 DNA bend angle (Koch et al, PNAS 2015). These studies RNA Polymerase Pauses at lac Repressor Obstacles indicate that full-length human XPA behaves differently than the protein do- Yan Yan, Wenxuan Xu, David D. Dunlap, Laura Finzi. mains in previous studies. Department of Physics, Emory University, Atlanta, GA, USA. Support from NIH 5R01ES019566 (B.V.H.) and T32GM088119 (E.C.B.). Transcription is regulated by accessory factors that bind to specific sites, which may overlap with the promoter so that binding inhibits transcriptional initiation, 476-Pos Board B246 or may be located at downstream sequences where they block the passage of Energetics of Nucleotide Translocation through HIV-1 CA Hexamer RNA polymerase. In this study we used magnetic tweezers to monitor the Chaoyi Xu, Juan Perilla. length of a DNA tethering a one-micron diameter, magnetic bead to an RNA Department of Chemistry and Biochemistry, University of Delaware, polymerase stuck on a glass surface. As the polymerase translocated during Newark, DE, USA. elongation, it drew in and shortened the DNA tether, which was under sub- The capsid is a proteinaceous container of the virus genome and other viral pro- picoNewton tension. This DNA tether included a high affinity (O1) binding teins. HIV-1 capsid is composed of 216 hexamers and 12 pentamers1. In the site for the lac repressor located downstream from the promoter. The lac HIV-1 replication cycle, the viral DNA is reverse-transcribed with the aid of repressor and it specific binding sites constitute a classic model of gene regu- the capsid. However, little is known about how the DNA nucleotides translo- lation in E. coli. When the RNA polymerase encountered obstacles constituted cate into the capsid or whether the capsid acts as a gating agent during by lac repressor, it paused for several minutes and the tether length remained HIV-1 early post-infection. Recently, Jacques et al.2 suggested that the center constant. The average pause lifetime was comparable to the lifetime of lac pore of HIV-1 capsid hexamer is a potential nucleotide channel and the R18 repressor binding measured on templates with no active transcription, which ring in the pore plays essential role in recruiting and binding with nucleotides. suggests that instead of dislodging the obstacle, RNA polymerase waits for Therefore, we designed and performed a serial of molecular dynamic simula- lac repressor to dissociate before proceeding. The pause times were similar tions and studied the translocations of single nucleotide through the pore of for elongation through lac repressor obstacles at binding sites placed either HIV-1 hexamer, including 4 ribonucleoside monophosphates (rNMPs), 4 ribo- 689 or 288 away from promoter. On occasion, either immediately or after a nucleoside triphosphate (rNTPs) and 4 deoxynucleoside triphosphates pause, the tether length abruptly returned to the initial length observed when (dNTPs). With umbrella sampling (US) - Hamiltonian replica exchange molec- RNA polymerase was poised at the promoter. This suggests that RNA polymer- ular dynamics (H-REMD) simulations, the potential-of-mean-force (PMF) pro- ase may somehow ‘‘slide’’ back to the promoter without dis-engaging from the files of nucleotide translocations were calculated. The PMF results indicated DNA template.

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Posters: Membrane Physical Chemistry I Lipid bilayer membranes are liquid crystals. The oppositely oriented head group dipoles in the lipid head groups makes the bilayer leaflets respond 479-Pos Board B249 differently to applied electrical potential. While the density of dipoles de- Peptide-Lipid Interactions and Lipid Lateral Diffusion Monitored via 31P creases in one leaflet, it increases in the other leaflet upon application of a CODEX NMR transmembrane electrical potential, thus resulting in bending of the bilayer. Angel Lai, Peter Macdonald. We investigated this flexoelectric phenomenon using coarse grained molecu- Chemical and Physical Sciences, University of Toronto, Mississauga, ON, lar dynamics simulations and show that increasing potentials bend the mem- Canada. brane to a larger extent. We demonstrate the implications of the phenomenon Recently, we demonstrated the use of 31P CODEX (Centerband-Only Detec- in the tubulation of giant unilamellar vesicles upon application of a calcium tion of Exchange) NMR to measure lateral diffusion of phospholipids. flux. Here we demonstrate the multiplexing capabilities of CODEX, i.e. simulta- neous measurement of multiple phospholipid lateral diffusion coefficients, 483-Pos Board B253 and compare the effects of polylysine and a model anti-microbial peptide, Bending Modulus and Edge Tension of Giant Unilamellar Vesicles (GUVS) KL-14 (KKLLK KAKKL LKKL), on lateral diffusion of various anionic Composed of Lipid Extracts from Erythrocytes Membranes phospholipids. Bruna R. Casadei1, Rumiana Dimova2, Karin A. Riske1. 1Biophysics, Universidade Federal de Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Department of Theory & Bio-systems, Max Planck Institute of Colloids and 480-Pos Board B250 Interfaces, Potsdam, Germany. Subdiffusive Motion of STIM1 at ER Membrane and ER-Plasma The erythrocyte membrane is a robust model for the study of biological Membrane Junction membranes because it is easy to obtain, isolate and to monitor the destabili- Xianan Qin1, Adolfo Alsina1,2, Sang Kwon Lee3, Chan Young Park3, zation (hemolysis) of its structure. Even though its composition and function Hyokeun Park1,4. are well known, there are few reports about the relationship between the or- 1Department of Physics, The Hong Kong University of Science and ganization of its components and its structure, stability and function. Here, Technology, Kowloon, Hong Kong, 2Departement de Physique, Ecole we measured the bending modulus and the edge tension of giant unilamellar Normale Superieure, Paris, France, 3Department of Biological Sciences, vesicles (GUVs) composed of lipids extracted from erythrocyte membranes Ulsan National Institute of Science and Technology, Ulsan, Korea, Republic (erythro-GUVs) and GUVs biomimetics composed of mixtures of 1-palmi- of, 4Division of Life Science, The Hong Kong University of Science and toyl-2-oleyl-sn-glycero-phosphatidylcholine (POPC), egg (chicken) sphingo- Technology, Kowloon, Hong Kong. myelin (SM), 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylserine (POPS) and The subdiffusive motion of transmembrane proteins on plasma membrane has cholesterol (chol). The bending modulus indicates the membrane mallea- been reported. However, it remains unclear whether subdiffusive processes bility and was measured from fluctuation analysis of GUVs. The edge exist on other cellular membranes structures, such as endoplasmic reticulum tension gives the energy cost per unit length to maintain frustrated lipids (ER) membrane and ER-plasma membrane junctions (the membrane structure at the pore rims and may be associated with membrane stability. The results formed by ER and plasma membrane at very close proximity). The stromal obtained show that the bending modulus is higher for cholesterol-containing interaction molecule (STIM) is an ER transmembrane protein, playing impor- membranes. Curiously, the bending modulus of erythro-GUVs (40.152.5 x tant roles in the regulation of store-operated calcium entry (SOCE). It is known 20 2þ 10 J) was lower than that of its biomimetic model POPC:SM:chol 4:2:4 to oligomerize, bind to Orai proteins (Ca channel located at plasma mem- 20 (54.153.1x10 J). The edge tension values were highly sensitive to brane) and form STIM-Orai complexes crossing ER-plasma membrane junc- þ the presence of cholesterol and of anionic lipids in the mixture. Erythro- tions upon the store depletion of Ca2 . We performed single-particle GUVs exhibited an edge tension value (6656 pN) intermediate between tracking (SPT) for STIM1 and found that STIM1 had subdiffusive motion POPC:SM:chol 4:2:4 (10959 pN) and POPC (50510 pN). The presence before and after oligomerization. The analyzed diffusion coefficients and the of 5 and 10 mol% POPS in the biomimetic mixture decreased the edge anomalous exponents from the SPT data showed significant heterogeneity. tension considerably, showing that the presence of surface charges destabi- By comparing the time-averaged and ensemble-averaged mean-squared lize the membrane structure. This work was supported by Fapesp (# 2015/ displacement (MSD), we also found that subdiffusive processes of STIM1 09948-2). and STIM1-Orai1 complexes were non-ergodic. The subdiffusive and non- ergodic processes were found on both ER membrane and ER-plasma membrane 484-Pos Board B254 junctions. Our findings shed a new light on protein diffusion on different Mimicking Cell Pinocytosis: Lipid Vesicles Engulfment of Oil-in-Water cellular membranes. Droplets Rafael B. Lira1, Lucia Benk2, Eleanor Ewins1, Joachin P. Spatz2, 481-Pos Board B251 Reinhard Lipowsky1, Ilia Platzman2, Rumiana Dimova1. Theoretical Modeling of Experimentally Determined Tilt Modulus of 1Max Planck Instute of Colloids and Interfaces, Potsdam, Germany, 2Max Lipid Bilayers Planck Institute of Intelligent Systems, Stuttgart, Germany. John F. Nagle. Pinocytosis, a type of endocytosis, is a process by which extracellular liquid Physics, Carnegie Mellon University, Pittsburgh, PA, USA. droplets are taken up by cells. It involves droplet adhesion and engulfment Values of the tilt moduli of single-component PC lipid bilayers recently deter- by the membrane. We develop a synthetic system that reproduces the essen- mined by analysis of diffuse x-ray scattering (Chem. Phys. Lipids 205, 18-24 tial steps of pinocytosis at the level of a single interacting pair - the mem- (2017)) show a strong correlation with how far the temperature was from the brane and the droplet. The system consists of an engulfing giant temperature TM of the main transition for each PC lipid. Consistent with this, unilamellar vesicle (GUV) and an engulfed polymer-stabilized oil-in-water the tilt modulus of DMPC decreased dramatically as temperature was lowered emulsion droplet. The interaction between the GUV and the droplet is medi- toward TM (Phys. Rev. E 96, 030401 (2017)). It is hypothesized that tilt is a ated by magnesium ions and is reversed by EDTA or Triton X-100. Adhe- symmetry breaking order parameter for the main transition of PC lipids that sion energy jWj, measured from the contact angle between a GUV have spontaneous tilt below TM. Consistently, POPS does not have spontaneous adhering to a droplet, either freestanding or manipulated with micropipettes, tilt below TM and its tilt modulus is not temperature sensitive. Theoretical is affected by the magnesium concentration. In the weak adhesion regime modeling will be presented that combines a tilt free energy functional with a (jWj0.4 mJ/m2, % 0.5 mM Mg2þ), GUVs adhere to the droplets and chain melting free energy functional based on an older exact statistical mechan- deform. Interaction is reversed by retracting the pipette-held GUV. In the ical theory that did not have tilt (Faraday Discussion 81, 151 (1986)). Coupling strong adhesion regime (g1.2 mJ/m2,0.5> mM Mg2þ), the GUV between the two terms obtains the experimental temperature dependence of the completely engulfs the droplet and the interaction is irreversible. The droplet tilt modulus of DMPC. becomes wrapped by a single bilayer in a process that increases membrane tension and leads to vesicle poration. Both adhesion and engulfment lead to a 482-Pos Board B252 decrease in lipid diffusion coefficient from 7 mm2/s on free-standing Measurements and Implications of How Electrical Potentials Can Bend bilayers to 1 mm2/s on the droplet-supported bilayer. Interestingly, the Membranes contact line of the membrane-droplet adhesion area is often connected to Dennis Bruhn, Weria Pezeshkian, Himanshu Khandelia. many lipid nanotubes. The developed synthetic pinocytosis system of MEMPHYS: Center for Biomembrane Physics, University of Southern GUV-droplet pairs allows detailed investigation of the endocytosis processes Denmark, Odense M, Denmark. of natural cells but with reduced complexity. Future surface modifications of

BPJ 8566_8569 Sunday, February 18, 2018 95a the membrane/droplet interaction have the potential to mimic specific brane peptides. Bicelles can be prepared through PC bilayer stabilisation pathogen-recognition interactions. by a variety of detergents, providing that physicochemical requirements This work is part of the MaxSynBio consortium which is jointly funded by the are met by adjusting detergent to lipid ratio, concentration, and temperature. Federal Ministry of Education and Research of Germany and the Max Planck In this work we propose an alternative to bicelles, obtained by softening PC Society. liposomes with a gentle surfactant. We show that polysorbate 80 (Tween 80) mixed to DMPC and DPPC bilayers leads to elongated oriented structures 485-Pos Board B255 which maintain their alignment over a wide range of lipid-to-detergent molar Dewetting-Induced Lipid Droplet Budding ratios (2

BPJ 8566_8569 96a Sunday, February 18, 2018 for understanding their fundamental voltage-sensing mechanism in future. isolated the population with the desired asymmetry. Efforts are underway to Taking advantage of the high stability, the reported platform is currently prepare asymmetric vesicles using additional types of cyclodextrins and to used also for mechanistic studies of lipid membrane ionophores, which expand the protocol to additional types of membrane lipids. Prior studies have relevance to medicine because of their antimicrobial and anti-cancer have employed methyl beta-CD, methyl alpha-CD and hydroxypropyl effects. alpha-CD. A comparison of additional CDs indicates that gamma-CD also has the ability to catalyze a high level of lipid exchange. Unmodified alpha- 490-Pos Board B260 CD is not as effective as gamma-CD. Use of gamma-CD, which is composed Silica-Supported Lipid Bilayers: Electrostatic Effects at Lipid Interfaces of eight glucose rings rather than seven in beta-CD and six in alpha-CD may as Reported by Spin-Labeling EPR extend the range of lipids that can be exchanged into vesicles because of its Erkang Ou, Maxim A. Voinov, Alex I. Smirnov, Tatyana I. Smirnova. larger cavity. Using gamma-CD, cholesterol-containing asymmetric vesicles Chemistry, North Carolina State University, Raleigh, NC, USA. have been prepared with DOPC in the outer leaflet and a POPS/POPE mixture Interfacing biological and artificial systems at the nano-scale level is in the inner leaflet. The ability of CDs to exchange cationic lipids is also under essential for developing novel living-nonliving biotechnology platforms investigation, in this case using CDs employed in previous studies. Using including biosensors based on biological systems. Despite an impressive methyl alpha-CD vesicles have been prepared in which ethyl PC, a cationic progress, the needs remain high to understand the influence of a nano- lipid which has a blocked phosphate group, is replaced in the outer leaflet structured support and nanoconfinement on structure and properties of by POPC. We also plan to investigate the formation of vesicles in which ethyl the membrane-protein interface. We have utilized novel pH-sensitive PC is introduced into the outer leaflet of zwitterionic vesicles. Asymmetric lipids IMTSL-PE and IKMTSL-PE to assess the phospholipid membrane vesicles with cationic lipids may have useful applications in delivery of mol- surface potential. It was shown that bilayers formed from zwitterionic ecules into cells. or mixed lipids on silica nanoparticle surfaces possess a higher negative electrostatic potential than the unsupported bilayers with the potential of Posters: Membrane Dynamics I mixed bilayers containing negatively charged lipids being significantly more sensitive to the silica support. pH-sensitive EPR probes were then 493-Pos Board B263 employed to label model WALP peptide known to form a-helix when in- Lateral Diffusivity of Cholesterol Depends on its Spatial Arrangement in tegrated into a lipid bilayer. The effects of silica support on the peptide Lipid Membranes dynamics and the effective pKa of the ionizable probe have been investi- Younghoon Oh, Bong June Sung. gated. It was demonstrated that the silica nanoparticles affect the peptide Sogang University, Seoul, Korea, Republic of. dynamics and shift the effective pKa of the ionizable nitroxide probe in a Lateral diffusion of cholesterol in lipid membranes plays crucial roles in membrane depth-dependent manner. Our data also show that upon proton- various biochemical reactions such as signal transduction. Cholesterol ation of the membrane-burred model ionisable sidechain the silica support frequently exhibits anomalous subdiffusion where its mean square displace- causes changes in the membrane association of WALP peptide that are not ment scales with time as hDr2(t)ita with 0 < a < 1. The timescale observed when WAPL is integrated into unilamellar phospholipid vesicles. and scaling exponent a of cholesterol subdiffusion depend on several Supported by NSF 1508607 to TIS. factors such as cholesterol concentration, lipid types and membrane phase. A previous simulation study [1] revealed that there are two distinctive time- 491-Pos Board B261 scales of subdiffusion with different exponent: the first subdiffusion regime Fractionation of Styrene and Maleic Acid Copolymers: The ‘‘Holy Grail’’ ( 1 ns) with exponent a and the second one ( 102 ns) with exponent a (0 of Membrane Solubilization 1 2 < a1< a2< 1). The second subdiffusion regime covers an unusually long Juan J. Dominguez Pardo, Josephine A. Killian. period. The physical origin of multiple regimes of cholesterol transport, Chemistry, Membrane Biochemistry and Biophysics, Utrecht, Netherlands. however, remains elusive. We employ Martini coarse-grained force field Styrene-and-maleic acid copolymers (SMA) are gaining interest in mem- and molecular dynamics simulations to study anomalous subdiffusion of brane protein research due to their ability of solubilizing lipid membranes cholesterol in binary component lipid membranes that are composed of satu- into nanodiscs. Using different commercially available SMA preparations rated lipid (dipalmitoylphosphatidylcholine) and cholesterol. We find from it was found that the average length and hydrophobicity are important pa- our simulations that cholesterol flip-flops rapidly from one leaflet to another, rameters in determining solubilization efficiency and properties of the re- which is consistent with previous studies. During the flip-flop, cholesterol sulting nanodiscs. However, our understanding of the underlying SMA- tilts first and then lies horizontally in between two leaflets. While cholesterol lipid interactions is hampered by the fact that SMA preparations are very stays at the center of the lipid bilayer in between two leaflets, the cholesterol polydisperse. Here, we fractionated widely used SMA preparations (Mw diffuses quite fast with the regime for the subdiffusion emerging at 10kD) into 4 fractions of polymers with different average length, based short time scales ( 1 ns). On the other hand, the cholesterol within a leaflet on their solubility in hexane/acetone mixtures and we investigated their diffusion slowly and its subdiffusion regime appears at long time scales interaction with lipid self-assemblies. We found that fractions with shorter ( 102 ns). We believe that the dependence of cholesterol diffusivity on SMA polymers solubilize phosphatidylcholine vesicles more rapidly than spatial arrangement should lead to the presence of multiple regimes in fractions with longer polymers. On the other hand, nanodiscs bounded by cholesterol transport. long SMA polymers were notably more stable than those bounded by shorter [1] Phys. Rev. Lett., 109, 188103 (2012) polymers, as indicated by a better retention of the native lipid thermotropic properties and by slower exchange rates between lipids in nanodiscs. When the SMA fractions were mixed with E.coli membranes, a somewhat 494-Pos Board B264 different behavior was observed, probably due to the complexity of these Scramblase Activities of Transmembrane Peptides Depend on Relative membranes. Here the fractions with relatively long polymers were found Position of Hydrophilic Amino Acid Residues and their Depth in the Mem- to be most efficient in solubilizing the tetrameric KcsA channel. Competi- brane tive solubilization experiments showed that short polymers even hinder Hiroyuki Nakao, Yuta Sugimoto, Keisuke Ikeda, Minoru Nakano. solubilization by long polymers. In conclusion, we here present a University of Toyama, Toyama, Japan. simple method to purify SMA fractions from commercial SMA in order Phospholipid transbilayer movement (flip-flop) is very rapid in the ER of eu- to improve solubilization of biomembranes as well as stability of the result- caryotes, where phospholipids in the cell are mainly synthesized only at the ing nanodiscs. cytoplasmic leaflet. However, the mechanism to promote the phospholipid flip-flop in the ER remains unknown. We have previously revealed that a pep- 492-Pos Board B262 tide with a membrane-spanning sequence of a human ER protein, EDEM1, Expanding the Preparation of Asymmetric Lipid Vesicles to Additional promotes the flip-flop, and that both Arg and His residues in the sequence Cyclodextrins and Cationic Lipids are necessary for the peptide’s activity. Interestingly, these hydrophilic resi- Sunjae Park, Bingchen Li, Erwin London. dues are located at the same side of an a-helix structure of EDEM1 peptide. Stony Brook University, Stony Brook, NY, USA. Therefore, we hypothesized that relative position of two hydrophilic residues Our lab has developed the use of cyclodextrins (CDs) to prepare asymmetric in the helix may be important for the flip-promotion ability of peptides. In this lipid vesicles with different lipids in the inner and outer leaflets (monolayers). study, we synthesized model transmembrane peptides containing two hydro- Asymmetric vesicles are prepared by using CDs to exchange lipids between philic residues at various positions in the sequences and evaluated their activ- two populations of vesicles with different lipid compositions, and then ity. We demonstrated that the activities of the peptides depended on the turn

BPJ 8566_8569 Sunday, February 18, 2018 97a angle between Arg and His in the helices, and significantly increased when that spontaneously oligomerize and form self-assembled structures on the two hydrophilic residues are located at the same side of the helix. The pep- host membrane cell, intake nutrition, inhibit the activity ultimately causing tides containing other high hydrophilic residues at the same side of the helices cell lysis and cell death. In this study, we investigate the influence of also had flip-promotion abilities, whereas the substitution of either Arg or His PFT’s on the dynamics of two mammalian cell membranes (DMPC and with a lower hydrophilic or hydrophobic residue inhibited the activities. DOPC) in the presence of cholesterol. We show that cholesterol fluidizes Therefore, the scramblase activities of the peptides were attributed to the hy- the membrane accelerating the dynamics. In the absence of cholesterol, drophilicity of the residues at the same side. We also found that the peptides the lipids form crystallized lipid-protein complexes causing a slowdown in with hydrophilic residues at the deeper position in the membrane showed dynamics in the vicinity of the protein. We also found that the dynamics higher flip-promotion ability. These results provided the possibility that the of lipids is strongly coupled to their local structure and also on the nature proteins whose transmembrane regions have similar physicochemical proper- of lipid-protein interactions. The lipid-protein interactions are influenced ties as those of the peptides used in this study might be involved in the flip- by the dominant structure of the transmembrane protein in the lipid bilayer flop promotion in the ER. and hence depends on the type of the PFT. There exists structural and dynamical heterogeneities in the lipid bilayer and in each leaflet very similar 495-Pos Board B265 to fluids approaching glass transition. We show that the upper leaflet from Cyclodextrin-Mediated Lipid Exchange Monitored with FRET where the toxin inserts into the membrane exhibits a slower dynamics Anna Weitzer1,2, John Katsaras3,4, Frederick A. Heberle5,6. 1 than the bottom leaflet and the alpha-PFT perturbs the membrane to a greater Institute of Molecular Biosciences, Biophysics Division, University of Graz, extent than the beta-PFT. This study will shed light on the toxicity level of Graz, Austria, 2BioTechMed-Graz, Graz, Austria, 3Dept. of Physics and 4 the PFT in influencing the lipid dynamics and the role of cholesterol on Astronomy, University of Tennessee, Knoxville, TN, USA, Neutron lipid-protein interactions. Sciences Directorate, Oak Ridge National Laboratory, Oak Ridge, TN, USA, 5Biology and Soft Matter Division, Oak Ridge National Laboratory, 6 498-Pos Board B268 Oak Ridge, TN, USA, Joint Institute for Neutron Sciences, Oak Ridge, Effect of Hypothermia on the Biophysical Performance of Pulmonary TN, USA. Surfactant from Neonates with and without Lung Injury We have studied cyclodextrin mediated exchange of phospholipids between Chiara Autilio1, Mercedes Echaide1, Daniele De Luca2, Jesu´sPerez-Gil1. unilamellar vesicles at different temperatures and cyclodextrin concentrations 1Department of Biochemistry, Faculty of Biology and Research Institut using Fo¨rster Resonance Energy Transfer (FRET). Lipid transfer between Hospital, Complutense University, Madrid, Spain, 2Division of Pediatrics and two vesicle pools was monitored through changes in the fluorescence intensity Neonatal Critical Care, ‘‘A. Becle`re’’ Medical Center, South Paris University of the donor lipid, NBD-DPPE, which is quenched in a concentration- Hospitals, APHP, Paris, France. dependent manner by an acceptor lipid, LR-DOPE. Experiments were per- Whole-body hypothermia (WBH) is used to improve neurological outcomes formed in which either: (a) separate donor-labelled and acceptor-labelled in perinatal asphyxia. Recent studies suggested a beneficial effect of cool- vesicle pools were mixed; or (b) doubly-labelled and unlabelled vesicles ing (33.5C) for some types of acute respiratory failure, and in a previous were mixed. The resulting data were analyzed with a kinetic model that ac- study, we showed improved interfacial accumulation of pulmonary surfac- counts for both lipid exchange and flip-flop. Temperature-dependent exchange tant at the air/liquid interface after 48h of hypothermia. Formation of highly rates obtained from the model were further analyzed with transition state theory dense lipid films at the interface is crucial to stabilize the respiratory sur- to determine thermodynamic parameters including the free energy and enthalpy face along breathing dynamics. In the current work, we have conducted a of activation. Our findings are supported by small angle x-ray scattering biophysical study by captive bubble surfactometry (CBS) of surfactant ob- (SAXS) and dynamic light scattering (DLS) measurements, as well as kinetic tained from asphyxiated neonates to further explore the effect of WBH on Monte Carlo simulations. surfactant function. Non-bronchoscopic bronchoalveolar lavage (BAL) was The information obtained from this study is important for understanding the collected from 10 asphyxiated neonates at different time-points (pre-WBH, stability of engineered asymmetric vesicles as well as other cyclodextrin- 24h, 48h, 72h of WBH and post-WBH), and surfactant was extracted and mediated processes. tested by CBS, at 37C and 33.5C. Minimum surface tension under 496-Pos Board B266 dynamic testing was significantly lower at 33.5 Cthanat37Cafterat Understanding Spatiotemporal Aspects of Cecropin a Attack on Single, least 48h of WBH and remained significant at 6h after rewarming. Total Live Bacteria using Time-Lapse Fluorescence Microscopy cholesterol content in surfactant showed a trend to decrease during WBH, Anurag Agrawal, James C. Weisshaar. suggesting that the WBH-promoted improvement of surfactant activity Chemistry, Univ Wisconsin-Madison, Madison, WI, USA. may be at least partially explained by a time-dependent structural reorgani- Antimicrobial peptides (AMPs) halt growth of bacterial cells without zation and/or a compositional adaptation of surfactant under hypothermia. causing substantial damage to eukaryotic cells. This specificity arises Similar changes, especially in cholesterol concentration, have been from charge-mediated electrostatic binding of AMPs with anionic bacte- already described during adaptive variations of surfactant in hibernating rial membranes. Previous studies in our group have explored the mem- animals. brane permeabilization caused by AMPs in detail. Low micromolar concentrations of AMPs can permeabilize exponentially growing cells 499-Pos Board B269 within 10 minutes. Here we investigate parameters that might influence The Hydrophobic Surfactant Proteins Reduce the Bending Modulus of the spatial and temporal aspects of Cecropin A action, as observed in our Phospholipid Bilayers Ryan W. Loney1, Zimo Yang2, Stephen B. Hall1, Stephanie Tristram-Nagle1. microscopy assays. The effects of lipopolysaccharide (LPS) core compo- 1 2 nents, i.e., negative charge from phosphorylation, and number of sugar Oregon Health & Science University, Portland, OR, USA, Carnegie Mellon layers was studied. Single cell, time resolved assays were performed on University, Portland, OR, USA. the K-12 BW25113 wild type strain and its deep rough mutants that The hydrophobic surfactant proteins, SP-B and SP-C, accelerate the adsorp- have reduced LPS layers in terms of the charge or the number of sugars. tion of the lipids in pulmonary surfactant to an air/water interface. Prior data A reduction in the length and/or the charge of the core LPS layer resulted suggest that the lipids form a negatively curved structure that extends from in faster permeabilization of the E. coli membranes. This suggests that the the adsorbing vesicle to the interface. The proteins could promote adsorption sugars and the phosphate groups are both hindrances for the permeabili- by reducing the energy of bending the lipids from their spontaneous curva- zation action of Cecropin A on E. coli. Additionally, the spatial pattern ture to the structure of the intermediate. Previous studies show that the pro- of permeabilization of any of the membranes did not change as a result teins shift spontaneous curvature toward the configuration of the of deep rough mutations, suggesting that a different factor may be hypothetical intermediate. The studies here tested whether the proteins could causing the pattern. also lower the energy of bending by making the lipids more flexible. We measured the diffuse X-ray scattering from oriented films of dioleoyl phos- 497-Pos Board B267 phatidylcholine, used as a mimic of the surfactant lipids, with the surfactant Role of Pore Forming Toxins in Modulating the Lipid Dynamics proteins in their physiological ratio. Analysis showed that the proteins Vadhana Varadarajan. dramatically reduced the modulus of bending. The effect occurred with Department of Chemical Engineering, Indian Institute of Science, Bangalore, small amounts of protein. As little as 0.3% (w:w) protein, well below the India. physiological level of 1.5%, approached the maximum reduction of 75%. Pore forming toxins (PFT’s) are important bacterial virulence factors The kinetics of adsorption, however, determined previously, fail to show secreted by bacteria and pathogens. These are water-soluble monomers such a precipitous effect by low concentrations of protein. The physiological

BPJ 8566_8569 98a Sunday, February 18, 2018 significance of the effect on the modulus of bending by the proteins therefore teins are regulated to enhance the collision with signaling molecules and remains uncertain. recruitment to clathrin coated pits in the vicinity of the Kv2.1-induced ER/PM junctions. 500-Pos Board B270 Loss of Carotenoids Impacts Membrane Protein and Lipid Distribution in 502-Pos Board B272 Pantoea sp. YR343 Roll to Roll Processing for Lipid Membranes Sushmitha Vijaya Kumar, Jennifer Morrell-Falvey. Bethany Reim. Genome Science and Technology, University of Tennessee, Knoxville, Keck Science, Claremont, CA, USA. Knoxville, TN, USA. While supported lipid bilayers have broad applications as biocompatible sur- Bacterial cell membranes are complex mixtures of lipids and proteins, the faces and biomimetic filters, they do not have a scalable manufacturing combination of which confers biophysical properties to the membrane and approach. Here we present a method for generating supported bio membranes allows it to respond to environmental conditions. Pantoea sp.YR343isa at almost arbitrarily large scales. We have adapted lipid surface-spreading to a plant-associated Gram-negative proteobacteria that produces carotenoids, roll-to-roll process on a flexible substrate. We demonstrate preliminary including zeaxanthin. We recently reported that Pantoea sp. YR343 mutants 10x10cm supported phospholipid bilayers, with an 80% coverage, as well lacking the crtB gene encoding phytoene synthase, which catalyzes the first as compare lipid-membrane compatibility of several potential flexible step in carotenoid biosynthesis, failed to produce carotenoids and displayed substrates. enhanced sensitivity to reactive oxygen species. The crtB mutant also dis- played unexpected defects in biofilm formation and root colonization compared to wildtype cells. Studies using in silico molecular simulations 503-Pos Board B273 suggest that carotenoids can act as modulators of membrane biophysical An Enhanced Platform for Bioelectrochemical Systems: A Novel properties, leading to the possibility that these phenotypes may result from Approach to Characterize Lipid Structure on Graphene 1 2 1 3 defects in membrane organization. We hypothesize that carotenoids modu- Megan E. Farell , Maxwell Wetherington , Inseok Chae , Manish Shankla , Seong Kim1, Aleksei Aksimentiev3, Manish Kumar1. late membrane dynamics by influencing the composition and organization 1 of membrane proteins and lipids. Fluorescence anisotropy measurements Chemical Engineering, The Pennsylvania State University, State College, PA, USA, 2Materials Science, The Pennsylvania State University, State on vesicles generated from lipids extracted from either wildtype or crtB 3 mutant cells revealed that the wildtype membrane vesicles were significantly College, PA, USA, Physics, University of Illinois at Urbana Champaign, more rigid than mutant membrane vesicles. In contrast, anisotropy measure- Illinois, IL, USA. ments on whole cells indicated that wildtype cells were less rigid than crtB Graphene is a two-dimensional material composed of a single carbon layer mutant cells. These observations were confirmed by atomic force micro- that offers several appealing properties including high conductivity, large scopy. Mass spectrometric based analysis of membrane proteins and lipids surface area, and flexibility. Its unique properties make graphene an ideal revealed differences in membrane protein and lipid profiles between the substrate for several applications, including energy storage, optical elec- mutant and wildtype cells. Among the different classes of proteins, signaling tronics, and medical devices. Functionalizing graphene with a lipid bilayer and transport protein classes were the most effected in the mutant. Similarly, both increases its biocompatibility and provides a platform for diverse bio- unsaturated and cyclopropane fatty acids classes differed in both cell types. electrochemical systems. However, characterization of lipids on graphene is These data suggest that loss of carotenoids in cell membranes changes the challenging since traditional fluorescent methods for characterization of abundance and distribution of membrane proteins and lipids which may be supported lipid structures are ineffective on graphene due to its highly the result of, or help compensate for, changes in membrane biophysical quenching nature. Furthermore, there are multiple conflicting models pub- properties. lished for the structure of lipids on graphene. We demonstrate that a novel technique using Raman spectroscopy and photoluminescence (PL) allows for characterization of lipids on graphene while providing additional bene- 501-Pos Board B271 fits over conventional setups. We use Raman-PL in conjunction with simu- Kv2.1-Induced ER/PM Junctions Modify the Cell Surface Diffusion lations to determine the structure, fluidity, and homogeneity of lipids on Landscape graphene. Laura Sole1, Yaping Moshier1, Sanaz Sadegh2, Patrick Mannion3, Diego Krapf2, Michael Tamkun1. 1Department of Biomedical Sciences, Colorado State University, Fort 504-Pos Board B274 Collins, CO, USA, 2Department of Electrical and Computer Engineering, Characterization of F2N12S in Cell Membranes using Time-Resolved Colorado State University, Fort Collins, CO, USA, 3School of Biomedical Fluorescence Techniques Engineering, Colorado State University, Fort Collins, CO, USA. Donald S. Anderson, Matthew J. Sydor, Harmen B. Steele, J.B.A. Ross, The Kv2.1 voltage-gated Kþ channel is widely expressed in the nervous Holian Andrij. system. In addition to its classical function as a delayed rectifier Kþ chan- University of Montana, University of Montana, Missoula, MT, USA. nel, Kv2.1 has a structural role where it interacts with the cortical endo- Methods are needed to determine the dynamics of cellular lipid membranes plasmic reticulum (ER) to form ER-plasma membrane (ER/PM) for measuring changes in lipid membrane characteristics (e.g., lipid order/ junctions in a variety of cell types. Within these micron-sized domains disorder) to ascertain the effects of engineered nanomaterials (ENM) on the ER membrane is held within 20 nm of the PM. Our previous work membranes. Previous studies of F2N12S have shown that this fluorescence has shown that ER/PM junctions serve as membrane trafficking hubs probe has potential for determining changes in membrane order when where endo/exocytosis events are localized. We also showed that large coupled with fluorescence lifetime imaging microscopy (FLIM). To our membrane proteins such as voltage-gated Naþ channels are excluded knowledge, F2N12S has yet to be used to investigate lipid membrane/nano- from the Kv2.1-induced ER/PM contact sites. In order to test the possibil- particle interactions. In this work, we used human red blood cells (RBC) as a ity that the Kv2.1-induced ER/PM junctions influence the overall cell sur- model membrane for interaction with ENM. FLIM was performed using a face diffusion landscape we used TIRF-based single molecule tracking to PicoQuant MicroTime 200. Methyl-beta cyclodextrin (MBCD) was used to examine the diffusion of CD4 and beta adrenergic receptors, two very extract cholesterol from the RBC membranes. Then 107 RBC were exposed different membrane proteins. Single-molecule tracking was performed to 25, 50 or 100 mg/ml of either titanium dioxide (TIO2), nickel oxide (NO) while simultaneously imaging ER/PM junctions induced either by Kv2.1, or zinc oxide (ZnO) nanospheres for four hours. F2N12S was added (100 Junctophilin-4 (JP4), or an artificial membrane bridge based on rapamycin nM), FLIM was performed on multiple fields of RBC, and intensity weighted ligands. The analysis of local mean square displacement (MSD) shows that average lifetimes, , were determined. In model solvent systems, the diffusion of these molecules is typically anomalous and the diffusive decreased with increasing dielectric constant (1.6 to 1.9 ns). Control RBC state displays a marked correlation to the proximity to ER/PM contact had = 3.9 ns, MBCD treated cells =3.2ns.TiO2 treated cells sites. This analysis indicates that Kv2.1 ER/PM junctions not only alter showed a dose-dependent = 4.7 ns (highest dose), while NiO exhibited protein diffusion in the vicinity of these membrane microdomains, but no change. ZnO at 25 mg/ml decreased to 1.9 ns with no further also concentrate PM proteins directly over the ER. Most interestingly, dif- decrease. The trends for the particle exposures are similar to pervious ferences between Kv2.1-induced junctions vs. junctions formed with Junc- work we have performed with a different probe. This work demonstrates tophilin or the rapamycin bridge indicate altered diffusion is specific to the the potential F2N12S has to determine changes in membrane order in a pri- presence of Kv2.1. These results suggest the dynamics of membrane pro- mary cell model. Funding: NSF MRI CHE-1531520, NIH R01ES023209,

BPJ 8566_8569 Sunday, February 18, 2018 99a

1F32ES027324, P20-GM103546, P30GM103338 and J Murdock Charitable ability. However, the high hemoglobin content inside the red blood cells may Trust. be a problem for the use of fluorescent membrane probes. Hemoglobin content inside erythrocytes is around 20 mM and the molecules near the inner mem- 505-Pos Board B275 brane surface would be the responsible for the quenching of fluorescent dyes Advanced STED Microscopy of the Membrane Organization in Activating such as 1,6-diphenyl-l,3,5-hexatriene (DPH) and 12-(9-anthroyl) stearic acid T-Cells (AS). Laurdan (6-lauroyl,1-2-dimethylamino naphthalene), a fluorescent 1,2 1 1 1 Iztok Urbancic , Erdinc Sezgin , Falk Schneider , Francesco Reina , probe commonly used to study membrane structure is being used in studies Christian Eggeling1. 1 2 in whole erythrocytes successfully. However, in order to avoid data misinter- University of Oxford, Oxford, United Kingdom, Jozef Stefan Institute, pretation due to the quenching effect of hemoglobin, authors either work at Ljubljana, Slovenia. constant hematocrit (constant hemoglobin concentration or they work with he- Nanoscale organization of the membranes of living cells plays crucial roles moglobin depleted erythrocytes (ghosts). Taking this into account, the aim of in numerous vital processes, including during the activation of T-cells and this work was to explore the use of Laurdan in the presence of hemoglobin their formation of the immunological synapse. However, the exact nature and during hemolysis. We studied the changes promoted in the membrane and function of reorganization of lipids during this key initiating event of rabbit red blood cells by the sucrose monoester of myristic acid, b-D-fruc- remain unclear. To gain further insight into this process, we employed tofuranosyl-6-O-myristoyl-a-D-glucopyranoside (MMS). We followed the two techniques that probe complementary properties of the membranes at interaction before and after hemolysis using FLIM (phasor plots) and Spectral the molecular level: 1) super-resolution STED-FCS to reveal detailed picture imaging (spectral phasor). Our data indicate that at sublytical concentration of of the diffusion of the lipids, with additional information on spatial hetero- surfactant (20 mM MMS), there is a decrease of about 35% in erythrocytes geneity provided by the scanning mode; and 2) spectral (super-resolution size, without changes in Laurdan lifetime or emission spectra. As hemolysis STED) imaging with environment-sensitive membrane probes, i.e. fluores- progress, Laurdan lifetime data are not informative due to the presence of he- cence microspectroscopy, to map differences in local molecular order within moglobin but Laurdan spectral phasor analyses clearly show an increase in the lipid bilayer. Using these methods, we monitored diffusion properties of membrane fluidity promoted by MMS. lipids and molecular order of the plasma membrane of (Jurkat) T-cells over Acknowledgements Fondecyt #1140454 (S.S.), Fondecyt #1080412 (G.G.), space and time upon their activation, revealing marked differences in lipid Beca de Doctorado Conicyt (C.S.). PICT 2657/2013 (VH). # FIS2015- diffusion and molecular order. For the most informative and robust descrip- 70339-C2-2-R (M.P.L.). LFD (8P41GM103540). tion of the latter, we systematically analyzed the benefits and pitfalls of the established representations, i.e. phasors, generalized polarization, and line- 508-Pos Board B278 shape description. Computational Insights into Fuels and Chemicals Extraction from Micro- 506-Pos Board B276 bial Biorefineries 1 2 1 STED-FCS Reveals Diffusional Heterogeneity of Lipids and GPI- Josh V. Vermaas , Gregg T. Beckham , Michael F. Crowley . 1Biosciences Division, National Renewable Energy Laboratory, Golden, CO, Anchored Proteins in the Plasma Membrane and Actin Cytoskeleton 2 Free Plasma Membrane Vesicles USA, National Bioenergy Center, National Renewable Energy Laboratory, Falk Schneider1, Dominic Waithe1, Mathias Porsmose Clausen2, Golden, CO, USA. Silvia Galiani1, Thomas Koller1, Gunes Ozhan3, Christian Eggeling1, Over the past two decades, substantial investments have been made in engi- Erdinc Sezgin1. neering microorganisms to produce specific fuels and chemicals as part of 1University of Oxford, Oxford, United Kingdom, 2University of Southern the global bioeconomy. Many target molecules accumulate intracellularly, Denmark, Odense, Denmark, 3Dokuz Eylul University Medical School, and a challenge is how to effectively extract the product from the cells Izmir, Turkey. without needing to destroy them due to the barrier imposed by the cell mem- The plasma membrane is a highly complex structure with nanoscale hetero- brane. For some hydrophobic compounds, an organic overlay is an effective geneities. The diffusion of proteins and lipids in the cellular membrane is strategy for nondestructive product extraction, although the relationship be- an important measure for its membrane heterogeneity and can be used to tween functional groups on the product and the rate of extraction are not well characterise transit interactions of molecules. In this study, we employ understood. Through both biased and unbiased molecular dynamics simula- super-resolution STED-microscopy in combination with fluorescence corre- tions for a range of fatty acyl compounds and terpenoids, we directly lation spectroscopy (STED-FCS) to investigate the diffusional properties of compute permeability coefficients for different steps of the extraction pro- lipid probes and fluorescently labelled GPI-anchored proteins (GPI-APs) in cess. Via comparative analysis between the calculated permeability coeffi- the plasma membrane of living cells and compare our findings to the diffu- cients and observed interactions between the compounds and the sional behaviour in actin cytoskeleton free cell-derived plasma membrane membrane, we determine how the rate limiting steps vary depending on vesicles (GPMVs). In the cellular plasma membrane, we find a variety of product chemistry. For instance, fatty aldehydes are found to transfer very different diffusion characteristics (hindered diffusion such as hop and rapidly across the membrane bilayer relative to alcohols, although their com- confined diffusion) but this hindered diffusion is mostly abolished in parable rate of extraction into the organic phase makes them equally effec- GPMVs for phospho- and sphingolipids. However, domain-like diffusion tive at extraction from the cell. In assessing the terpenoids, it is found that in of the ganglioside GM1 persists in GPMVs. STED-FCS measurements of general a modestly hydrophilic product improves desorption rates into an GPI-AP revealed also actin-dependent domain-like diffusion in the live organic phase sufficiently to make up for their lower bilayer crossing rate. cell plasma membrane (free diffusion in GPMVs), yet fluorescence cross- With this new insight, we can more effectively engineer microorganisms to- correlation spectroscopy (FCCS) indicated no co-diffusing GPI-APs in wards the production of these modestly hydrophilic fuel precursors or cells. This study underlines the strong influence of the cortical actin cyto- chemicals. skeleton on the plasma membrane organisation of most (but not all) of the membrane molecules. 509-Pos Board B279 Interactions of Poly(Ionic Liquid) Nanoparticles with Giant Unilamellar 507-Pos Board B277 Vesicles Using Laurdan and Spectral Phasor Analysis to Study Erythrocytes Eleanor Ewins1, Tom Robinson1, Rafael B. Lira1, Weiyi Zhang2, Membrane Solubilization Jiayin Yuan2, Markus Antonietti3, Rumiana Dimova1. Susana A. Sanchez1, Vanesa Herlax2, M. Pilar Lillo3, Catalina Sandoval4, 1Theory and Bio-Systems, Max Planck Institute, Potsdam, Germany, Joao Aguilar1, German Gunther4. 2Chemistry & Biomolecular Science, Clarkson University, Potsdam, NY, 1Facultad de Ciencias Quimicas, Departamento de Polı´meros, Facultad de USA, 3Colloid Chemistry, Max Planck Institute, Potsdam, Germany. Ciencias Quı´micas, Universidad de Concepcio´n, Concepcion, Chile, When approaching the complex field of drug delivery, one crucial aspect to 2Instituto de Investigaciones Bioquı´micas de La Plata (INIBIOLP), CCT—La consider is the interaction that occurs between particles and cellular mem- Plata, CONICET, Facultad de Ciencias Medicas, Universidad Nacional de La branes, as this is the first point of contact between cells and external material. Plata., La Plata, Argentina, 3Facultad de Ciencias Quimicas, Departamento The same can also be said of anti-microbial/bacterial agents. To progress de Quı´mica Fı´sica Biolo´gica, Instituto de Quı´mica-Fı´sica Rocasolano, within these bio-medical fields, new interactions between particles and mem- (CSIC), Madrid, Spain, 4Laboratorio de Cinetica y Fotoquı´mica, Facultad de branes must be explored. One such novel system is the poly(ionic liquid) Ciencias Quı´micas y Farmaceuticas, Universidad de Chile, Santiago, Chile. (PIL) nanoparticles, first reported by in JACS 133:17556, 2011. Polymer- Erythrocytes are widely used as a model system for membrane studies due to based nanoparticles have an increasing presence in research due to their their relatively simple structure (they lack nuclei and organelles having only attractive properties such as flexible functionality; when this is coupled the plasma membrane), their convenient experimental manipulation and avail- with the additional properties afforded by ionic liquids, a new realm of

BPJ 8566_8569 100a Sunday, February 18, 2018 possibilities becomes available. These nanoparticles form via dispersion poly- dispersions of POPE in either aqueous solution of TMAO or Urea, we can merisation of vinylimidazolium-type ionic liquid monomers into concentric see their antagonistic effect from a structural perspective; their influence on onion-like layered structures. It is thought that pharmacologically active ma- the organization of the self-assembled units. Both osmolytes seem to require terial could be incorporated between these layers, and that upon contact with a high temperatures to effectively interact with the POPE (headgroups) and influ- lipid membrane, the particle could ‘‘unwrap’’, delivering the encapsulated ma- ence the dispersion. TMAO favours the lamellar layer structures, while urea terial to the cell/vesicle interior. In addition, PILs have also been shown to tends to hinder the macro organization of the system. have anti-bacterial properties when coated on surfaces. Here, we present the characterisation of PILs interactions with phospholipid giant unilamellar ves- 512-Pos Board B282 icles (GUVs) of various surface charge density. We employ GUVs as a model Membrane Elasticity: Understanding the Gaussian Curvature Modulus system, as these closed lipid bilayers mimic the size and curvature of plasma from Lipid Tilt Theory membranes. We find that incubation of the PILs with GUVs results in pora- M. Mert Terzi, Markus Deserno. tion of the lipid membrane in a concentration-dependent manner as followed Physics, Carnegie Mellon University, Pittsburgh, PA, USA. with a microfluidic approach using a novel GUV trapping design. We show The elasticity of fluid lipid membranes is classically described by Helfrich the incurring morphological changes and observe a critical ‘‘poration’’ con- theory: a membrane is modeled as a structureless surface whose energy centration of PILs. At sub-micromolar concentrations, the vesicles loose only depends on geometry—specifically, mean and Gaussian curvature. contrast as sugars exchange across the membrane. At higher concentrations, The energy contribution of the Gaussian curvature only changes when a the vesicles collapse. Using fluorescently labelled PILs, we quantify the mem- membrane undergoes a topological change via a fission or fusion event. brane coverage. But once that happens, the associated energy change can be very large, given by 4p times the Gaussian curvature modulus. Due to its topological nature, measuring the Gaussian curvature modulus in experiment or simulation is un- Posters: Membrane Structure I fortunately difficult. We have recently revisited [J. Chem. Phys. 147, 084702 (2017)] a well-known tilt theory due to Hamm and Kozlov and shown that 510-Pos Board B280 the Gaussian curvature modulus appears in front of a novel tilt-curvature High-Speed Force Spectroscopy of Lipid Bilayer Rupture coupling term. This allows us to access it without the need for a topology Lorena Redondo-Morata, Felix Rico. change—even in experiments—by measuring the membrane undulation spec- Institut National de la Sante et la Recherche Medical (INSERM), Marseille, trum, where the Gaussian curvature modulus now enters together with France. bending and tilt modulus in such a way that all three of them can in principle Supported Lipid Bilayers (SLBs) are simple biological membrane models be disentangled. Beyond this, we emphasize the importance of a connection widely used for fundamental studies. Atomic Force Microscopy (AFM)- between the Gaussian curvature modulus, the twist modulus, and the second based Force Spectroscopy is an ideal technique to investigate the mechanical moment of the stress profile, which was already noted by Hamm and Kozov, properties of SLBs at the nanoscale, their elastic constants but also their and which may help us to understand some discrepancies raised earlier in the resistance to failure. The mechanical rupture of the lipid bilayer by the literature. AFM tip has been used extensively as a hallmark of its mechanical stability, usually by measuring the forces required to break the bilayer probed at 513-Pos Board B283 different velocities. Being a stochastic process, rupture forces generally in- Kinetics of Membrane Bending by Protein Crowding creases linearly with the logarithm of the loading rate, defining a character- Gokul Raghunath, Brian Dyer. istic dynamic force spectrum. Thanks to the miniaturization of the cantilever Chemistry, Emory University, Atlanta, GA, USA. and piezoelectric elements, High-Speed AFM (HS-AFM) allows reaching tip Dynamic shape changes in phospholipid membranes are considered critical to velocities in the millimeter per second range with microsecond time resolu- a number of important cellular processes. Such structural changes are thought tion (1), covering a wider dynamic force spectrum. Here, we apply HS-AFM to be driven by a number of possible mechanisms. Recently, the effects of force spectroscopy to access six decades of indentation velocities on sup- protein crowding on shaping and remodeling membrane surfaces has attracted ported lipid bilayers. We also assess the possible contribution of the canti- a lot of interest due to the ubiquity of crowded interfaces in biological sys- lever resonance frequency to the force spectrum. The preliminary dynamic tems. While a number of recent studies have confirmed the ability of proteins force spectrum of DOPC can be interpreted with a single energy barrier to drive curvature solely via a colligative, crowding-based mechanism- a num- although possibly followed by an outer state. The outcomes allow us to eval- ber of key fundamental mechanistic details remain unsolved. Specifically, uate the validity of general theoretical developments applied in the field (2). very little is known about the kinetics of this process. What are the timescales 1.Rico F, Gonzalez L, Casuso I, Puig-Vidal M, & Scheuring S (2013) High- of membrane deformation process? How kinetically separated are protein Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular binding and the membrane bending events? Is membrane deformation pre- Dynamics Simulations. Science 342(6159):741-743. 2.Butt HJ & Franz ceded by a previously unresolved intermediate? To address these questions, V (2002) Rupture of molecular thin films observed in atomic force micro- we provide a comprehensive study on the kinetics of these events through scopy. I. Theory. Physical Review E 66(3). stopped-flow fluorescence using FRET as the spectroscopic handle. A fluorophore-quencher pair was used to study the protein binding kinetics to 511-Pos Board B281 the bilayer, while a donor-acceptor pair was used as a reporter for membrane Urea and TMAO on Lipid Bilayers area expansion. We report a complex multi-phasic kinetic behavior for both Sergio S. Funari1, Bernstorff Sigrid2, Joana Valerio1. the protein association and membrane expansion process. The association 1 2 HASYLAB, Hamburg, Germany, Elettra-Sincrotrone Trieste, Trieste, Italy. rates follow a surprising negative trend with respect to increasing protein con- We studied the influence of osmolytes Urea and TMAO (Trimethylamine centration presumably due to saturation of membrane binding sites owing to N-oxide), which have antagonistic effects on the fluidity and stability of protein crowding. We find that the membrane deformation happens at signif- lipid membranes. In red blood cells, urea slightly increases the gel-phase do- icantly slower timescales relative to the protein binding events. The mains, but this effect is counteracted by TMAO. We used a combination of complexity of the fits suggest that the membrane bending process might be simultaneous SAXS/WAXS/DSC measurements to identify the structures, their preceded by an unresolved intermediate. Taken together with a predictive dimensions and phase transition temperatures. Thermal scans show morphol- theoretical model that is currently underway, we believe that our observations ogies similar to pure lipids, although with different dimensions and transition will advance our knowledge about membrane bending driven by protein temperatures. In POPE, we observe structures with high curvature, and upon crowding. temperature cycling, the induction of possible cubic phases. Fully hydrated POPE prepared in 400 mM TMAO surprisingly lacks diffraction over a wide 514-Pos Board B284 temperature range on cooling from the hexagonal phase. The diffraction peaks Super Resolution Imaging of Highly Curved Membrane Structures in in the last patterns are much less intense than at the beginning of the scan. DSC Giant Unilamellar Vesicles Encapsulating Polymer Solutions shows a higher transition temperature on heating than pure aqueous POPE, Ziliang Zhao, Debjit Roy, Jan Steinkuhler,€ Tom Robinson, Roland Knorr, while during cooling this is not seen. DSC of POPE in 200 mM Urea shows Reinhard Lipowsky, Rumiana Dimova. a transition at a temperature higher than POPE and POPE in 400 mM Max Planck Institute of Colloids and Interfaces, Potsdam, Germany. TMAO, however involving less heat than the previous system. The SAXS Giant vesicles encapsulating polymer solutions can undergo morphological scan indicates a less organized stack of bilayers and the hexagonal phase, could transformations upon deflation (Adv. Mater. Interfaces 4:1600451, 2017). not be seen up to 85oC. Moreover, no organized structure was formed during The vesicle membrane can initiate budding and nanotube formation. The nano- cooling. This evidences that Urea destabilizes bilayers due to the interaction tube diameter is related to the membrane spontaneous curvature. Based on our on the headgroup interface region. Combining SAXS/WAXS and DSC from previous findings (ACS Nano 10:463, 2016), nanotubes with membrane

BPJ 8566_8569 Sunday, February 18, 2018 101a composition of the liquid-disordered phase have a diameter below optical res- lipids on nanoscale curvature sites in supported lipid bilayers (SLBs). Giant olution. Direct imaging of the intrinsic contact angle formed by the membrane unilamellar vesicles (GUV) were burst over 100 nm diameter nanoparticles and the two phases in budded vesicles (PRL, 103:238103, 2009) is also hin- (NP) on glass coverslips. Membrane composition variations yielded minimal dered by the poor axial resolution in confocal microscopy. Here, we use a super changes to the effects of curvature, including mixtures of POPC, lysoPC, resolution technique, stimulated emission depletion (STED) microscopy in DOPC, and DLPC. However, membrane curvature had a widely varying ef- both 2D and 3D mode combined with microfluidics to study these remarkable fect on different fluorescent lipids. Lipid head labeled TexasRed-DHPE membrane morphologies. We first designed a microfluidic device which can diffused half of the speed on curvature site compared to lipid tail labeled Top- dramatically increase the trapping efficiency of giant unilamellar vesicles FluorPC or TopFluorPIP2 yet two time faster in flat lipid bilayer. The curva- (GUVs) and improve the solution exchange rate. Then, with a resolution less ture affected these fluorescent lipids in drastically different ways. We than 35nm from STED microscopy, we visualize the membrane nanotube struc- hypothesize that diffuser tail properties affects diffusion at curvature sites tures with unprecedented detail, and compare the directly measured nanotube more than head groups. Further head-labeled lipids and tail-labeled lipids diameters with previously reported theoretical and experimental ones. Addi- will be studied to examine the generality of this observation. Additionally, tionally, by manipulating the height of the microfluidic channels, we pinch the site of the fluorescent label affected the sorting of the fluorescently labeled and orient the budded vesicles to image the intrinsic contact angle with lateral lipid to the curvature site. Understanding and comparing the molecular sorting STED resolution. These highly curved membrane structures imaged with super in lipid bilayers can further our understanding of the curvature sensing lipids resolution microscopy will serve to deepen and expand our understanding of properties such as tail saturate/ unsaturated preference and head group types. biomembranes. This work is part of the MaxSynBio consortium, jointly funded These studies will contribute to the greater biophysical understanding of by the Federal Ministry of Education and Research of Germany and the Max membrane curvature. Planck Society. 518-Pos Board B288 515-Pos Board B285 Cardiolipin-Induced Phase Separation in Biomimetic Mitochondrial Intrinsic Curvature Effects of Oxidized Lipids on Spatial Lipid Membranes and Cardiac Vesicles is Dependent on Cardiolipin Concentra- Organization tion and Acyl Chain Composition Radha Ranganathan, Intisar Alshammri. Edward R. Pennington1,2, E. Madison Sullivan1,2, David A. Brown3, Physics, California State University Northridge, Northridge, CA, USA. Saame Raza Shaikh1,4. The oxidized lipid, PGPC, is a curvature inducing lipid. It has distinctly 1Department of Biochemistry & Molecular Biology, Brody School of different effects on the lipid organizations in POPC, DOPC, and DPPC bilayers Medicine, East Carolina University, Greenville, NC, USA, 2East Carolina that are observable by average Laurdan fluorescence generalized polarization Diabetes & Obesity Institute, Greenville, NC, USA, 3Department of Human measurements. These bilayer lipids themselves differ in their individual curva- Nutrition, Foods, and Exercise, Virginia Tech Corporate Research Center, tures. Oxidized lipid- induced spatial lipid reorganization was investigated in Virginia Tech, Blacksburg, VA, USA, 4Department of Nutrition, Gillings model bilayer lipid membranes using Dynamic Light Scattering and Laurdan School of Public Health and School of Medicine, University of North Fluorescence. Distinct differences were observed between the effects of the Carolina at Chapel Hill, Chapel Hill, NC, USA. oxidized lipid, PGPC, on the three bilayer lipid membranes of POPC, DPPC, Cardiolipin (CL) is critical for inner mitochondrial membrane (IMM) bio- and DOPC. The OxPL, PGPC, increases the polarity of POPC and of the gel physical organization and protein activity. It is hypothesized that localized phase DPPC and decreases the polarity of DOPC and of the liquid phase domains of CL form within the IMM to regulate the formation and function DPPC. DLS experiments showed that mixed vesicles are formed by POPC / of respiratory proteins complexes. However, it is poorly understood how PGPC and by PGPC / gel phase DPPC. On the other hand DOPC / PGPC mitochondrial CL domains are organized. To address this, we used biochem- and PGPC / liquid phase DPPC separate into coexisting vesicles and micelles. ical and biophysical techniques to study CL microdomains. We first used clas- The DLS and Laurdan fluorescence experiments interpreted together suggest sical detergent extraction to isolate CL microdomains. Studies with three that POPC mixes randomly with PGPC; but DOPC and DPPC do not. The different detergents, at varying concentrations, showed increased non- different lipid organizations are hypothesized to be driven by intrinsic curva- specific solubilization of all mitochondrial phospholipids and protein. This ture differences between the mixing molecules. demonstrated that CL microdomains couldn’t be isolated via detergent extrac- 516-Pos Board B286 tion methods. Subsequently, we studied CL microdomains by constructing, Vitamin E Bends Model Cell Membranes to Promote its Antioxidant and quantitatively imaging, giant unilamellar vesicles (GUVs) modeling the Function IMM. In addition, we constructed giant mitochondrial vesicles (GMVs) Andres T. Cavazos, Morris I. Bank, Michaela E. Bell, Zachary L. Leach, from isolated rodent cardiac mitochondrial phospholipids. Results from imag- Jacob J. Kinnun, Stephen R. Wassall. ing experiments indicated that in the absence of protein, the physiochemical Department of Physics, IUPUI, Indianapolis, IN, USA. properties of lipid-lipid interactions were not sufficient for formation of phase The primary role of vitamin E in membranes is to protect phospholipids from separated microdomains. However, reconstitution of cytochrome c at oxidation. A longstanding question has been whether there is a structural differing concentrations induced strong morphological changes concomitant component in support of its function. Here we focus on the effect of vitamin with formation of distinct phase separated structures that were driven by a E on the structure of model membranes with phospholipids containing PC decrease in phospholipid packing and increase in the membrane elasticity and PE headgroups. Using solid-state 2H NMR spectroscopy, complemented modulus. Lastly, we studied the role of CL concentration and acyl chain by DSC, we compare the molecular organization of 1-palmitoyl-2-oleoyl-sn- composition on membrane domain organization and phase separation with cy- glycerophosphatidylethanolamine (16:0-18:1PE, POPE) and 1-palmitoyl-2- tochrome c. Decreased CL concentration, as observed in several metabolic oleoyl-sn-glycerophosphatidylcholine (16:0-18:1PC, POPC) mixed with diseases, prevented morphological phase separation. Similarly, the incorpora- increasing concentration of vitamin E. Somewhat akin to cholesterol, the gen- tion of docosahexaenoic acid (DHA) into CL, also associated with differing eral effect on both POPE and POPC is to broaden and depress the gel to liquid metabolic disorders, prevented phase separation due to differences in cyto- crystalline phase transition - vitamin E disrupts chain packing in the gel phase chrome c-acyl chain cofactor interactions. Altogether, our data demonstrate and restricts chain reorientation in the liquid crystalline phase. The spectra that CL microdomain organization is driven by lipid-protein interactions and likely exists in specific regions of high membrane curvature and protein observed with POPE-d31 consist of a superposition of two components at higher temperatures that we ascribe to vitamin E promoting a transition from lamellar concentration. (La) to inverted hexagonal phase (HII). In contrast, POPC-d31 maintains La phase in the presence of vitamin E. The tendency to induce negative curvature 519-Pos Board B289 Impact of Phospholipid Acyl Chain Length Mismatch on Sterol Affinity indicated by the formation of HII phase suggests vitamin E would make mem- branes less permeable to free radicals. and Lateral Segregation Oskar Engberg1, Victor Hautala1, Hiroshi Tsuchikawa2,3, 517-Pos Board B287 Thomas K.M. Nyholm1, Michio Murata2,3, J. Peter Slotte1. Single-Lipids Diffusion and Lipid Sorting at Nanoscale Curvature Sites 1Faculty of Science and Engineering, A˚ bo Akademi University, Turku, Xinxin Woodward, Christopher V. Kelly. Finland, 2Department of Chemistry, Graduate School of Science, Osaka Physics, Wayne State University, Detroit, MI, USA. University, Osaka, Japan, 3Lipid Active Structure Project, Japan Science and Membrane curvature may cause changes to the local effective membrane vis- Technology Agency, Erato, Toyonaka, Osaka, Japan. cosity and induce a lateral sorting of membrane components. However, the Lateral segregation into ordered and disordered domains may occur when mix- relationships between curvature, diffusion, and lipid shape have not been es- ing phospholipids (PLs) with high and low gel-fluid transition temperatures. tablished. We used single-particle tracking to observe fluorescently labeled Cholesterol is known to influence this lateral domain formation in model

BPJ 8566_8569 102a Sunday, February 18, 2018 membranes, which likely resembles the formation of cholesterol rich nanodo- structural and thermodynamics characteristics of homogenous lipid species, mains in biological membranes. In previous studies the sterol affinity difference but also to properly reproduce experimental phase separation. Several iterations between unsaturated and saturated PLs was found to influence the ability of of the parameters were tested across the entire phase diagram, and at different cholesterol to promote formation of ordered domains. temperatures. Our optimized parameters possess greatly improved fidelity to Our aim was to study the formation of ordered domains when there is an acyl experimental phase properties. This approach can be applied to other, biologi- chain length mismatch between unsaturated PCs and SM before segregation. cally relevant lipid mixtures. Preliminary sterol partitioning results suggested that the sterol affinity for the This work performed under the auspices of the U.S. Department of Energy by di-monounsaturated-PC bilayers correlated with acyl chain length. Sterol affin- Lawrence Livermore National Laboratory under Contract DE-AC52- ity for palmitoyl-sphingomyelin (PSM) in these PC bilayers was also exam- 07NA27344 and by Los Alamos National Laboratory under Contract DE- ined. 2H nuclear magnetic resonance (NMR) spectroscopy results showed AC52-06NA25396. Release numbers: LLNL-ABS-739386. that the acyl chain order of deuterated PSM in binary bilayers correlated with the acyl chain length of the di-monounsaturated-PC included in the 522-Pos Board B292 bilayer. Lateral segregation was explored using time resolved fluorescence of Lipid Domain Boundary as Universal Attractor 1 2,3 2,3 trans-parinaric acid. To compare to what degree cholesterol influenced lateral Veronika V. Alexandrova , Sergey A. Akimov , Timur R. Galimzyanov . 1Medical physics, Moscow State University, Faculty of Physics, Moscow, segregation we determined the amount of saturated PLs required to form or- 2 dered domains in fluid bilayers containing 0 or 20 mol% cholesterol. Russian Federation, Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and Electrochemistry RAS, Moscow, Russian Federation, General observations about SM and cholesterol interaction with unsaturated 3 mismatched acyl chain length PCs, and how it leads to formation of ordered Theoretical Physics & Quantum Technologies, National University of domains will be presented. Science and Technology ‘‘MISiS’’, Moscow, Russian Federation. We show that boundary of liquid-ordered domains (rafts) works as the univer- 520-Pos Board B290 sal attractor for a wide variety of membrane minor components, such as various Are Vitamin E and PUFA Driven Together by Choleterol? Computer peptides and non-bilayer lipids. Using elasticity theory approach developed for Simulation Studies lipid membranes, we show that these kinds of impurities tend to distribute to the Samuel W. Canner, Xiaoling Leng, Fangqiang Zhu, Stephen R. Wassall. narrow intermediate region at the liquid-ordered domain boundary. Moreover, Physics, Indiana University-Purdue University Indianapolis, Greenwood, IN, redistribution of these components dramatically varies the morphology and size USA. of liquid-ordered domains, which is achieved by changing the domain bound- Vitamin E (a-tocopherol) is the principal antioxidant in cell membranes ary energy. This effect was predicted theoretically and confirmed experimen- and is believed to protect PUFA (polyunsaturated fatty acids) from oxida- tally for the ganglioside GM1 [1]. tion. Since a-tocopherol is in low concentration in cell membranes, Despite the low concentration, minor components play a crucial role in cell life: generally less than 0.1 mol%, co-localization with PUFA-containing phos- whether it amphipathic peptide, cancer-related ganglioside or fusion peptides pholipids would be advantageous to protect them from oxidative damage. sensible to raft boundary. Liquid-ordered lipid domains are assumed to be We hypothesize that cholesterol, ubiquitous in the plasma membrane of an- important actors in diverse cellular processes, mainly signal transduction and imals, drives a-tocopherol and PUFA-containing phospholipids together. membrane trafficking. They are thicker than the disordered part of the mem- All-atom, umbrella sampling molecular dynamics (MD) simulations that brane, thus compensating the hydrophobic mismatch between transmembrane were run on bilayers composed of SOPC (1-stearoyl-2-oleoyl-sn-glycero- proteins and the disordered lipid environment. That leads to the main cause 3-phosphocholine) and SOPC with cholesterol (50 mol%) demonstrate that of the boundary energy, a strained asymmetric structure of the boundary, which the presence of cholesterol reduces membrane affinity for a-tocopherol. can be relaxed by the line-active components. With the use of coarse graining (CG) methods, a new model for a-tocopherol Therefore the found attractive activity of the domain boundary can explain the was created to better mimic the physical properties of the branched phytyl mechanisms and suggest the new pathways of the strong influence of the low side chain. This model was then employed in CG simulations on a bilayer concentration of membrane impurities on various physiological processes composed of a-tocopherol mixed with SM (sphingomyelin), cholesterol involving rafts. The work was supported by the Russian Science Foundation and polyunsaturated PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3- (project #17-79-20440) phosphocholine) that separates into SM-rich/cholesterol-rich (raft) and 1.Galimzyanov et al., Langmuir 33(14) (2017). PDPC-rich/cholesterol-poor (non-raft) domains. The results of these studies 2.Galimzyanov et al., Physical Review Letters 115.8 (2015). will be presented. 523-Pos Board B293 Interactions between Sterols and Phospholipids with Different Head- 521-Pos Board B291 groups - Influence on Lateral Segregation Accurate Phase Separation of Complex Lipid Mixtures (DPPC/DOPC/ Shisihir Jaikishan, Oskar Engberg, Victor Hautala, J. Peter Slotte, CHOL) with a Refined Coarse Grained Martini Model Thomas K.M. Nyholm. Timothy S. Carpenter1, Cesar A. Lopez2, Chris Neale3, Helgi I. Ingolfsson1, Bioscienses - Biochemistry, A˚ bo Akademi University, Turku, Finland. Cameron Montour4, Sandrasegaram Gnanakaran2, Felice C. Lightstone1. 1 Cholesterol is an important modulator of the structure and function of Biosciences and Biotechnology Division, Lawrence Livermore National mammalian cell membranes. The effect of cholesterol is mediated through Laboratory, Livermore, CA, USA, 2Theoretical Biophysics and Biology 3 the interactions between the sterol and the phospholipid components of the Group, Los Alamos National Laboratory, Los Alamos, NM, USA, Center for membrane. Recently, we have reported a correlation between the relative af- Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, NM, USA, 4 finity cholesterol have for different phospholipid components and the degree Biochemistry and Molecular and Cellular Biology, Georgetown University, to which the sterol can facilitate lateral domain formation. In the current proj- Washington, DC, USA. ect, the aim is to determine the affinity of cholesterol and other sterols for Ternary mixtures containing a high melting temperature lipid (such as di- phospholipids with different headgroups, and to analyze how the measured af- palmitoyl-phosphatidylcholine, DPPC), a low melting temperature lipid (such finities relate to the formation of lateral membrane structure. The affinity of as di-oleoyl-phosphatidylcholine, DOPC), and cholesterol (CHOL) form bila- the sterols for phospholipid bilayers is measured using different fluorescence yers consisting of up to three different lipid phases. The lipid phases that can based partitioning assays, and the formation of lateral domains in the bilayers form are liquid-disordered (Ld), liquid-ordered (Lo), gel-like (Lb), or any com- is determined by measuring the fluorescence lifetimes of trans-parinaric acid bination of the three. The phase(s) present within these membranes are depen- as a function of lipids composition. The results from these experiments offer dent on both temperature and the specific percentage composition of the three insights into the mechanisms driving the formation of lateral domain forma- components within the membrane. These phases have been well mapped exper- tion that will help us understand the processes that occurs in cellular imentally to construct detailed phase diagrams. membranes. Previous efforts to use Molecular Dynamics (MD) simulations to reproduce the distribution of phases have proved somewhat challenging. This is either due to 524-Pos Board B294 the size/timescale sampling restrictions of all-atom approaches, or some of the Lipid Domains at the Plasma Membrane of Red Blood Cells: Organization inherent limitations of Coarse Grain (CG) forcefields such as limited lipid pa- and Involvement in Deformation rameters that were made to reproduce homogenous membranes, but can behave Louise Conrard. erroneously when in lipid mixtures. Cell Biology, Institut de Duve, Brussels, Belgium. In this work, the existing CG MARTINI DPPC and DOPC lipid parameters Erythrocytes are highly deformable cells that go through capillaries eight were iteratively optimized by fitting to extensive all-atom simulations run using times-narrower than their diameter to deliver oxygen throughout the body. the CHARMM36 forcefield. The parameters were tested not only to preserve This deformability is linked to erythrocytes specific features such as their

BPJ 8566_8569 Sunday, February 18, 2018 103a biconcave shape, their highly regulated hemoglobin concentration and the 527-Pos Board B297 strong anchorage of their membrane to the underlying cytoskeleton. Based DNA Origami as an Experimental Tool to Probe Critical Casimir Forces in on our recent evidence for submicrometric lipid domains upon labeling of Membranes cholesterol and polar lipids (sphingomyelin, phosphatidylcholine, ganglioside Anshul V. Puli, Sarah Veatch. GM1) in erythrocyte outer plasma membrane leaflet, we here explore the map- Biophysics, University of Michigan, Ann Arbor, MI, USA. ping of lipid domains in erythrocytes at resting state and under deformation. DNA origami are nanoscale assemblies of DNA designed to fold into two or Thanks to differential properties between cholesterol- and polar lipid- three dimensional 10-100 nm sized structures designed by the user. Further- enriched domains (curvature association, lipid order, temperature dependence, more, it is possible to assemble larger DNA objects by encoding interactions cholesterol content dependence) and partial spatial dissociation, we suggest the between origami. Past work has taken this approach to assemble nanotracks coexistence of at least two types of lipid domains at the erythrocyte surface at or nanogrids in solution or have used coupling to homogeneous bilayers to resting state: those enriched in cholesterol and preferentially associated with facilitate two dimensional assembly over large areas. In membranes near a high-curvature membranes and those mostly enriched in phosphatidylcholine miscibility phase transition, theoretical investigations predict that inclusions and ganglioside GM1 and associated with low-curvature membranes. Biophys- feel effective potentials based on their preference for a specific local membrane ical properties of these domains are currently explored by atomic force micro- environment. Similar critical Casimir forces have been shown experimentally scopy and force correlation spectroscopy. Lipid domains abundance, to facilitate phase transitions in colloidal systems in three dimensions. In this composition and biophysical properties are differentially modulated by (i) poster, we will present ongoing experimental work aimed at exploring if and impairment of erythrocyte deformability (energy depletion, modulation of how coupling origami to membranes near a miscibility transition facilitates anchorage to the cytoskeleton by pharmacological and genetic approaches), their assembly into extended structures. (ii) simulation of deformation (calcium channel activation, stretching in silicon chambers) and (iii) simulation of shape restoration after deformation (stimula- 528-Pos Board B298 tion of calcium efflux). Indeed, high-curvature cholesterol-enriched domains Looking for Groundbreaking Structural and Functional Features in the gather under deformation while low-curvature domains become enriched in Lung Surfactant System using a Surface-Active Agent Purified from Hu- sphingomyelin and lose some of their ganglioside GM1 enrichment. We hy- man Amniotic Fluid 1,2 1,2 ´ 1,2 pothesize that cholesterol-enriched domains can help to stabilize high curvature Jose C. Castillo-Sanchez , Nuria Roldan , Begon˜a Garcı´a-Alvarez , 3 2,3 1,2 membranes during deformation while polar lipid-enriched domains, especially Emma Batllori-Badia , Alberto Galindo , Antonio Cruz , Jesu´sPerez-Gil1,2. those enriched in sphingomyelin, are involved into calcium balance regulation 1 during or after deformation. This hypothesis is under investigation. Biochemistry and Molecular Biology Department, Complutense University, Madrid, Spain, 2Research Institute Hospital Universitario ‘‘12 de Octubre’’ 525-Pos Board B295 (imas12) and Complutense University, Madrid, Spain, 3Department of Lateral Phase Behavior of Human Skin Lipids Obstetrics and Gynaecology, Hospital Universitario ‘‘12 de Octubre’’, Michael J. Counihan, Shelli L. Frey. Madrid, Spain. Chemistry Department, Gettysburg College, Gettysburg, PA, USA. Pulmonary surfactant is a surface-active agent essential to facilitate respiratory The human stratum corneum (SC) is the outermost layer of the epidermis, the dynamics by covering the alveolar air-liquid interface and decreasing surface first barrier against the environment. This largely impenetrable layer consists of tension. Typical surfactant preparations have usually been isolated from animal anucleated, flattened, protein-rich cells called corneocytes suspended in an lungs, and lipids and hydrophobic proteins are obtained by organic extraction extracellular lipid matrix. This lipid matrix is composed of ceramides (Cers), and reconstituted in aqueous suspensions to be used in clinical applications. cholesterol (Chol), and free fatty acids (FFAs). The SC lipids are unique These procedures disrupt the native organization of lipids and proteins in sur- from other biologically relevant lipids commonly found in cell membranes in factant complexes. However, lung surfactant is synthesized and packed into that the Cers and FFAs have very long (>20 carbons) fatty acid chains, nearly specialized organelles of alveolar type II cells, the lamellar bodies, whose all of which are fully saturated. Lateral packing and domain formation within structural features determine the optimal surface activity of freshly secreted Langmuir monolayers of the individual Cer, Chol, and FFA components and complexes. Several approaches have tried to obtain large amounts of surfactant their binary and ternary mixtures were investigating using surface pressure assembled into lamellar body structures, to be used not only in research but also vs. molecular area isotherms and fluorescence microscopy to determine the in clinical applications. Here, we propose that amniotic fluid can be a source of role of each component in the material properties of the SC layer. Particular a lung surfactant whose structural features are similar to those reported and ex- attention was paid to the effect of FFA length on membrane fluidity – FFA pected for freshly secreted lamellar bodies. In this work, we compare the struc- chains ranging from 16 to 30 carbons (24 carbons being the most common in ture and functional behavior of surfactant obtained from amniotic fluid with the SC) were studied. A physiologically accurate mixture of several FFAs that of surfactant purified from bronchoalveolar lavages of porcine lungs. Am- with different chain lengths (FFAmix) is shown to be more stable to niotic fluid surfactant contains highly packed multilamellar structures similar to compression-expansion cycling than any single FFA. Longer chain FFAs lamellar bodies as observed by Cryo-Electron Microscopy. These complexes have a condensing effect in Cer monolayers, while Chol tends to disrupt seem to assemble as highly dehydrated membranes where phospholipid acyl the Cer-FFA organization. The trans double bond in the sphingosine chains are highly immobilized and non-lamellar phases are present as sensed backbone of certain SC Cers allows close lateral lipid packing, stiffening the by Laurdan fluorescence, Electron Spin Resonance and 31-Phosphorus Nuclear layer. Additionally, there are significant differences between the canonical Magnetic Resonance. Moreover, amniotic fluid surfactant is able to reduce effi- 1:1:1 Cer:Chol:FFA mixture and a more physiologically accurate 10:9:4 ciently surface tension along compression-expansion cycling simulating respi- mixture with more representative Cers and FFAmix. Including a higher ratio ratory dynamics, exhibiting a better resistance to inhibition by agents such as of Cers and a lower one of FFAs in such mixtures condenses the monolayer serum or meconium. despite FFAs being more incompressible than Cers alone, indicating that ternary SC lipid mixtures experience non-additive complex lipid interactions. 529-Pos Board B299 Modeling Formation of Caveolar Superstructures 526-Pos Board B296 Gonen Golani, Michael M. Kozlov. Lipid Bilayer Modulation using DNA Origami Mimics of Clathrin Tel Aviv University, Tel Aviv, Israel. Vivek Ramakrishna1,2, Celine Journot3, Andrew J. Turberfield3, Caveolae are small, 60-80 nm, flask-like structures formed, abundantly, on the Mark Ian Wallace1. plasma membranes of mammalian cells by membrane proteins, caveolins, cyto- 1Department of Chemistry, King’s College London, London, United solic proteins and cavins. Caveolae are remarkably uniform in their shapes and Kingdom, 2Physical and Theoretical Chemistry, University of Oxford, dimensions and can cover up to 50% of the membrane surface. Whereas cav- Oxford, United Kingdom, 3Department of Physics, University of Oxford, eolae have been suggested to participate in different processes of intracellular Oxford, United Kingdom. trafficking, the most prominent proposed role of these structures is to serve as a The assembly of clathrin coats on vesicles is a major endocytotic pathway, and membrane reservoir, which buffers the changes of membrane tension in the effects of clathrin’s structure on vesicle formation are still unclear. Here we response to cell swelling or stretching. The mechanism of the latter phenome- exploit DNA origami nanostructures designed as structural mimics of clathrin. non must be closely related to the interplay between the caveolae organization Using DNA clathrin mimics we seek to isolate the effects of shape and structure on the plasma membrane and the membrane tension. Our study addresses, on membrane properties. DNA nanostructures associate with lipid membranes theoretically, this interplay and its possible consequences for the caveolar func- via cholesterol anchors and can be triggered to polymerise into a meshwork on tion. We analyze the membrane-mediated and tension dependent interaction the membrane. We explore how this origami network can be used to induce between caveolae, and model the related formation of the caveolae superstruc- structural changes in giant unilamellar vesicles. tures described as caveolar oligomers or rosettes. We show that self-assembly

BPJ 8566_8569 104a Sunday, February 18, 2018 and stabilization of such caveolar oligomers are driven by low membrane ten- bility of TF is diminished leading to enhanced film breakup. The aqueous TF is sions and can serve as a mechanism of buffering the tension variations. We stabilized by presence of a relatively thin layer of lipids coating its outermost analyze the formation of caveolar doublets, triplets, quadruplets and pentaplets, surface, so called tear film lipid layer (TFLL). This layer is typically altered in compare their oligomerization energies and kinetic stability. We demonstrate DED but the details of its behavior are not fully understood. that the numerically recovered morphologies of these caveolar oligomers are In order to reveal basic properties of TF and the role of TFLL, we apply a in a close agreement with experimental observations predict the probability multi-level approach. First, we employ in silico modeling using large-scale of formation of each kind of oligomers and estimate their relative contributions coarse grain molecular dynamics simulations of TF models at the molecular to the tension buffering. level. Second, we experimentally assess biophysical properties of TF mimics with compositions corresponding to those used in simulations. These exper- 530-Pos Board B300 iments include characterization of surface films by Langmuir balance tech- Characteristics of Bulk Endocytosis within Chromaffin Cells nique combined with fluorescence microscopy. Third, we evaluate behavior Seth A. Villarreal, Gianvito Arpino, Wonchul Shin. of human TF extracts employing experimental approaches parallel to those National Institute of Neurological Disorders and Stroke, Bethesda, MD, used for film mimics. The combination of these three levels of description, USA. spanning from behavior of individual molecules to collective macroscopic Rapid vesicle fusion with the plasma membrane for signaling or cargo properties of human tear extracts, allows us for reliable in-depth description release allows large and sudden signaling shifts by a cell. In readiness for of basic TF properties. Here, we apply our multi-level approach to study the repeating heavy requirements in signaling, bulk endocytosis fulfils this role of two molecules, cetalkonium chloride (CKC) and poloxamer 188 role, and is an area of study to understand how vesicle proteins are concen- (P188) in stabilization of TF. It was earlier observed that these exogenous trated and the bulk endosomes partitioned into new vesicles. In this work, we agents stabilize TF in DED in mice and humans. We demonstrate that reduc- examine the recovery of vesicles to maintain a ready supply for bovine chro- tion of polar lipids content, similar to that occurring in DED, leads to desta- maffin cells. Four general routes exist for vesicle recovery: Clathrin Medi- bilization of TF by enhanced water-nonpolar lipids contacts. We explain how ated Endocytosis (CME), Clathrin Independent Endocytosis (CIE), Bulk molecular-level interactions of CKC and P188 with TF prevent this destabi- Endocytosis (BE), and Kiss & Run. The prioritization of these mechanisms lization both at microscopic and macroscopic levels. varies depending on the level of stimulation. This work examined the recy- cling of the plasma membrane into vesicles after stimulation within the BE 533-Pos Board B303 pathway, as visualized by electron microscopy (EM). Negative stain EM al- Artificial Diamidophospholipids: Monitoring Bilayer Properties using lows a complete and detailed cross-sectional view of a cell, readily allowing Membrane Array Technique before and after stimulation analysis for tallying a population of events. In Ekaterina Zaitseva1,2, Ibrahim Halimeh2, Gerhard Baaken2, Renate Reiter3, this case, the distribution of recovered membrane for recycling into vesicles. Andreas Zumbuehl4, Jan Behrends1. A common problem with single layer sections can be determining when a 1Institute of Physiology II, University of Freiburg, Freiburg, Germany, bulk endocytotic event has completed fission from the plasma membrane. 2Ionera Technologies GmbH, Freiburg, Germany, 3Experimental Polymer In this case, it is alleviated by using serial sections to determine the overall Physics, University of Freiburg, Freiburg, Germany, 4Department of nature of bulk endocytotic structures as a cellular process for the recovery of Chemistry, University of Fribourg, Freiburg, Switzerland. vesicles. Artificial diamidophospholipids are known to form faceted liposomes with 531-Pos Board B301 interdigitated membrane structure. Here we report a formation and character- Morphogenesis of Small Intestinal Villus ization of planar lipid bilayers from an artificial C16-1,3-diamidophospholipid Yuki Umemura, Shigeyuki Komura, Takuma Hoshino. utilizing aparallel lipid bilayer platform based on a microelectrode cavity array Department of Chemistry, Tokyo Metropolitan University, Hachioji-shi, (MECA) chip. Japan. Membrane specific capacitances, voltage threshold for irreversible electropora- In recent years, morphogenesis of organism has been actively studied in terms tion (Vep) as well as conductance and single channel kinetics of the ion channel of physics and/or mechanics. In particular, periodic patterns such as wrinkled gramicidin A were monitored in diamidophospholipid (Pad-PC-Pad) mem- structures have attracted great interest both from experimental and theoretical branes with different cholesterol content and at different temperatures and point of view. The small intestine is covered by a single layer of epithelial cells. compared with Di-phytanoyl-phosphatidylcholine membranes. Buckling instability of the monolayer leads to the formation of periodic arrays Planar Pad-PC-Pad membranes showed appearance of membrane lesions and 5 of villus and crypts. In vivo, villus assemble together to form various patterns in generally low Vep (203 15 mV). Stabilization of Gramicidin A ion channels different parts of the small intestine. It forms stripes at the entrance of the in- in an open conformation together with high specific capacitance of the Pad-PC- testine (near the stomach), while it gradually changes into a finger-like pattern Pad membranes are compatible with lipid interdigitation. These electrophysio- in more distal parts. logical results corroborate a previous finding that cholesterol and temperatures We theoretically study undulating structures of a cell monolayer. We present a above a phase transition are able to prevent lipid interdigitation in Pad-PC-Pad model for such structures based on the buckling instability and the phase sep- membranes. aration. We assume that the monolayer consists of two types of cells. By The present combination of the high resolution on-chip electrophysiology on considering the coupling between the buckling and the phase separation, the membrane arrays with implementation of Gramicidin A ion channel as a re- monolayer transforms into folded structures in concert with the cell separation. porter of lipid bilayer properties allows for rapid characterization and moni- We suggest a free energy of the system and discuss not only the structure in the toring of the lipid-lipid interactions in artificial lipid membranes. steady state but also the dynamics of the pattern formation. Moreover, an effect of cellular differentiation is included in the model in order to express the cell 534-Pos Board B304 growth and to control the cell composition in the final stage of the process. Substrate for Supported Lipid Bilayers Affects Domain Mobility and Our simple model enables us to understand the morphogenesis of the small in- Phase Behaviour testine, and can be applied to other layered system. We expect that our model is James A. Goodchild, Simon D. Connell. also useful for medical practice. Dept. of Physics and Astronomy, University of Leeds, Leeds, United Kingdom. 532-Pos Board B302 A wide range of biophysical techniques have enabled a detailed structure of the Improving Stability of Tear Film Lipid Layer via Concerted Action of Two phospholipid bilayer to be established. In particular, Fluorescence Microscopy, Drug Molecules: A Biophysical View AFM, NMR and XRD have been used to elucidate the lateral heterogeneity of Yana Nencheva1, Agnieszka Olzynska2, Adela Melcrova2, phase separating membranes. Different techniques can use different model Georgi As Georgiev1, Philippe Daull3, Jean-Sebastien Garrigue4, membranes, such as Giant Unilamellar Vesicles (GUVs), Black Lipid Mem- Lukasz Cwiklik2. branes and Supported Lipid Bilayers (SLBs). Heterogeneity in SLBs can be 1Department of Optics and Spectroscopy, Faculty of Physics, St. Kliment investigated using many surface sensitive techniques, but different substrates Ohridski University of Sofia, Sofia, Bulgaria, 2J. Heyrovsky Institute of are often used. AFM uses Mica, Fluorescence Microscopy/FCS/Fluorescence Physical Chemistry, Czech Acedemy of Sciences, Prague, Czech Republic, Recovery after Photobleaching (FRAP) generally use glass, and QCM-D 3J. Heyrovsky Institute of Physical Chemistry, Novagali Innovation Center, uses Silicon Oxide. Another substrate of recent interest for SLBs is polydime- Santen SAS, Evry, France, 4Novagali Innovation Center, Santen SAS, Evry, thylsiloxane (PDMS). This cheap and relatively inert polymer is simple to France. mould into different patterns and can be used to design a wide-range of biotech- A thin tear film (TF) which covers and protects the surface of cornea is a dy- nological devices. These include drug-screening chips and microfluidic namic system evolving between eye blinks. In Dry Eye Disease (DED) the sta- devices.

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We are interested in how substrates affect the phase behaviour of SLBs. We port the phospholipid membrane (DPPC). Phospholipids were deposited on the have investigated how different substrates affect 1) the hydrophobic and ther- Chitosan/TiN coating by Physical Vapor Deposition (PVD). The phospholipid modynamic drive for bilayers to form, 2) the diffusion of individual lipids, bilayer on our Chitosan/TiN coating was studied with AFM. By heating the domain formation and hydrodynamic motion of domains, and 3) the lipid sample, we were able to observe phase transitions of the lipid bilayer. The for- ordering and melting transition. We find that whilst molecular diffusion is mation of artificial membranes was studied by AFM, measuring the topography hardly affected by changes in substrate, domain mobility is significantly hin- and performing force curves. A calorimetric assay was used to demonstrate that dered on glass and PDMS compared to mica, likely due to a combination of the TiN samples maintain the biocompatibility of the un-treated titanium with increased surface roughness and increased hydrophobicity. We also show its native oxide film. Supported by FONDECYT grant 1170261 (HB), 1141105 that nanoscale domains on PDMS can only be observed using ‘Partial Penetra- (UGV), Postdoctoral Fellowship FONDECYT 3160803 (MJR) and 3160179 tion AFM’, where imaging force is controlled to selectively break through (PS), FONDECYT INICIACION 11160664 (TPC), CONICYT-PIA ACT different phases. 1108 and 1409, Conicyt Master and PhD fellowships (MC). 535-Pos Board B305 537-Pos Board B307 Formation and Morphology of Single Phospholipid Bilayers Formed by AFM Study of Elastic Module of Physical-Vapor-Deposited Phospholipid Velocity-Controlled Dip-Coating Membranes Tomas P. Corrales1, Diego Diaz2,3, Rodrigo Catalan2,3, Ulrich G. Volkmann1,2, Rodrigo Catalan1,2, Maria J. Retamal2,3, Maria Jose Retamal2,3, Marcelo A. Cisternas2,3, Nicolas Moraga2,3, Marcelo A. Cisternas1,2, Nicolas Moraga1,2, Diego Diaz1,2, Marco Soto-Arriaza3,4, Ulrich G. Volkmann2,3. Tomas P. Corrales4, Tomas Perez-Acle5, Marco Soto-Arriaza2,3, 1Physics Department, Universidad Tecnica Federico Santa Marı´a, Patrick Huber6. Valparaı´so, Chile, 2Physics Institute, PUC Chile, Santiago, Chile, 3CIEN-UC, 1Instituto de Fı´sica, Pontificia Universidad Cato´lica de Chile, Santiago, Chile, Santiago, Chile, 4Chemistry, PUC Chile, Santiago, Chile. 2CIEN-UC, Santiago, Chile, 3Facultad de Quı´mica, Pontificia Universidad The formation of controlled biological membranes on supported surfaces is of Cato´lica de Chile, Santiago, Chile, 4Departamento de Fı´sica, Universidad importance for the development of novel biosensors and the study of protein Tecnica Federico Santa Maria, Valparaiso, Chile, 5Computational Biology membrane interactions. In particular, we are interested in the study of prepara- Lab (DLab), Fundacio´n Ciencia y Vida, Santiago, Chile, 6Institute of tion parameters, such as solvent evaporation rates and pulling velocities, that Materials Physics and Technology, TUHH, Hamburg, Germany. lead to controlled self-assembled patterns over solid surfaces [1,2]. Here we The physical study of artificial phospholipid membranes on solid substrates has show the possibility of self-assembling single bilayers of DPPC over silicon become a relevant way to gain insights into the physical behavior of cell mem- by dip-coating at controlled velocities. We find that by changing the pulling ve- branes. The study of mechanical properties of artificial membranes (lab-made) locity from a chloroform solution we can obtain a single phospholipid bilayer has become possible with the use of atomic force microscopy. Here, we analyze with different morphologies. Our resulting bilayers were imaged by Atomic the Young’s modulus and adhesion force of several phospholipidic membranes Force Microscopy and measured quantitatively with Surface Force Spectros- as a function of temperature using Scanning Force Spectroscopy (SFS). Phos- copy (SFS). We find that the structures obtained by pulling from solution above pholipids were deposited onto double-polished silicon substrates by physical a critical velocity, called the Landau-Levich regime, are circular clusters of vapor deposition (PVD), as we have recently reported [1, 2]. We have used around 5 nanometers in height. Below this critical velocity, these clusters Raman spectroscopy to show that the chemical structure of our deposited phos- merge forming a network of clusters with a constant height which matched pholipid remains unaltered after PVD. After PVD we performed AFM and SFS the bilayer height. Furthermore SFS studies as a function of temperature measurements on the following phospholipids: DPPC, DMPC and DSPC. Mea- show that our dip-coated bilayers exhibit phase transitions consistent with liter- surements using AFM in liquid confirm the self-assembly of the phospholipid ature values for DPPC membranes. Supported by FONDECYT grant # bilayer. By using SFS of the deposited membrane in liquid, we observe 2 phase 1141105 (UGV), FONDECYT INICIACION grant #11160664 (TPC), CONI- transitions, both in Youngs Modulus, as well as adhesion channels. The CYT Fellowships (RC and MC), Postdoctoral Fellowship FONDECYT measured phase transitions are consistent with the ripple-gel transition and 3160803 (MJR) and CONICYT-PIA ACT 1409. [1] Maria Jose Retamal, the gel-liquid crystalline phases. Furthermore, we have studied the frequency Tomas P. Corrales, Marcelo A. Cisternas, Nicolas H. Moraga, Diego I. Diaz, dependence of these phase transitions by changing the tip velocity in SFS ex- Rodrigo E. Catalan, Birger Seifert, Patrick Huber, and Ulrich G. Volkmann. periments. Supported by FONDECYT grant # 1141105 (UGV), FONDECYT Biomacromolecules 17 (3), 1142 (2016). [2] Tomas P. Corrales, Mengjun INICIACION grant # 11160664 (TPC), CONICYT Fellowships (RC and Bai, Valeria del Campo, Pia Homm, Piero Ferrari, Armand Diama, Christian MC), Postdoctoral Fellowship FONDECYT 3160803 (MJR) and CONICYT- Wagner, Haskell Taub, Klaus Knorr, Moshe Deutsch, Maria Jose Retamal, Ul- PIA ACT 1409. [1] Marı´a J. Retamal, Marcelo A. Cisternas, Sebastian E. rich G. Volkmann, and Patrick Huber ACS Nano 2014 8 (10), 9954-9963. Gutierrez-Maldonado, Tomas Perez-Acle, Birger Seifert, Mark Busch, Patrick Huber and Ulrich G. Volkmann, J. Chem. Phys. 141, 104201 (2014). [2] Marı´a 536-Pos Board B306 Jose Retamal, Tomas P. Corrales, Marcelo A. Cisternas, Nicolas H. Moraga, Study of Phospholipid Bilayers Supported on Chitosan-Titanium Nitride Diego I. Diaz, Rodrigo E. Catalan, Birger Seifert, Patrick Huber, and Ulrich Coatings Produced by Plasma Immersion Ion Implantation (PIII) G. Volkmann. Biomacromolecules 17 (3), 1142 (2016). Marcelo A. Cisternas1, Maria Jose Retamal2, Partha Saikia1, Nathalie Casanova1, Nicolas Moraga1, America Chandia3, 538-Pos Board B308 Alejandra Alvarez3, Donovan E. Diaz-Droguett1, Fernando Guzman4, Enhanced Ordering in Monolayers Containing Glycosphingolipids: Stephan M€andl5, Darina Manova5, Tomas P. Corrales6, Ulrich G. Volkmann1, Impact of Carbohydrate Structure Mario Favre1, Heman Bhuyan1. Shelli L. Frey1, Erik B. Watkins2, Eva Y. Chi3, Kathleen D. Cao4, 1Instituto de Fisica, Pontificia Universidad Catolica de Chile, Santiago, Chile, Tadeusz Pacuszka5, Jaroslaw Majewski2, Ka Yee C. Lee4. 2Instituto de Quı´mica, Pontificia Universidad Catolica de Chile, Santiago, 1Chemistry Department, Gettysburg College, Gettysburg, PA, USA, 2MPA/ Chile, 3Facultad de Ciencias Biolo´gicas, Pontificia Universidad Catolica de CINT/Manual Lujan Jr. Neutron Scattering Center, Los Alamos National Chile, Santiago, Chile, 4Facultad de Ciencias Fı´sicas y Matema´ticas, Laboratory, Los Alamos, NM, USA, 3Department of Chemical and Universidad de Chile, Santiago, Chile, 5Leibniz-Institut fur€ Biological Engineering and the Center for Biomedical Engineering, Oberfl€achenmodifizierung e.V., D-04318 Leipzig, Germany, 6Departamento University of New Mexico, Albuquerque, NM, USA, 4Department of de Fisica, Universidad Tecnica Federico Santa Marı´a, Valparaiso, Chile. Chemistry, Institute for Biophysical Dynamics, and James Franck Institute, The study of biocompatibility of an artificial membrane on a suitable substrate University of Chicago, Chicago, IL, USA, 5Department of Biochemistry, is an important aspect for the construction of a biosensor. A good candidate for Medical Center of Postgraduate Education, Warsaw, Poland. this kind of substrate is titanium nitride (TiN), which is harder and more corro- The influence of carbohydrate structure on the ordering of glycosphingolipids sion resistant than titanium. With this motivation, we synthesize coatings of (GSLs) and surrounding phospholipids was investigated in monolayers at the TiN on titanium substrate using a plasma immersion ion implantation (PIII) air-water interface. Binary mixtures composed of GSLs, chosen to span a technique, which allows us to optimize substrate hardness and biocompati- range of carbohydrate complexity, and zwitterionic dipalmitoylphosphatidy- bility. Here we fabricate and analyze the system, starting from pure titanium, choline (DPPC) phospholipid were studied. X-ray reflectivity was used to treated with the PIII technique to form a biocompatible coating composed of measure the out-of-plane structure of the monolayers and characterize the TiO2 and TiN. Our TiN coatings where characterized by Vickers micro- extension and conformation of the GSL carbohydrates. Using synchrotron hardness test, Atomic Force Microscopy (AFM), X-ray Photo-electron Spec- grazing incidence x-ray diffraction, the in-plane packing of the lipid acyl troscopy (XPS) and Secondary Ions Mass Spectroscopy (SIMS). After TiN chains and the area per molecule within ordered domains were characterized coating, we deposit a chitosan (CH) layer, which acts as hydrated layer to sup- at different mole ratios of the two components. Our findings indicate that

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GSL containing mixtures, regardless of the carbohydrate size, enhance the beta-2 adrenergic receptor (B2AR), an important transducer of sympathetic ordering of the surrounding lipids, resulting in a larger fraction of ordered stimulation.HYPOTHESIS: Inhibition of PDE5 with sildenafil will phase of the monolayer and greater dimensions of the ordered domains. restore cyclic guanosine monophosphate (cGMP) produced by B2AR, as Reduction of the averaged area per molecule within the ordered domains well as cyclic adenosine monophosphate (cAMP) due to an increase in was also observed but only in the cases where there was a size mismatch be- PDE5/PDE3 interaction. Sildenafil will lead to improvement in BAR- tween the phospholipid headgroups and GSL components, suggesting that the dependent cardiac excitation-contraction coupling (ECC) and overall heart condensation mechanism involves the relief of steric interactions between function via increased PKG and PKA activity. METHODS: To explore headgroups in mixtures. this mechanism, we fed wild type and B2AR knockout mice high fat diet until wild type mice had reduced ejection fraction. At this point, mice Posters: Membrane Receptors and Signal were acutely injected with or treated for one month with sildenafil, and echocardiogram analysis with isoproterenol injection was performed. Cardi- Transduction I omyocytes were isolated and used for ECC analysis; fluorescent resonant energy transfer (FRET) analysis of cGMP, cAMP and PKA; and Western 539-Pos Board B309 blot analysis of ECC proteins. RESULTS: Both acute and sustained treat- Allosteric Modulation and Thermodynamic Constraints in Occupancy ment with sildenafil improved in vivo response to sympathetic stimulation Models of Oligomeric G Protein-Coupled Receptors in a B2AR-dependent manner. Similarly, when cells were treated acutely 1 2 Gregory D. Conradi Smith , Richard Hammack . ex vivo and when mice were sustainably treated with sildenafil, sildenafil 1Department of Applied Science, College of William and Mary, 2 improved isolated myocyte ECC response to isoproterenol. Inhibition of Williamsburg, VA, USA, Department of Mathematics and Applied PKG with DT2, and PKA with myrPKI confirmed improvements were Mathematics, Virginia Commonwealth University, Richmond, VA, USA. PKG- and PKA-dependent.FRET analysis indicated sildenafil improved Terrell Leslie Hill diagrammatic method of quantifying free energy trans- BAR-stimulated cAMP and PKA activity in adult diabetic mouse cardio- duction in biochemical cycle kinetics (e.g., the sliding filament model of myocytes. Through this study, we have begun to elucidate mechanisms by muscle contraction) is readily applied to stochastic models of ligand- which PDE5 inhibition is an effective treatment for diabetics with heart receptor binding. Hill’s method uses the Markov Chain Tree Theorem to ex- hypertrophy. press the steady-state probability distribution of a receptor occupancy model in terms of the unimolecular rate constants associated to its state-transition 542-Pos Board B312 graph G, with each spanning tree of G rooted in a given state contributing Kinetic Details of the Interplay between Sodium Binding and Opioid to that state’s occupancy measure. To facilitate application of Hill’s dia- Receptor Signaling grammatic method to receptor oligomers, we have characterized the struc- ^ Xiaohu Hu, Yibo Wang, Davide Provasi, Marta Filizola. tural properties of ‘‘reduced graph powers’’ - denoted G (N) - that are the Department of Pharmacological Sciences, Icahn School of Medicine at transition graphs for the master Markov chain for N identical (but not inde- Mount Sinai, New York, NY, USA. pendent) M-state receptor models with transition graph G of size |G|=M. Sodium cations have been known to control ligand binding to and signaling of Most significantly, we provide a construction of minimum cycle bases of ^ various rhodopsin-like G Protein-Coupled Receptors (GPCRs) for more than G (N) that elucidates the combinatorics of allosteric modulation and ther- four decades, but the molecular details of the sodium binding site have been modynamic constraints on equilibrium association constants in occupancy revealed only very recently by means of high-resolution crystal structures of models of oligomeric G protein-coupled receptors. receptors. Notwithstanding this important information, a complete understand- ing of the role of sodium on ligand binding and efficacy, requires additional 540-Pos Board B310 thermodynamic and kinetic details. Dynamic Palmitoylation is a Critical Regulator of b-Adrenergic Signaling Here, we analyze micro-second scale all-atom molecular dynamics simula- in Cardiomyocytes tions of sodium binding to the active or inactive crystallographic conforma- Jie Chen, Askar Akimzhanov, Darren Boehning. tions of the m-opioid receptor (MOR) embedded in an explicit 1-palmitoyl- Biochemistry and Molecular Biology, UT Health Science Center, Houston, 2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol bilayer. Using TX, USA. information from time-independent component analysis (tICA) of unbiased S-palmitoylation is a reversible posttranslational modification that plays an simulations and multi-ensemble Markov State modeling combining unbi- important role in regulating protein localization, trafficking, and stability. ased and umbrella sampling simulations, we estimate the thermodynamics Previous work from our group showed that rapid and transient palmitoyla- and kinetics parameters of the binding of sodium cations to MOR. While tion of the tyrosine kinase Lck after death receptor stimulation is essential an energetically favorable ion pathway is identified across the active confor- for downstream signaling events and apoptosis. Here we investigated mation of MOR based on calculations of time-averaged ion spatial distribu- whether b-adrenergic stimulation of neonatal rat cardiomyocytes led to rapid tion, this pathway is occluded in the inactive receptor. Key residues palmitoylation of signaling proteins. We found that isoproterenol stimulation involved in ion binding kinetics emerge from this analysis, and the mutual led to robust but transient GasandGai palmitoylation. The kinetics of pal- effects of the ion on both the conformation and solvation of the protein are mitoylation was temporally consistent with the downstream cAMP genera- also characterized. These results generate testable hypotheses of the molec- tion, PKA activation and positive inotropic responses. Additionally, we ular determinants of ion-receptor binding kinetics, and their role in receptor identified novel palmitoylated substrates in cardiomyocytes including G function. protein-coupled receptor kinase 2. Each of these substrates displayed unique palmitoylation kinetics suggesting substrate specificity for the palmitoyla- 543-Pos Board B313 tion/depalmitoylation machinery. Finally, live cell imaging revealed that Signaling through a m-Opioid - Cannabinoid CB1 Receptor Heteromer, palmitoylation is critical for the assembly of the macromolecular signaling a Novel Analgesic Target complexes necessary for b-adrenergic signaling and downstream contractile Guoqing Xiang1,2, Takeharu Kawano2, Apostolia Baki2, responses. Our results suggest that palmitoylation is a major regulator of Diomedes Logothetis2. b-adrenergic signaling in cardiomyocytes with significant implications for 1Physiology and Biophysics, Virginia Commonwealth University, cardiac function. Richmond, VA, USA, 2Pharmaceutical Sciences, Northeastern University, Boston, MA, USA. 541-Pos Board B311 Opioids are the most common analgesics in the clinic despite severe side ef- Sildenafil: A Beta-2 Adrenergic Receptor-, Protein Kinase G-, and Protein fects, including respiratory depression, tolerance, dependence and constipa- Kinase A-Dependent Treatment for Adverse Cardiac Function and tion. The main target for opioids is the mu-opioid receptor (MOR), a 7 Excitation-Contraction Coupling Associated with Diabetes transmembrane G-protein coupled receptor (GPCR). Cannabinoids can Toni M. West1, Qingtong Wang2, Yongming Wang1, Federica Barbagallo3, also produce analgesic effects targeting the cannabinoid 1 receptor Dana Chen1, Yang K. Xiang1. (CB1R). Clinical and animal behavioral studies, using selective agonists 1Pharmacology, UC Davis, Davis, CA, USA, 2Pharmacology, Anhi Medical or knockout animals, suggest a functional interaction between MOR and University, Hefei, China, 3Sapienza University of Rome, Rome, Italy. CB1R. Utilizing electron microscope immunocytochemistry, MOR was INTRODUCTION: In humans with type 2 diabetes and heart hypertrophy, found to co-localize with CB1R in striatum GABAergic neurons. Further- the inhibition of phosphodiesterase 5 (PDE5) by sildenafil improves cardiac more, bioluminescence resonance energy transfer (BRET) studies further outcomes. However, the mechanism by which sildenafil improves cardiac support heteromerization of MOR and CB1R. Decoding the signaling of function is unclear.We have observed a relationship between PDE5 and MOR-CB1R heteromer complex will help understand the mechanism of

BPJ 8566_8569 Sunday, February 18, 2018 107a functional interaction between opioids and cannabinoids allowing design of GIRK channels. When GIRK channels were pre-activated via endogenous bivalent ligands targeting at MOR-CB1 heteromer, which may show potent M2 receptors, stimulation of CaSRs caused pronounced inhibition of analgesic effects but be devoid of side effects. We use G-protein sensitive GIRK currents. This effect was specific to CaSR activation: GIRK current inwardly rectifying potassium channels (GIRK) as reporters of Gi signaling inhibition was sensitive to NPS-2143, a negative allosteric modulator of of MOR and CB1R in the Xenopus oocyte heterologous expression system. CaSRs, and abrogated by FR900359, a direct inhibitor of Gq proteins. GIRK current is monitored by two-electrode voltage clamp (TEVC) to assess GIRK current inhibition was also sensitive to the PKC inhibitor chelerythr- receptor activation. We show that co-expression of CB1R enhances the Gi ine, suggesting that following activation of CaSR and Gq, GIRK currents signaling of MOR. To corroborate our finding, we have also utilized a cal- are modulated by PKC phosphorylation. We conclude from this data that cium mobilization assay. HEK293 cells were transiently transfected with cardiac CaSRs do not activate Gi proteins and regulate GIRK currents pref- the GPCR of interest and the G alpha 16/o chimera which biases signaling erentially via the Gq/PKC pathway. towards Gq leading to phospholipase C activation and calcium mobilization. Intracellular calcium level was monitored by GCaMP 6s in a BMG plate 546-Pos Board B316 reader. Our data support the results we obtained in the oocyte system. We Unique Molecular Determinants that Contribute to Melanopsin’s (OPN4) are in the process of testing the effects of co-expression of MOR on the Capability to Sustain Light Responses 1 1 2 signaling of CB1R as well as their functional crosstalk. Juan C. Valdez-Lopez , Stephen Petr , Matthew P. Donohue , Veronika Szalai2, Phyllis R. Robinson1. 544-Pos Board B314 1Biological Sciences, University of Maryland Baltimore County, Baltimore, 2 Voltage-Dependent Activation of M2 Muscarinic Receptors-IKACH by the MD, USA, National Institutes of Standards and Technology, Gaithersburg, Superagonist Iperoxo in Cardiac Myocytes MD, USA. Ana L. Lopez-Serrano1, Martin Tristani-Firouzi2, Melanopsin is a unique visual pigment and G-protein-coupled receptor Eloy G. Moreno-Galindo1, Ricardo A. Navarro-Polanco1. (GPCR) expressed in a small subset of retinal ganglion cells in the mamma- 1Centro Universitario de Investigaciones Biomedicas, Universidad de lian retina, and is involved in non-image forming tasks such as circadian Colima, Colima, Mexico, 2Nora Eccles Harrison Cardiovascular Research photoentrainment and pupil constriction. Data from electrophysiology and Training Institute, University of Utah, Salt Lake City, UT, USA. calcium imaging assays suggest that the kinetics of melanopsin’s photo- Recently, M2 muscarinic receptor (M2R) have been shown to exhibit transduction cascade are sluggish, and the response has a unique sustained intrinsic voltage sensitivity in an agonist-specific fashion, since the mem- property unlike the transient rod and cone responses. While several molec- brane depolarization may decrease (with ACh), increase (with pilocarpine ular determinants of melanopsin’s signaling cascade have been described and choline) or not change (with bethanechol) the M2R activation. In the (couplingtoGRKs&b-arrestins), there are additional properties that other hand, there are compounds with both higher affinity and efficacy remain to be elucidated. First, we aim to describe the phosphorylation pro- (and thus with outstanding potency) than physiological agonists, which file of melanopsin’s long C-terminus tail, which we hypothesize is contrib- have been termed superagonists. Iperoxo (IP) is a superagonist for musca- uting significantly to both receptor activation and deactivation. Second, we rinic receptors and it has acquired increasing interest for inducing distinctive hypothesize an intracellular conformation that might contribute to regu- conformational changes on the M2R. Here, we evaluated in atrial myocytes lating G-protein signaling. Third, we hypothesize that melanopsin interacts the effect of the membrane voltage on the IP-M2R interaction, using the acti- with a serine/threonine phosphatase of the protein phosphatase family, and þ vation of the ACh-gated K current (IKACh) as readout. We recorded macro- this interaction is important for resensitization. We aim to test these hy- scopic currents in the whole-cell configuration of the patch-clamp technique, potheses through expression in HEK293 cells, in vitro calcium imaging performing concentration-response curves and current-voltage relationships. to measure signaling kinetics, immuno-purification for electron paramag- We found that IP activation of IKACh was modified by membrane potential netic resonance (EPR) spectroscopy, RT-PCR and immunohistochemistry in a similar manner to ACh, albeit with remarkable higher potency at of retinal samples. We have found that there are six serines and threonines both studied voltages. With depolarization, IP produced a striking rightward on the proximal C-terminus, combined with six additional putative shift of the concentration-response curves: EC50=0.2250.04 nM phosphosites in either proximal or distal direction, properly deactivate and 1.1350.19 nM at 80 and þ40 mV, respectively. Also, with IP the melanopsin. Additionally, we have evidence suggesting an intracellular deactivation time course of IKACh was significantly slower at 80 mV conformation where the C-terminus is proximal to intracellular loop 3. (t=14.852.3 s) than at þ40 mV (t=6.751.2 s), suggesting a greater affinity This conformation plays a role in receptor activation and is stabilized by at hyperpolarized voltage. Likewise, consistent with ACh, IP induced IKACh C-terminus phosphorylation, involving a tyrosine residue (Y382). Finally, to display the ‘relaxation’ property, which denotes a slow decrease or in- we have found diverse expression of protein phosphatases within the mu- crease in current magnitude with depolarization or hyperpolarization, respec- rine retina, and in vitro data suggests PP2A-mediated resensitization. We tively. Therefore, our results provide additional evidence to support the present these findings as contributors to melanopsin’s unique signaling agonist-specific voltage sensitivity of M2R. This work was supported by properties within the mammalian retina. SEP-CONACYT, Mexico (Grant No. 2011-01-167109 to E.G.M.-G and 2013-01-220742 to R.A.N.-P). 547-Pos Board B317 Mechanisms of Chimeric Antigen Receptor (CAR) Signaling during T Cell 545-Pos Board B315 Activation D G Protein Signaling of Ca2 -Sensing Receptors (CaSRs) in Cardiac Xiaolei Su1,2, Ronald Vale1. Myocytes 1University of California, San Francisco, San Francisco, CA, USA, 2Yale Marie-Cecile Kienitz1, Jennifer Schmidt1, Gabriele Ko¨nig2, Evi Kostenis2, School of Medicine, New Haven, CT, USA. Lutz Pott1, Andreas Rinne1. The chimeric antigen receptor (CAR) enables T cells to specifically target 1Institute of Physiology, Bochum, Germany, 2Institute of Pharmaceutical and kill cancer cells. Despite of its success in clinical trials, the cellular Biology, Bonn, Germany. mechanism of how CAR is activated to trigger downstream signaling path- Ca2þ-sensing receptors (CaSRs) belong to the class C of G protein-coupled ways remains unclear. Here we use a supported lipid bilayer system together receptors (GPCRs) and are activated by extracellular Ca2þ. CaSRs can with TIRF imaging to investigate the dynamics of CAR signaling during T couple to different classes of heterotrimeric G proteins depending on cell activation. We chose CD19-CAR, which targets CD19 molecules on the agonist and cell type, therefore they display biased G protein signaling. B cell surface, as a model since it has been wildly used in cancer immune In this study we used fluorescent biosensors to directly analyze G protein therapy. We found that, similar to endogenous TCR, CAR forms micro- coupling to CaSRs and downstream signaling in living cells. In HEK 293 clusters as a signaling platform right after ligand engagement. These micro- cells CaSRs displayed biased signaling: elevation of extracellular Ca2þ clusters undergo a centripetal movement to form the center part of from 0 mM to 5 mM or application of the alternative agonist spermine immunological synapse (cSMAC). However, once formed, the CAR synapse (5 mM) caused activation of Gi-andGq-proteins. Adult cardiac myocytes either disassembles very quickly or becomes mobile as cells start to migrate, express endogenous CaSRs, which have been implicated in regulating suggesting an organization pattern different from TCR. What is more, the Ca2þ signaling and contractility. Biased signaling of CaSRs has not been CAR ligand does not induce the formation of TCR microcluster, and vice investigated in these cells. To evaluate efficiencies of Gi- and Gq signaling versa. CAR and TCR microclusters do not overlap with each other when via CaSRs in rat atrial myocytes, we measured G protein-activated Kþ cells are activated by both ligands of TCR and CAR. These data suggest (GIRK) channels. Activation of GIRK requires binding of Gbg subunits that CAR and TCR can be activated and transduce signaling independently. released from Gi proteins, whereas Gq signaling inhibits GIRK channel ac- In addition, CAR localizes to the plasma membrane and ER membrane, tivity. Stimulation of CaSRs by Ca2þ or spermine failed to directly activate but is largely absent from endosomes. This contrasts to TCR, of which

BPJ 8566_8569 108a Sunday, February 18, 2018 endosome trafficking plays an important role in regulating its activity. We As part of the adaptive immune system T-cells play an essential role in cell- will discuss how the structure difference between CAR and TCR could mediated immunity. Interaction of T-cell receptors (TCR) with peptide- lead to their different signaling kinetics and regulatory mechanism. Our loaded major histocompatibility complexes on antigen presenting cells triggers work might also provide insights in designing new CARs for optimizing T a cascade of events ultimately leading to T- cell activation. Besides protein re- cells’ killing of tumors. arrangement, enrichment of certain lipids within the immunological synapse (IS) is thought to either directly modulate TCR function or alter give an expla- 548-Pos Board B318 nation for the spatial re-organization of proteins and lipids at the plasma mem- Monomeric TCR-CD3 Complexes Drive T-Cell Antigen Recognition brane in the course of T-cell activation and how antigen recognition is Mario Brameshuber1, Florian Kellner2, Benedikt K. Rossboth1, Haisen Ta3, translated into a cellular response, but particularly the role of lipids in this pro- Kevin Alge2, Eva Sevcsik1, Markus Axmann4, Nicholas R.J Gascoigne5, cess is not well understood. Here, we employ a novel experimental approach Simon J. Davis6, Hannes Stockinger2, Gerhard J. Schuetz1, [2] to measure lipid interactions of the TCR during T-cell activation. Specif- Johannes B. Huppa2. ically, we use protein micropatterning to immobilize and enrich the TCR within 1 Institute of Applied Physics - Biophysics, TU Wien, Vienna, Austria, well-defined areas of the T-cell plasma membrane. Thus, by reducing the de- 2 Institute for Hygiene and Applied Immunology, Medical University of gree of freedom for one interaction partner (the TCR), co-localisation and inter- 3 Vienna, Vienna, Austria, Department of NanoBiophotonics, Max-Planck- action times of well-chosen lipids can be measured by single molecule tracking. 4 Institute for Biophysical Chemistry, Goettingen, Germany, Institute of From the diffusion behaviour of lipids at different area densities of TCRs in Medical Chemistry and Pathobiochemistry, Medical University of Vienna, activated and non-activated T-cells [3], we were able to extract quantitative in- 5 Vienna, Austria, Department of Microbiology and Immunology, National formation on the association of the TCR with selected lipids in the plasma 6 University of Singapore, Singapore, Singapore, Radcliffe Department of membrane of living cells. [1] J.B. Huppa, M.M. Davis, Advances in Immu- Medicine and MRC Human Immunology Unit, University of Oxford, Oxford, nology, Volume 119, 1-50 [2] E. Sevcsik, M. Brameshuber, M. Fo¨lser, J. We- United Kingdom. ghuber, A. Honigmann, G.J. Schutz,€ Nature Communications, Volume 6 [3] T-cell antigen recognition requires T-cell antigen receptors (TCRs) binding A.M. Arnold, E. Sevcsik, G.J. Schutz,€ Journal of Physics, Volume 49. to MHC-embedded antigenic peptides (pMHCs) within the contact region of a T-cell with its conjugated antigen-presenting cell. Despite 551-Pos Board B321 micromolar TCR:pMHC affinities, T-cells respond to even a single anti- Dynamic Interactions of Stimulated IgE-FcεRI Receptor with Lyn and Syk genic pMHC, and higher order TCR-structures have been postulated to Kinases at the Plasma Membrane Measured by Imaging Fluorescence maintain high antigen sensitivity and trigger TCR-proximal signaling. We Correlation Spectroscopy interrogated the stoichiometry of TCRs and their associated CD3 signaling Nirmalya Bag, David Holowka, Barbara Baird. chains on the surface of living primary T-cells with the use of (i) single Cornell University, Ithaca, NY, USA. molecule brightness and (ii) single molecule co-localization analysis, (iii) Spatio-temporal organization of the plasma membrane (PM) plays a signifi- photon-antibunching based fluorescence correlation spectroscopy and (iv) cant role in cell signaling. However, the PM is complex, including lipids Fo¨rster Resonance Energy Transfer measurements. While all four ap- that exhibit phase-like behavior, membrane associated proteins, and cytoskel- proaches unambiguously confirmed the accepted subunit ratio within etal linkages. Our laboratory, using super-resolution fluorescence imaging, TCR/CD3 complexes, we found exclusively monomeric TCR/CD3 com- previously showed that immunoglobin E (IgE) complexed to its receptor, plexes driving the recognition of antigenic pMHCs. Our findings underscore FcεRI, (IgE-FcεRI) undergoes time-dependent redistribution on the membrane the exceptional capacity of single TCR/CD3 complexes to elicit robust surface after multivalent ligand stimulation, upon which the receptor is phos- intracellular signaling, which may prove critical for optimizing T-cell-based phorylated by membrane-anchored Lyn tyrosine kinase. This initial coupling immunotherapies. is followed by recruitment of soluble Syk tyrosine kinase to the cross-linked receptor for signal amplification and subsequent downstream processing. 549-Pos Board B319 Considerable evidence suggests that this interaction of IgE-FcεRI and Lyn oc- Contribution of Adhesion to Early T Cell Signaling curs in the ordered-lipid regions of the PM in mast cells. We are examining Martin Fo¨lser, Julia Appenroth, Viktoria Motsch, Gerhard J. Schutz.€ the nature and dynamics of plasma membrane organization as experienced TU Wien, Wien, Austria. by IgE-FcεRI, Lyn, and Syk in resting state and after antigen stimulation Antigenic peptides are recognized by T cells within the interaction inter- by Imaging Total Internal Reflection Fluorescence Correlation Spectroscopy face with an antigen presenting cell. Specifically, the binding of the T cell (ITIR-FCS), which maps lateral diffusion with pixel resolution. We also receptor (TCR) to antigenic peptide loaded MHCII (pMHC) is the critical conduct spot variation FCS (svFCS), which analyzes space-dependence of step to trigger T cell activation. While many signaling proteins contrib- diffusivity on the same set of ITIR-FCS data, to reveal the existence of nano- uting to early TCR signaling have been known for years, the molecular scopic obstacles (e.g., nanodomains) that influence the diffusivity of these mechanisms that translate binding to biochemical signals have not been proteins. To evaluate features of PM organization in this mast cell response, clearly identified yet. Moreover, it is up for debate how multiple signaling we pharmacologically modify actin cytoskeleton polymerizaton and PM or- events are added up and what a putative regulatory system would look der. We monitor the influence of these perturbations on diffusion properties like. of key components in the unstimulated state and the extent of antigen- T cell signaling is governed by two entangled processes - by the build-up crosslinking of IgE-FcεRI and recruitment of kinases to the cross-linked re- of cell-cell adhesion and by the recognition of pathogenic peptides. To ceptors. These results, in conjunction with super-resolution imaging, which dissect the contribution of the two processes, we use supported lipid bila- determines the size and density of these complexes, will provide a new level yers decorated with pMHC and the adhesion ligand ICAM 1 to mimic an- of insight into PM organization and its dynamic remodeling upon ligand- tigen presenting cells. By omitting ICAM 1 from activating surfaces the receptor stimulation. role of adhesion to T cell calcium signaling can be investigated. To ensure the close contact between T cells and bilayer even in the absence of adhe- 552-Pos Board B322 sion proteins, T cells are gently pushed to the bilayer with the cantilever of Triggering of the High-Affinity IgE Receptor in an Aggregation- an atomic force microscope (AFM). Furthermore, the investigation of T Independent Manner cell activation kinetics in conventional seeding experiments is limited in James Felce, Erdinc Sezgin, Madina Wane, Michael Dustin, termsofaccuracybytheslowspreading of T cells at the bilayer. An Christian Eggeling, Simon Davis. advantage of the AFM based method is the instantaneous establishment University of Oxford, Oxford, United Kingdom. of the interface area. Therefore, activation time can be determined with The high-affinity IgE receptor, FcεR1, is responsible for the sensitization of high precision. mast cells and basophils to IgE-targeted antigens. The current model of FcεR1 triggering rests on the principle of aggregation-driven phosphorylation, 550-Pos Board B320 yet several recent observations have indicated that this is incomplete. Here we Probing Lipid Interactions of the T-cell Receptor Complex: A Micropat- re-examine the minimal requirements for FcεR1 triggering and observe that it is terning Approach induced without receptor aggregation by surface-associated antigen in a size- Joschka Hellmeier1, Florian Kellner2, Gerhard Schuetz1, Johannes Huppa2, dependent manner. Using confocal microscopy and fluorescence correlation Eva Sevcsik1. spectroscopy we determine that this is driven by exclusion of the large inhibi- 1Technical University of Vienna, Institute of Applied Physics, Vienna, tory phosphatase CD45 from the receptor-antigen contact, abrogation of which Austria, 2Medical University of Vienna, Institute for Hygiene and Applied inhibits triggering. Such kinetic segregation (KS) of CD45 and FcεR1 also oc- Immunology, Vienna, Austria. curs in triggering-deficient cells and basophil-derived giant plasma membrane

BPJ 8566_8569 Sunday, February 18, 2018 109a vesicles, and hence is a passive, steric process. Partial KS also occurs in cells tured glucan was abolished by pre-treating the glucan under conditions that forming surface contacts in the absence of FcεR1 ligand, leading to ligand- melt its triple helical structure. Finally, we found that co-transfection of independent triggering that is responsible for the conventional morphology Dectin-1A/1B diminishes signaling responses to highly structured glucans. of basophilic cell lines in culture. The potential for both aggregation and KS These results indicate that Dectin-1 differentially senses the solution confor- mechanisms broadens the range of antigen capable of inducing triggering, mation of b-glucans through its A and B isoforms, and that Dectin-1A and so affords the system substantially increased versatility. and 1B engage in cross-talk that can tune cellular signaling responses to b-glucans. 553-Pos Board B323 Functional Organization of Plasma Membrane Adaptor Proteins in B Cell Receptor Signaling 555-Pos Board B325 Sarah A. Shelby, Sarah L. Veatch. Cardiolipin Acts as an Agonist or an Antagonist of Toll Like Receptor Biophysics, University of Michigan, Ann Arbor, MI, USA. (TLR4) As part of the adaptive immune system, B cells mount a cellular response when Jean-Marie Ruysschaert, Caroline Lonez, Malvina Pizzuto. Free University of Brussels, Brussels, Belgium. the B cell receptor (BCR) is clustered through binding of a foreign antigen. Un- Toll like receptors (TLRs) are the sentinels of our cells; located at the cell sur- derstanding how interactions between clustered BCR and its signaling partners are spatially regulated on the plasma membrane is a challenge because face they alert the immune system of the presence of viruses and bacteria. In signaling complexes may be weakly assembled, transient, and/or too small to damaged mitochondria, cardiolipin (CL) is redistributed and externalized to detect with conventional microscopy. We have utilized live-cell super-resolu- the outer mitochondrial membrane. This externalization has an important tion localization microscopy to simultaneously image BCR and other mem- and not deciphered role in mitochondria signaling. We investigate here the pro-and anti-inflammatory properties of CL. We show that unsaturated brane species during BCR stimulation. This quantitative technique can mammalian cardiolipins act as competitive TLR4 antagonists by occupying measure interactions between species with enough sensitivity to detect subtle effects such as lipid-mediated forces. Previously, we found that minimal the binding site of LPS which is TLR4 natural ligand. By contrast saturated membrane-anchored probes that partition into ordered domains of model mem- cardiolipins are TLR4 agonists and induce pro-inflammatory cytokine secre- branes co-localize with BCR clusters, while disorder-preferring anchors are tion in macrophages. Our results strongly suggest that only linear saturated excluded from clusters, suggesting that BCR clusters locally enrich a specific CL mimics LPS interaction and induces TLR4:MD2 dimerization and lipid composition. As an extension, we are exploring the effects of signaling. Unsaturated CL adopts a folded conformation that buries the mole- cule deeper into the MD2 pocket so preventing interaction with TLR4.The membrane-driven sorting on the organization and function of BCR signaling proteins. Transmembrane adaptor proteins play key roles in BCR signaling data presented here contribute to reinforce a new concept based on the obser- as hubs that recruit various regulatory proteins and integrate distinct signaling vation that TLRs recognize non bacterial patterns. This means that the actual pathways. Localization or compartmentalization of adaptor proteins are poten- minimal requirements for TLRs recognition have to be reevaluated. It also tially crucial mechanisms for regulation of the overall response. We have found suggest that any molecule with two or three saturated hydrophobic chains that the palmitoylated transmembrane adaptor proteins LAT2, LIME, and PAG, and a polar head and whatever its origin is a potential activator of the innate system. which serve distinct adaptor functions in BCR signaling, are differentially re- cruited to clustered BCR. This differential organization is due to a combination of interactions between the transmembrane domains of these proteins with the 555.1-Pos Board B325.1 membrane itself and specific protein-protein interactions. We are investigating G Protein-GPCR Interaction Studied by SANS 1 2 3 3 the relative contributions of lipid- and protein-mediated interactions of adaptors Olivier Soubias , Jonathan D. Nickels , Alexei Yeliseev , Kirk G. Hines , 3 3 2 3 with membrane structures, including lipid domains, caveolae, adhesion struc- Walter E. Teague , John Northup , John Katsaras , Klaus Gawrisch . 1NCI, NIH, Bethesda, MD, USA, 2BSMD, ORNL, Oak Ridge, TN, USA, tures, and the cytoskeleton. These experiments will help determine how the or- 3 ganization of a given adaptor is linked to specific signaling functions. LMBB, NIAAA, NIH, Bethesda, MD, USA. G protein-coupled membrane receptors (GPCR) transmit extracellular sig- 554-Pos Board B324 nals elicited by compounds like neural transmitters, hormones, odorants, Differential Signaling and Cross-Talk of Dectin-1A and 1B after Activa- or light to the cell interior, where they activate GTP-binding proteins (G pro- tion with Soluble Beta-Glucans teins). It is desirable to study GPCR and G protein under functional Eduardo U. Anaya. conditions in their natural environment, namely a fluid lipid matrix. Exper- Pathology, University of New Mexico, Albuquerque, NM, USA. iments were conducted using the GPCR bovine rhodopsin and the G protein Dectin-1 is a C-type Lectin innate immunoreceptor that recognizes b-(1,3)- transducin. We have recently shown that SANS is capable of measuring the glucan, a structural component of Candida species cell walls. Dectin-1 has state of GPCR homo-oligomerization in a lipid matrix under functional con- eight splice isoforms including, A and B. Dectin-1A has a long stalk region ditions. Moreover, these experiments proved that the resolution is sufficient in the ectodomain while Dectin-1B lacks this stalk. Innate immunocytes can to obtain information about the size and shape of membrane associated pro- express one or both isoforms, but neither their relative responsiveness to tein-protein complexes. We conducted detailed, time-resolved functional b-glucan nor the potential for inter-isoform signaling cross-talk is well un- studies of G protein activation by GPCR via measurement of GDP to radio- derstood. Determining how different types of soluble b-glucans affect both active [S35] GTP-g-S exchange. The experiments revealed experimental isoforms will unravel how differences in glucan structure impact the earliest conditions at which homogeneity and stability of the G protein-GPCR com- events in Dectin-1 signaling. In this study, we focused on the impact of liga- plex are sufficient for conducting experiments on the timescale of several tion by soluble b-glucans varying in size and quaternary structure on the hours as required for small angle neutron scattering (SANS) and at a pro- Ca2þ signaling responses of the Dectin-1 isoforms. We measured Ca2þ tein/lipid molar ratio that is expected yielding lowest perturbation of signaling using Fluo-2 leak resistant probe in HEK-293 cell lines expressing SANS from the presence of a lipid matrix. The following questions are ad- Dectin-1A or-1B after stimulation with various glucans. S. cerevisiae b-glu- dressed: Does the water-soluble G protein bind directly to GPCR in lipo- cans of medium and high molecular weight have a large degree of triple he- somes from solution or does it bind to the lipid-water interface before lical structure in aqueous solution. In contrast, the small algal b-glucan interacting with GPCR? Is GPCR-G protein interaction sensitive to the laminarin and phosphorylated S. cerevisae glucans have much less triple he- conformational state of GPCR switched by ligands? Does G protein interact lical structure. Stimulation with highly structured b-glucans resulted in with individual GPCR molecules or with GPCR dimers? Interaction of greater amplitude of calcium influx in cells expressing Dectin-1A, relative G protein with agonist bound GPCR triggers a GDP-to-GTP exchange at to less structured glucans. Meanwhile, Dectin-1B expressing cells had the Ga subunit of the GPCR and a dissociation of Ga and Gbg subunits. decreased signaling compared to Dectin-1A and were less responsive to glu- What is the affinity of Ga and Gbg subunits for each other, for interaction cans’ triple helical content. The responsiveness of Dectin-1A to highly struc- with the lipid matrix and the GPCR?

BPJ 8566_8569 110a Sunday, February 18, 2018

Posters: Mechanosensation visited (Marcotti et al. 2014). Using rat and mouse auditory hair cells, a new high-speed imaging technique, different pharmacological approaches, and in- ternal Ca2þ manipulations, we investigated the role of ATPase activity, 556-Pos Board B326 2þ Matrix Stiffness Contributes to Pathological Activation of Cardiac Fibro- including myosin motors, in Ca -dependent adaptation, set point regulation, blasts and gating of MET channels. 1 2 1 Tova Christensen , Kristi Anseth , Leslie Leinwand . 559-Pos Board B329 1Molecular, Cellular, and Developmental Biology, University of Colorado, 2 Electrophysiological Characterization of Mechano-Activated Currents in Boulder, Boulder, CO, USA, Chemical Engineering, University of Human Endothelial Cells Colorado, Boulder, Boulder, CO, USA. Luis O. Romero, Julio F. Cordero-Morales, Valeria Va´squez. In the heart, prolonged activation of the healing response causes cardiac Physiology, College of Medicine, University of Tennessee Health Science fibrosis. Fibrosis is characterized by collagen deposition that causes cardiac Center, Memphis, TN, USA. stiffening, diastolic dysfunction, and arrhythmias. Using hydrogels to model Mechanosensitive channels are force sensors essential for cardiovascular the mechanical changes that occur going from a healthy to fibrotic heart, we function, acting in endothelial and smooth muscle cells to control vascular observe that cardiac fibroblasts respond to changes in stiffness by changing structure and blood pressure. Several cation channels, including members morphology, proliferation, stress fiber formation, and gene expression. Addi- of the transient receptor potential (TRP) and Piezo channel families, have tionally, fibroblasts secrete factors in an activated state on stiff substrates been implicated in endothelial cells response to mechanical stimulation that promotes activation of surrounding fibroblasts. These studies provide (i.e., shear flow). However, the precise mechanisms responsible for increase insight into the way that the stiffness of the extracellular matrix contributes in Ca2þ permeability upon mechanical stimulation in endothelial cells to persistent fibrosis in the heart. remain largely unknown. To characterize the native mechano-activated cur- rents of human microvascular endothelial cells (HMVECs), we record in 557-Pos Board B327 the whole-cell patch clamp configuration while indenting the plasma mem- Dynamics of Stretch-Dependent Calcium Signaling in Heart brane with a piezoelectric-driven blunt glass pipette, at a constant voltage. Humberto C. Joca, George S.B. Williams, W. Jonathan Lederer, þ Our pharmacological data shows that Gd3 completely abolishes the Christopher W. Ward. HMVEC indentation-dependent currents; whereas 50% of the currents are in- Center for Biomedical Engineering and Technology, University of Maryland hibited by L-GsMTx4. Moreover, TRPC3, TRPC6, TRPA1, and TRPV4 an- Baltimore, Baltimore, MD, USA. tagonists do not significantly decrease current amplitudes. The inactivation In heart, the uninterrupted cycles of cardiac filling and ejection compels the car- rates are voltage dependent, speeding-up significantly with membrane hyper- diac cells into a constant state of mechanical stress. Our group previously polarization; interestingly, inactivation rates are not reminiscent of Piezo described a microtubule (MT) dependent mechano-chemo-transduction channels transiently expressed in HEK293 cells. Molecular, biochemical, pathway that links this constant mechanical stress to reactive oxygen species and biophysical characterizations are underway to determine the mechanism (ROS) production by NADPH oxidase 2 (NOX2). During physiological stretch- whereby endothelial cells respond to mechanical stimuli. (Supported by ing of ventricular cardiomyocytes this NOX2-dependent ROS (X-ROS) sensi- 2þ 2þ AHA). tizes the cardiac ryanodine receptors (RYR2s) to cytosolic Ca ([Ca ]i) and 2þ is readily observed experimentally via increased Ca spark frequency. While 560-Pos Board B330 2þ sensitized RYR2s can promote the synchronized Ca release in healthy cardi- Role of Piezo Channels in Urothelial Cell Mechanotransduction omyocytes, the mechanical stimulus and X-ROS can also trigger arrhythmo- Marianela G. Dalghi. 2þ genic events like Ca waves in pathological conditions such as Duchenne University of Pittsburgh, Pittsburgh, PA, USA. muscular dystrophy (DMD). Here we use a systems biology approach, During the micturition cycle, the ability to sense and transduce the degree of combining experimental tests with state-of-the-art computational modeling, tension in the wall of the bladder allows the bladder to accommodate to 2þ to further define how MT dependent X-ROS regulates cardiac Ca signaling changing urine volumes and to transmit the filling state to the CNS. The ur- under both physiological and pathological conditions. Expanding upon our ex- othelium, and in particular the umbrella cells (UC), is proposed to play an isting mechanistic model of cardiac excitation-contraction coupling (ECC), we important role in mechanotransduction: upon bladder filling, UC sense mem- have implemented a simple yet realistic formulation of stretch-induced sarco- brane stretch and transduce it into a number of cellular responses, including 2þ lemmal Ca influx, ROS production and sensitization of RYR2s. This novel exocytosis of subapical discoidal/fusiform vesicles and release of neurotrans- 2þ model effectively reproduces the stretch-dependent increase in Ca spark fre- mitters. The mechanosensor present in the UC that directly responds to mem- 2þ quency in healthy cells and also captures the development of ‘‘unstable’’ Ca brane stretch is unknown. Piezo channels are a newly discovered family of 2þ events during DMD conditions. The model predicts that these abnormal Ca mechanosensitive channels associated with the physiological responses to events are partially related to the increase in MT density observed in DMD. pressure, shear stress and tension. In this study, we sought to determine the Combined, our single cell experiments and computational modeling provide involvement of Piezo channels in UC mechanosensation. We found that rat 2þ quantitative details regarding changes in Ca spark frequency, duration, UC express both Piezo1 and Piezo2 channels which exhibit different subcel- amplitude, and mass during cellular stretch due to ROS-sensitized RYR2s. lular localizations: Piezo1 is expressed at the apical region and at tight junc- 2þ Our work provides critical new insights into Ca signaling dynamics during tion sites, whereas Piezo2 is found in the cytosol. Silencing Piezo1 expression mechanical stress endured by cardiomyocytes during their constant work, in by in-situ transduction of rat bladders with adenoviruses encoding Piezo1- healthy or disease conditions. shRNA showed a significant decrease in apical exocytosis (61 5 9%) compared to control upon ex vivo bladder filling. We also assessed the impact 558-Pos Board B328 of knocking-down Piezo1 in bladder function by monitoring continuously the Role of Calcium and ATPase Activity in Slow Adaptation and Set Point intravesical pressure on anesthetized rats previously transduced with either Regulation in Cochlear Mechanotransduction scrambled- or Piezo1-shRNA. We observed that loss of Piezo1 leads to a Giusy A. Caprara, Anthony W. Peng. lower voiding frequency and also affects bladder compliance (defined as Physiology and Biophysics, University of Colorado, Aurora, CO, USA. DV/DP during filling). All together, these results show the involvement of Hair cells of the inner ear are mechanosensors that transduce mechanical forces Piezo1 in sensing mechanical stretch required for normal bladder function. arising from sound waves and head movement to provide our senses of hearing Future directions involve establishing the role of Piezo2 in rat bladder func- and balance. Sound deflects the hair bundle, a crosslinked cluster of stereocilia tion as well as detailed characterization of the cascade of events triggered on the apical surface of a hair cell, and causes the opening of mechanically sen- by Piezo1 activation. sitive ion channels to start the MET process. A variety of unconventional my- osins are present within the MET complex, but, in mammals, their precise role 561-Pos Board B331 in cochlear mechanotransduction is unclear. The current model to explain an Probing the Mechanosensing Mechanism of GPR56 to Understand Adhe- MET Ca2þ-dependent slow adaptation mechanism proposes a direct link be- sion GPCR-Related Disease Pathogenesis tween Ca2þ influx through MET channels and the movement of unconven- Kassidy J. Tompkins. tional myosin 1C (Myo1C) to release tension and close the channel. Recent Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, evidence shows that in mammalian species, MET channels and Myo1C have Minneapolis, MN, USA. a different localization and that the expression during development of Mechanosensing proteins respond to piconewton-sized forces, resulting in Myo1C does not match the onset of slow adaptation (Beurg, M. et al. 2009, cell-signaling processes in a process known as mechanotransduction. Dysre- Grati, M. et al 2011, Waguespack, J. et al. 2007). Also, other models that pro- gulation of these force-sensing pathways promotes aberrant cell migration posed the role of myosin motors in mammalian auditory system need to be re- and development, which contribute to disease pathogenesis. Adhesion GPCRs

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(aGPCRs) are transmembrane proteins predicted to signal in response to 564-Pos Board B334 mechanical force generated by cell-cell or cell-matrix interactions. However, A Protein Interaction Mechanism for Suppressing the Mechanosensitive the molecular mechanisms used by aGPCRs to sense and respond to Piezo Channels tension in the cellular environment remain unknown. My hypothesis is that Shaopeng Chi. the GAIN (GPCR auto-proteolysis inducing) domain is the putative mechano- Tsinghua, Haidian, China. sensing domain in aGPCRs. This domain is self-cleaved during translation Piezo proteins are bona fide mammalian mechanotransduction channels for and exists as a noncovalent heterodimer located just extracellular of the various cell types including endothelial cells. The mouse Piezo1 of 2547 membrane. Thus, mechanical forces derived from the extracellular residues forms a three-bladed, propeller-like homo-trimer comprising a environment induce ‘‘shedding’’ of the GAIN domain, exposing the aGPCR central pore-module and three propeller-structures that might serve as ligand-binding site to induce cellular signaling pathways. I will present our mechanotransduction-modules. However, the mechanogating and regulation work studying the biophysical and mechanical properties of the GAIN domain of Piezo channels remain unclear. Here we identify the Sarcoplasmic /Endo- þ of GPR56, an aGPCR that interacts with collagen III in the extracellular plasmic-Reticulum Ca2 ATPase (SERCA), including the widely expressed matrix. My work using a cell-based ‘‘urea dissociation assay’’ shows that SERCA2, as Piezo interacting proteins. SERCA2 strategically suppresses the GAIN domain dissociates between 2 and 3M urea, making it more Piezo1 via acting on a 14-residue-constituted intracellular linker connecting sensitive to dissociation than Notch and dystroglycan non-covalent associa- the pore-module and mechanotransduction-module. Mutating the linker im- tions. I am using microenvironment-dependent luciferase assays and in vitro pairs mechanogating and SERCA2-mediated modulation of Piezo1. Further- single-molecule magnetic tweezer experiments to determine the extent to more, the synthetic linker-peptide disrupts the modulatory effects of which external forces contribute to GPR56 signaling at the auto-proteolysis SERCA2, demonstrating the key role of the linker in mechanogating and site. regulation. Importantly, the SERCA2-mediated regulation affects Piezo1-dependent 562-Pos Board B332 migration of endothelial cells. Collectively, we identify SERCA-mediated Cytoskeleton-Associated Proteins Modulate the Tension Sensitivity of regulation of Piezos and the functional significance of the linker, providing Piezo1 important insights into the mechanogating and regulation mechanisms of Piezo Charles D. Cox, Navid Bavi, Boris Martinac. channels. Victor Chang Cardiac Research Institute, Sydney, Australia. Mechanosensitive channels are essential molecular components of mechano- 565-Pos Board B335 sensory systems in all organisms. It has long been recognized that Functional Investigation of Dystroglycan’s Proteolysis Domain bacterial mechanosensitive channels are gated by bilayer tension. However Amanda Hayward. it is likely that tension may also be the relevant gating parameter for a host BMBB, University of Minnesota-Twin Cities, Minneapolis, MN, USA. of eukaryotic mechanosensitive channels (e.g. TREK-1/2, Piezo1). Here we Dystroglycan is an extracellular matrix receptor that provides a critical me- assess the tension required for activation of Piezo1 in the presence of a chanical link between the extracellular matrix (ECM) and the actin cytoskel- number of cytoskeleton-associated proteins using patch fluorometry. In eton to help muscle cells withstand contraction and to help neural cells addition we apply protocols that enable us to also measure the mechanical maintain the blood-brain barrier. When this link is disrupted, diseases properties of the cells expressing these channels. In the presence of the such as cancer and muscular dystrophy arise. Dystroglycan contains a sensitizer STOML3 the tension required to gate Piezo1 is <0.5 mN/m. In domain sandwiched between its ligand binding and transmembrane do- contrast the actin cross-linker FilaminA made the channel less sensitive to mains, which we term the ‘‘proteolysis domain’’, that contains proteolytic applied force. In addition for the first time we estimated the pre-tension sites that may act as additional ‘‘breakpoints’’ of the mechanical link it pro- that arises from the interaction between the cell membrane and glass vides. Notably, cleavage of dystroglycan by MMP-2 and MMP-9 in the pipette. We find that between patches this value can vary by more than 10x 40 amino acids prior to the transmembrane domain is often observed in (0.13 - 1.35 mN/m) and along with geometry provides a significant experi- pathogenic states. Thus understanding how the MMP cleavage is regulated mental variable when patching mechanically-gated channels. In addition, we is of clinical interest and may shed light on new mechanisms of disease find that the effects of cytoskeleton-associated proteins on the sensitivity of pathogenesis. We first mapped the boundaries of dystroglycan’s proteolysis Piezo1 seem to be independent of the bulk mechanical properties of the domain, which consists of an Ig-like and an SEA-like domain. We next cell, in this case Young’s modulus. The fact that cytoskeleton-associated showed that dystroglycan’s proteolysis domain is normally resistant to proteins can change the gating tension by more than threefold provides MMP cleavage. However, muscular dystrophy disease mutations found spatio-temporal means to modify the sensitivity of any channel to membrane within this domain or truncations destabilize the proteolysis domain and tension. sensitize it to MMP cleavage. We found that these mutations/truncations 563-Pos Board B333 enhance cell migration in wound healing assays in the context of full- Molecular Dynamics Study for Exploring the Force Transmission Pathway length receptors, and this phenotype can be rescued by MMP inhibitors or in the Bacterial Mechanosensitive Channel MscL deletion of the putative protease sites. Finally, we show that the dystrogly- Yasuyuki Sawada1, Takeshi Nomura2, Masahiro Sokabe3. can proteolysis domain can functionally substitute for the analogous proteo- 1Nutrition Science, Nagoya Univ Economics Fac Human Life Science, lytic switch domain in Notch receptors, hinting that increased mechanical Inuyama, Japan, 2Physical Therapy, Sch Health Sciences, Kyushu Nutrition forces in the cellular environment may provide a stimulus for MMP cleav- Welfare Univ, Kitakyushu, Japan, 3Mechanobiology Lab, Nagoya Univ Grad age in pathogenic states. Sch Medicine, Nagoya, Japan. The bacterial mechanosensitive channel MscL is constituted of homopentamer 566-Pos Board B336 of a subunit with TM1 inner and TM2 outer transmembrane helix. The major Fully Automated Ultrasound-Based Touch Assay for Small Model issue on MscL is to understand the gating mechanism driven by membrane ten- Organisms sion. Upon membrane stretch, the helices are dragged by lipids at the tension Miriam B. Goodman, Holger Fehlauer. sensor F78 and tilted, accompanied by outward sliding, leading to a gate expan- Stanford University, Stanford, CA, USA. sion. To get insights into the relationship between F78 and the gate including The sense of touch can be studied in small animals like C. elegans round- G22, we performed MD simulations of G22N GOF and F78N LOF MscLs worms and Drosophila larvae. A fundamental experiment for these studies and G22N showed spontaneous opening without membrane stretch, while is the classical touch assay: a worm is stroked with an eyebrow hair causing F78N could not be opened even under strong membrane tension. To assess the animal to reverse its movement. Two factors decrease the robustness of the role of Asn22 for the spontaneous opening, the double mutant G22N/ this assay: 1) small forces (microNewtons) are applied manually and are F78N MscL simulation was performed with and without membrane stretch highly variable1 and 2) behavioral changes are scored by observation. To in- and found that G22N/F78N MscL cannot open the gate upon membrane stretch crease reproducibility and throughput, we present a fully automated set-up under the both conditions. Furthermore, using some mutated MscL models, we delivering well-defined mechanical stimuli and performing automated found that the closed MscL can transmit resting tension to the gate via the inter- scoring of responses. In this set-up the stimulus is an ultrasound burst. It action between F78 in TM2 and I32-L36- I40 in the neighboring TM1 and the was shown previously that ultrasound induces behavioral changes in C. ele- transmitted force can lead to opening of the pore. It is suggested that the sub- gans by activating a mec-4-dependent gentle touch sensation pathway2.For stitution of F78 with Asn loses the transmission pathway, leading to be harder analyzing these behavioral changes in our set-up the animal is tracked, to open than G22N regardless of the substituted Asn22 in the hydrophobic gate recording changes in direction and speed following automate stimulus deliv- of G22N/F78N. ery. Such responses are classified as either null or an avoidance response.

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Our data shows that this set-up can be used to acquire highly reproducible cell membrane and actin. Previous studies suggest that a-catenin contains a touch assay results. Furthermore, the ultrasound parameters (frequency, pres- domain that normally masks vinculin binding sites, which can become sure, pulse length, inter-stimulus interval) can be varied to determine how accessible after protein unfolding induced by an external force. However, they affect touch sensation. This also allows to study mutants having an the molecular mechanisms remain largely unexplored as an obstacle in these increased touch sensation which is not possible in the classical touch assay. works is the lack of a single molecule technique capable of investigating We designed the system from commercially-available parts, enabling others actin-protein interactions at different forces with sufficient temporal and to replicate the apparatus and we will present not only behavioral results, but spatial resolution. also build instructions. Work supported by the Stanford Neurosciences Insti- Here, we use ultrafast force-clamp spectroscopy, a single-molecule technique tute NeuroCircuit program. based on laser tweezers, for investigating actin-protein interactions under con- 1 Nekimken, A. et al., PLOS ONE, 12 (5) (2017), 2 Kubanek, J. et al., bioRxiv, stant force with sub-millisecond and sub-nanometer resolution. We apply our 104463 (2017) technique to study the a-catenin-actin interaction in a range of physiological forces (1-17 pN). More specifically, we studied single a-catenin homodimers 567-Pos Board B337 and single a/b-catenin heterodimers under force. While single homodimers A Computational Study Towards Engineering an MscL Nanovalve exhibit a resistance to force with increasing lifetime with force, the single het- Adam D. Martinac1, Navid Bavi2, Omid Bavi3, Boris Martinac1. erodimers rapidly detach and reattach to actin, with increasing detachment rates 1Victor Chang Cardiac Research Institute, Sydney, Australia, 2Department of as the applied force increases. Notably, above few pN force, the a-catenin ho- Physiology, School of Medical Sciences, University of New South Wales, modimer displays 12 nm steps during its interaction with actin that are likely Sydney, Australia, 3Department of Physics, University of Tehran, Tehran, due to reversible unfolding of the protein. Such steps are not observed in the Islamic Republic of Iran. case of single heterodimers. However, when multiple heterodimers interact There are great opportunities in the manipulation of bacterial mechanosensi- with actin, both 12 nm steps and increased lifetimes of the catenin-actin tive (MS) ion channels for specific and targeted drug delivery purposes. bond are again observed. Recent research has shown that these ion channels have the potential to be converted into nanovalves through clever use of magnetic nanoparticles 570-Pos Board B340 and magnetic fields. Using a combination of molecular dynamics (MD) sim- The Anrep Effect: Role of Titin Strain ulations and the finite element (FE) modelling, this study investigates the Younss Ait-Mou1, Jody Martin2, Marion Greaser3, Mengjie Zhang1, feasibility of opening the MscL channel of E. coli by applying mechanical Pieter de Tombe2. force to its N-terminus, since this region has already been reported to func- 1Physiology, Loyola University Chicago, Maywood, IL, USA, 2Physiology tion as a major mechanosensor. The stress-strain behaviour of each MscL he- and Biophysics, University of Illinois at Chicago, Chicago, IL, USA, 3Animal lix was obtained using all atom MD of the wild-type (WT) MscL and G22N Sciences, University of Wisconsin at Madison, Madison, WI, USA. MscL mutant. In addition to indicating the main interacting residues at the Stretch of myocardium results in an immediate increase in contractile force hydrophobic pore, their pairwise interaction energies were monitored during (Frank-Starling response), followed by a slower increase that develops over the channel gating. We implemented these inputs into our FE model of MscL the course of several minutes (the Anrep effect). The cellular basis for the using curve-fitting codes and continuum mechanics equations. In the FE Anrep effect is an increase in cellular calcium loading; the molecular mech- model, the channel could be fully opened via pulling directly on the N-ter- anisms underlying this phenomenon, however, are incomplete understood. In minus and bottom of TM1 by mutating dominant van der Waals interactions this study we employed right ventricular trabeculae isolated from the hearts in the channel pore. This is an important finding suggesting that applying of either wild-type (WT) or homozygous giant titin mutant (HM) rats. Sarco- force in this manner is sufficient to open an MscL nanovalve delivering mere length was measured by high-speed laser diffraction; temperature was various drugs used, for example, in cancer chemotherapy. In summary, 25C. The sensitivity of twitch force towards extracellular [Ca2þ] was similar our computational modelling indicates that to fully operate an MscL nano- between HM and WT muscles. In contrast, presence of the giant titan iso- valve by pulling directly on the N-terminus and bottom of TM1, gain-of- form was associated with reduced passive force upon stretch and a blunting function (GOF) mutants would have to be employed rather than the WT of the the immediate Frank-Starling response. Moreover, the slow force MscL channel. response (Anrep) was blunted (by 50%) in the HM muscles, while the time-course of its development was not affected by titin strain. Twitch 568-Pos Board B338 timing increased similarly in both groups immediately upon stretch, but Mechanical Tension Serves as a Late G1 Cell Cycle Checkpoint did not alter during the subsequent slow force response in either muscle Nash D. Rochman, Nicolas Perez, Jiaxiang Tao, Sean Sun. group. The applied sarcomere strain upon stretch, and thus by inference Johns Hopkins, Baltimore, MD, USA. also membrane strain, was identical in WT and HM trabeculae. Our results, Cell cycle regulation seems a very complex process from the perspective of therefore, indicate that membrane strain per se may not be the principal the observer; but how does it appear to the cell? What quantities can the cell mechanism underlying the Anrep effect. measure and regulate to govern its own cycle? We investigate the role of cortical mechanics, and the interplay between mechanical stimulus and 571-Pos Board B341 YAP expression throughout cell cycle progression. Our evidence suggests Dissecting the Fast Hypoosmotic Permeability Response in E. coli a tension-mediated cell cycle checkpoint in late G1. We find that in non- Anthony Schams1, Madolyn Britt1, Ian Rowe1, Ugur Cetiner2, cancerous cells, a critical level of tension (reported through the measurement Andriy Anishkin1, Sergei Sukharev2. of pMLC content) must be achieved before DNA copying occurs. In 1Biology, University of Maryland, College Park, MD, USA, 2Biology and contrast, cancerous cells show much less well ordered behavior. YAP IPST, University of Maryland, College Park, MD, USA. expression also shows dependence on pMLC levels and we evaluate the Enteric bacteria and pathogens transmitted through fresh water easily survive role of YAP, downstream to increased cortical tension, in the progression drastic osmolarity changes. Upon a sudden dilution of external medium, from G1 to G2. bacteria evade mechanical rupture by engaging tension-activated channels MscS and MscL acting as major osmolyte release valves. Despite the prog- 569-Pos Board B339 ress in biophysical studies of these mechanosensitive channels, little is Load-Dependent Interaction of Single a- and a/b-Catenin Complexes with known about the actual release process in vivo. In the present work we utilize Actin early logarithmic MJF465 (mscS-, mscL-, mscK-) cultures expressing individ- Marios Sergides1,2, Claudia Arbore1,2, Francesco Saverio Pavone1,2, ual MscS or MscL channels and compare them with Frag1 (wt) in stopped- Marco Capitanio1,2. flow light scattering experiments. The scattering traces obtained from fast 1European Laboratory for Non-Linear Spectroscopy, Sesto Fiorentino, Italy, mixing of cells grown at 1200 mOsm with low-osmotic (0-700 mOsm) media 2Department of Physics and Astronomy, University of Florence, Sesto indicate a 30 ms swelling phase followed by fast (20-120 ms) osmolyte Fiorentino, Italy. release. At all shocks, the bending point designating the onset of release Mechanical signals occurring at the interface between cell membrane and (when half of the channel population opens) comes at slightly shorter times extracellular matrix and at intercellular junctions trigger biochemical signals for MscS than for MscL, consistent with their respective 7 and 12 mN/m that are fundamental for cell growth, development and regulation. Adaptor midpoint tensions determined in patch-clamp. According to estimations of proteins, which link the cell membrane to the actin cytoskeleton, seem to refractive indexes with the Rayleigh-Gans theory, cells undergoing osmotic partake in the process of mechanotransduction. Catenins play a key role in shock, may lose up to 15% of their dry mass in the form of permeable osmo- intercellular (adherens) junctions, where they act as a bridge between the lytes, within 50 ms. The lowest loss of osmolyte mass is observed in the

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MscS-expressing cells, consistent with the smaller pore of the channel. Par- the statistical distribution of antigen-antibody bond lifetimes. We demon- allel plate count experiments have shown that the cell viability starts to strate the use of our techniques on a library of co-evolved variant human an- decline at shock magnitudes where the osmolyte release rate, mediated by tibodies - both specific and broadly neutralising - and HIV gp120 envelope particular channels, saturates. Simulations of the competing processes of proteins. swelling and osmolyte release with the kinetic suite COPASI predict shock magnitudes at which the release system will be overwhelmed by water influx 574-Pos Board B344 2D and membrane tension will exceed the lytic threshold. The data indicate that Mechanical Stress Modulates Cytosolic Ca in MLO-Y4 Osteocyte-Like the cells require both MscS (capable of inactivation) and MscL for full Cells survival. Sheenah Lynn Bryant1, Elizabeth T. Leung2, Daniel R. Prather3, Conner Patricelli4, Daniel Fologea3. 1 2 572-Pos Board B342 Biomolecular Sciences, Boise State University, Boise, ID, USA, Health 3 Ultrasound Modulates Piezo1-Mediated Mechanotransduction in Neu- Science, Boise State University, Boise, ID, USA, Physics, Boise State 4 ro2A Cells University, Boise, ID, USA, Mechanical Engineering, Boise State Wei-Wen Liu, Pai-Chi Li. University, Boise, ID, USA. National Taiwan University, Taipei, Taiwan. Understanding the molecular processes that lead to accelerated bone loss for Mechanotransduction has been demonstrated to play an important role in astronauts (1-2% each month) is crucial for mitigating risk factors associated mediating cellular or tissue adaption. Ultrasound stimulation can trigger me- with extended spaceflight and mission operations. In this endeavor, we hypoth- 2þ chanosensing bioeffects in several biological systems through the mechanical esized that abnormal Ca transport through mechanosensitive Transient Re- pressure wave. It is well documented that the ultrasound-induced mechano- ceptor Potential Vanilloid (TRPV) ion channels is a major contributor to sensing bioeffects have been widely applied for several kinds of noninvasive accelerated bone loss during mechanical stress and/or radiation. We succeeded 2þ therapies, such as bone fracture healing, lithotripsy, nerve regeneration, deep- in monitoring Ca influx of osteocyte-like MLO-Y4 cells using Cal520-AM as 2þ brain stimulation for brain disorder treatment, or noninvasive functional aCa detection reagent and ionomycin as a specific Ca2þ transporter positive neurosurgery. However, the gene regulatory mechanism of ultrasound- control. Spectrofluorometry and image analysis performed after exposure of 2þ induced mechanosensing bioeffects is not fully understood. Recently, Piezo1 cells to hypotonic conditions clearly demonstrated a significant Ca influx, was characterized as the mechanically activated ion channels through cell- which presumably originated from TRP channel activation. Using a custom de- attached patch clamp assay, poke assay, atomic force microscopy-based assay, signed stretch system, we confirmed that 5-20% uniaxial stretch induced 2þ high-pressure perfusion assay, or stretch assay. It also has been demonstrated similar changes in cytosolic Ca . that Piezo1 channel activity was mediated in a substrate stiffness-, shear 575-Pos Board B345 stress-, osmotic pressure-, or compression-dependent manner. However, Mechanosensation through Radicals in Tensed Collagen whether Piezo1 can play a role in ultrasound-induced mechanotransduction Christopher Zapp1, Agnieszka Obarska-Kosinska1, Csaba Daday1, pathway is not clear. Here, we applied a technique by using focused ultra- Reinhard Kappl2, Frauke Gr€ater1. sound to deliver a non-contact mechanical stimulus to cells labeled fluorescent 1Molecular Biomechanics, HITS, Heidelberg, Germany, 2FR2.5 Biophysik, calcium dye. Our study demonstrated that the calcium influx was transiently Universit€at des Saarlandes, Homburg, Germany. increased in Piezo1-overexpressed Neuro2A cells following the ultrasound Biological tissues are subjected to vastly varying levels of mechanical load. stimulation. Using siRNAs against Piezo1 in Neuro2A cells showed decreased Protein regulation by force-induced conformational changes has been estab- calcium influx following the ultrasound stimulation. Compared to cells treated lished as a crucial mode of mechanosensing. However, protein-based biological with or without ultrasound, cell viability was not affected. In conclusion, our materials like skin, cartilage, and tendon can experience excessive stresses all study demonstrated that ultrasound could emerge as a way to deliver mechan- the way up to tissue rupture. Whether and how covalent bond scission in ical force to trigger Piezo1 channel activity by using our experimental system. stressed protein materials could contribute to mechanosensing remains un- The system can serve as a basis for further investigation of the Piezo1- known. We addressed this question for collagen, the most abundant protein mediated mechanotransduction mechanism in cells following ultrasound in humans, the major force-bearing component of essentially all tissues, and stimulation. a heavily crosslinked biopolymer. Using combined rheology and electron paramagnetic resonance (EPR) experi- 573-Pos Board B343 ments, we show that physiological levels of stress on collagen fibrils from rat Biomechanics of HIV and Immune Cell Interactions Investigated using tail tendon lead to the formation of mechano-radicals by covalent bond rupture. Magnetic Tweezer Force Spectroscopy Radical formation increases with the level of applied stress and occurs already James L. Flewellen1,2, Pavel Tolar1,2. 1 2 much before fiber rupture. Imperial College London, London, United Kingdom, The Francis Crick We identify specific bonds in the lysine-based crosslinks as points of stress Institute, London, United Kingdom. concentration in tensed fibers through classical molecular dynamics simula- Antibodies are essential to the immune response to infections. They are pro- tions. Using high-level quantum calculations, we establish the chemical duced naturally by B lymphocytes upon exposure to a pathogen, or introduced nature of the scission to be homolytic. The radicals subsequently undergo through vaccination. Pathogens with high variability, including human immu- rapid reactions with water, yielding reactive oxygen species. Taken nodeficiency virus (HIV) and influenza, can evade the typical antibody together, our study proposes a new mode of coupling between mechanical response. However, broadly neutralising antibodies capable of counteracting and oxidative stress in collagen-based tissues as a missing link between these pathogens do develop naturally: usually only in chronically infected pa- mechanical load and biological processes, from pain sensation to tients, and in a minority of cases. Such broadly neutralising antibodies are typi- inflammation. cally selected against by normal cellular differentiation processes throughout the immune response. This selection is thought to be influenced by the strength 576-Pos Board B346 and nature of physical binding between the B cell receptor and antigen proteins A Lever-Like Transduction Pathway for Long-Distance Chemical- and from the pathogen. A greater understanding of the biomechanics of immune Mechano-Gating of the Mechanosensitive Piezo1 Channel cell interaction with pathogens will inform the development of next generation Yanfeng Wang1, Shaopeng Chi1, Qiancheng Zhao1, Jianhua Wang2, vaccines. Tingxin Zhang1, Jie Geng1, Guang Li1, Li Wang1, Kun Wu1, Yu Rao1, Magnetic tweezers offer a straightforward, non-invasive technique for high- Liansuo Zu1, Wei He1, Huifang Guo3, Meng-Qiu Dong2, Bailong Xiao1. throughput force spectroscopy experiments on the microscale. We present a 1Tsinghua university, Beijing, China, 2National Institute of Biological bespoke magnetic tweezer system and molecular biology assays capable of Science, Beijing, China, 3Chinese Academy of Medical Science, Beijing, probing the biomechanical interactions between HIV envelope proteins China. and antibodies derived from co-evolved B cells. DNA tethers, bound to a The three-bladed, propeller-like Piezo1 channel represents a prototype of glass surface at one end, are conjugated to antigens of interest at the other. mammalian mechanosensitive cation channels critical for various mechano- The antigens bind to complementary antibodies, which in turn are fixed to transduction processes. However, its mechanogating mechanisms remain un- superparamagnetic microspheres. Voice coil technology is employed to clear. Here we identify a novel set of Piezo1 chemical activators, termed move permanent magnets to the proximity of the sample and the introduced Jedi, distinct from the known activator Yoda1. Both Jedi and Yoda1 bind to magnetic field provides an exquisitely controllable force clamp. Through the N-terminal propeller-resembling structure instead of the C-terminal pore re- video microscopy, our apparatus facilitates high-throughput recording of gion located 100 A˚ away, indicating long-distance allosteric gating. Deleting many single molecule interactions simultaneously, allowing us to determine two extracellular loop regions that might reside in the distal blade abolishes

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Jedi-, but not Yoda1-induced activation and diminishes mechanogating of observed expected tension changes. We also encoded it into the mechano- Piezo1. We have mapped the region that forms the 80 A˚ -long intracellular sensing cell-surface receptor Notch. Upon presentation of Notch’s ligand beam-like structures, which bridge the blades to the pore. Mutating two single on a neighboring cell, we detected a decrease in BRET signal corresponding residues in the proximal end of the beam abolishes Jedi- and Yoda1-induced to a stretching of the sensor due to the force induced by endocytosis of the activation and impairs mechanogating. We propose that Piezo1 utilizes a ligand. This signal can be measured in bulk by a luminometer and in individ- lever-like mechanism for effectively transducing force from the mechanosens- ual cells via imaging. We anticipate our BRET-based molecular tension ing blades to the pore, enabling long-distance gating. sensor will be well suited to identify novel mechanosensing proteins and characterize the magnitude of change in tension sensed during disease 577-Pos Board B347 pathogenesis. Recovery of Equilibrium Free Energy from Non-Equilibrium Thermody- namics with Mechanosensitive Ion Channels in E. coli 580-Pos Board B350 Ugur Cetiner1,2, Oren Raz3, Sergei Sukharev1,2, Christopher Jarzynski1,4. Using a Fluctuation Analysis of Limit Cycle Oscillations in Inner Ear Hair 1Institute for Physical Science and Technology, University of Maryland, Bundles as a New Test of Low Dimensional Dynamical Models College Park, MD, USA, 2Department of Biology, Maryland Biophysics Janaki K. Sheth. Program, University of Maryland, College Park, MD, USA, 3Department of Physics and Astronomy, University of California, Los Angeles, Los Angeles, Physics of Complex Systems, Weizmann Institute of Science, Rehovot, CA, USA. Israel, 4Department of Chemistry and Biochemistry, Department of Physics, The transduction of pressure waves into electrical signals in the inner ear is an Maryland Biophysics Program, University of Maryland, College Park, MD, active process involving the complex interplay of elastic elements, molecular USA. motors, and mechanically sensitive ion channels. One dramatic manifestation Bacterial mechanosensitive channels are major players in cells’ ability to cope of the active process in inner ear hair cells is the occurrence of spontaneous with hypo-osmotic stress. Excess turgor pressure due to fast water influx is bundle oscillation, which has been observed in vitro in a number of species. reduced as the channels, triggered by membrane tension, open and release os- Several systems of nonlinear differential equations have been proposed as molytes. However, in vitro measurements of the free energy difference between models of this limit cycle oscillation, including the simple two-dimensional the open and closed states of ion channels are challenging due to hysteresis ef- Hopf oscillator and more complex variants that explicitly incorporate physio- fects and inactivation. Exploiting recent developments in statistical physics, we logical variables such as membrane potential, bundle deflection, and molecular present a general formalism to extract the free energy difference between the motor activity. All such models that admit spontaneous oscillation exhibit a sta- closed and open states of mechanosensitive ion channels from non- ble limit cycle in some parameter regime. Additionally, all of these lower equilibrium work distributions associated with the channels’ gating recorded dimensional models include stochastic noise in one or more variables repre- in native patches under ramp stimulation protocols. We show that the work dis- senting implicitly the effect of other degrees of freedom. Examples include tributions obtained from the gating of MscS channels in E. coli membrane ion channel noise, Brownian motion of the bundle, and stochastic molecular satisfy the strong symmetry relations predicted by the fluctuation theorems motor activity. We study the effects of noise on these stable limit cycle dy- and recover the equilibrium free energy difference between the closed and namics using both analytic calculations and numerical simulations. We show open states of the channel within 1 kBT of its best estimate obtained from an that d-dimensional noisy limit cycle oscillators generically display phase diffu- independent experiment. sion at long time scales and (d-1) degrees of freedom that behave roughly as overdamped oscillators with characteristic Lorentzian power spectra. We 578-Pos Board B348 then compare our theoretical results to data from Rana catesbeiana hair cells, Deciphering Binding Capabilities of Human Antibodies to Defined Pat- showing that the physiological system indeed displays evidence of the terns of Antigens nonlinear coupling of the fluctuations predicted by the proposed models. We Bjo¨rn Ho¨gberg. also address the fundamental question of how experiments observing only a Karolinska Institutet, Stockholm, Sweden. subset of the dynamical variables may use a fluctuation-based analysis to Human antibodies in the families IgG and IgM are highly competent extract the maximal information regarding these remaining ‘‘hidden binders utilized by the immune system. The molecules in their monomeric variables.’’ form are capable of forming bivalent binding due to the nature of the double Fab domains. Traditionally it has been hard to precisely compare 581-Pos Board B351 the binding capabilities of the different IgGs. Here we present a comprehen- Activation of Endogenous Piezo1 Channels by Shear Stress in Excised sive study building on a new method where molecularly precise nanoscale Membrane Patches patterns of antigens are presented using DNA orgami immobilized in an Jian Shi, Baptiste Rode, David J. Beech. SPR setup. Variants of human IgG1, 2, 3, 4 and IgM bearing the exact University of Leeds, Leeds, United Kingdom. same ligand binding domains are compared and the binding analyzed with The sensing of shear stress arising because of blood flow is critical in vascular respect to both distance between antigens and arrangements of multimeric an- development and maintenance of a healthy vasculature in the adult. The iden- tigen patterns. These results might have implications for exact nanoscale tity of molecules which sense and transduce this force into appropriate arrangement of antibodies on biological functions and their relation to com- vascular anatomy and function is therefore keenly sought. A central question plement pathway activation and antibody dependent cellular cytotoxicity is is whether there is a force sensor protein (‘‘receptor’’) which directly detects discussed. the force, acting either alone or in complex with other proteins. Piezo1 chan- nels are Ca2þ-permeable non-selective cationic channels which are activated 579-Pos Board B349 by membrane stretch. These channels are important for shear stress-sensing Development of a BRET-Based Molecular Tension Sensor to Study Altered and vascular function in embryonic and adult mice (Li et al 2014 Nature Tensions in Disease Pathogenesis 515, 279-; Rode et al 2017 Nature Communications 8, 350-). We have used Eric J. Aird, Kassidy J. Tompkins, Wendy R. Gordon. patch-clamp recording to study the channels in freshly isolated endothelium Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, of second-order mesenteric arteries of adult mice. The pipette solution con- Minneapolis, MN, USA. tained (mM): 145 KCl, 1 MgCl2, 0.5 EGTA, 10 HEPES. Whole-cell perfo- Alteration of mechanical forces is an emerging factor in diseases like cancer. rated patch recordings from endothelial fragments revealed a resting Changes in macroscopic stiffness in disease are accompanied by a wealth of membrane potential of 43 mV which was depolarized by fluid flow. changes in a cell’s tensional homeostasis at a molecular level where mecha- When Piezo1 was conditionally deleted in endothelium the resting potential notransduction signaling pathways are aberrantly activated. However, the was hyperpolarized and fluid flow-evoked depolarization was absent (Rode lack of tools to measure tensions at a molecular level in the cellular environ- et al 2017). Outside-out patches were then excised from endothelium and ment has crippled the identification of mechanosensing proteins and charac- found to contain constitutive single channel activity with the expected 25- terization of magnitudes of physiologic force in normal and disease contexts. pS unitary conductance of Piezo1 channels; the channel activity could be We have developed a new bioluminescence resonance energy transfer further enhanced by fluid flow and inhibited by the non-selective Piezo1 chan- þ (BRET)-based molecular tension sensor that responds predictably to changes nel inhibitor, Gd3 , and was absent in Piezo1 knockout mice (Rode et al in forces and exhibits an enhanced dynamic range compared to other FRET- 2017). The data suggest Piezo1 channels of the endothelium can be activated based molecular tension sensors. We show that our tension sensor exhibits a by shear stress in a sparse excised membrane environment, consistent with distance-dependent change in BRETthrough the use of rigid alpha-helical direct sensing of shear stress or a closely-associated force. The research linkers of varying length. As a proof of concept, we inserted our sensor was supported by grants from the British Heart Foundation, MRC, Wellcome into the canonical mechanosensing focal adhesion protein vinculin, and Trust.

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582-Pos Board B352 Together our results describe, at the molecular level, a potential mechanism Strained Collagen Resists Bacterial Collagenase Degradation by which transduction is tuned permitting the detection of a variety of mecha- Karanvir Saini, Manorama Tiwari, Jerome Irianto, Charlotte Pfeifer, nosensory stimuli. Cory Alvey, Dennis E. Discher. Univ of Pennsylvania, Univ of Pennsylvania, Philadelphia, PA, USA. Background: Collagen, a triple-helical, self-organizing protein, is the predom- Posters: Intracellular Calcium Channels and inant structural protein in mammals, found in tendon, bone, ligament, carti- lage, intervertebral disc, skin, blood vessel, and cornea. Tendons have 70- Calcium Sparks and Waves I 80% dry weight as collagen, and function as dynamic structures which 585-Pos Board B355 respond to the magnitude, direction, frequency, and duration of physiologic Understanding the Molecular Mechanism of Cation Permeation in the as well as pathologic mechanical loads via complex interactions between Cardiac Ryanodine Receptor (RyR2) Channel using Computational cellular pathways and the highly specialized extracellular matrix. This extra- Electrophysiology cellular matrix of tendon is hierarchical structure having collagen fibrils ori- Williams E. Miranda1, Van A. Ngo1, S.R. Wayne Chen2, Sergei Y. Noskov1. ented along longitudinal axis of tendon. The theory suggests that the 1Department of Biological Sciences, Centre for Molecular Simulations, mechanisms which drive the preferential accumulation of collagen of loaded University of Calgary, Calgary, AB, Canada, 2Department of Physiology and tissue are not only cell-driven but operate at the molecular level. The concept Pharmacology, Libin Cardiovascular Institute of Alberta, University of reduces control of matrix morphology in degrading chemical environment via Calgary, Calgary, AB, Canada. mechanical strain. Methodology: The investigation was carried out in an The ryanodine receptor isoform 2 (RyR2) is a key component in the cardiac environmentally-controlled microbioreactor in which fascicle extracted from þ excitation-contraction coupling. This channel regulates the release of Ca2 mice tail tendon was subjected to strain gradients using three-point bending. from the sarcoplasmic reticulum to the cytosol, a critical event for cardiomyo- The unstrained fascicle was labelled with dye and stripes were created on it cyte contraction. Interestingly, RyR2 shows poor selectivity for calcium via photobleaching. The deformation of fascicle lead to change in the þ þ þ þ (Ca2 /K 7-fold), while conducting K 6-fold faster than Ca2 . Most displacement between stripes which resulted in quantification of strain magni- of combined mutagenesis and electrophysiology studies for mapping key res- tude. Thereafter, deformed fascicle was exposed to bacterial collagenase (BC) þ idues modulating cation permeation in RyR2 are based on K conductance. and its degradation was tracked using Second Harmonic Generation (SHG) þ Nevertheless, the molecular basis of K permeation mechanism and thermo- signal produced by collagen fibrils present in it. It was found that mechanical dynamics in RyR2 is still lacking. In this work, we used computational elec- strain significantly increased degradation time of Collagen fibrils present in trophysiology approaches in all-atom molecular dynamics (MD) simulations the regions of fascicle having high tensile strain compared to the ones having þ to study the permeation of K ions through the wild-type form of the channel low strain magnitudes. Conclusions/Significance:Our study demonstrates (RyR2-WT) and cytoplasmic-vestibule mutants (RyR2-G4871C/E4872C and for the first time that applied mechanical strain preferentially preserves RyR2-G4871H). Starting from the open-state channel structure solved by collagen fibrils forming tendon tissue in the presence of a pathologically- Cryo-EM at 4.2 A˚ resolution, we generated 10x100 ns trajectories for a total important BC. Our results have the potential to extend our understanding of of 1 ms aggregated simulation time. A constant electric field was applied to phenomena like development, adaptation, remodeling and disease of many þ drive K through RyR2-variants from the luminal- to the cytosolic- side collagen matrices. under symmetric concentration [KCl] = 810 mM. The calculated average 583-Pos Board B353 conductance (635, 606, and 557 pS) fairly agree with experimental- Lipid-Gel Model of Biological Membranes electrophysiology single-channel measurements (763, 706, and 516 pS), 1 2 2 3 respectively. Also, our simulations highlight important differences between Zheng Shi , Zachary T. Graber , Tobias Baumgart , Howard A. Stone , þ Adam E. Cohen1. the pores of canonical K channels and RyR2. We identified two major cation 1Chemistry and Chemical Biology, Harvard University, Cambridge, MA, binding sites at the selectivity filter and the constriction point near the USA, 2Chemistry, University of Pennsylvania, Philadelphia, PA, USA, cytoplasmic-vestibule. Finally, we found that mutations at the cytoplasmic- 3Mechanical and Aerospace Engineering, Princeton University, Princeton, vestibule increase the free energy barriers for ion permeation towards the NJ, USA. cytoplasmic region, resulting in reduced conductance compared to the WT. 2þ Membrane tension affects cell migration, cell cycle, cell signaling, and me- This work provides the basis for future all-atom simulations of Ca perme- ation to understand the molecular underpinnings of low Ca2þ/Kþ selectivity chanosensation. The fluid-mosaic model posits a liquid-like plasma mem- þ brane, which can flow in response to tension gradients. It is widely and higher K conductance in RyR2. assumed that membrane flow transmits local changes in membrane tension across the cell in milliseconds. This conjectured signaling mechanism has 586-Pos Board B356 been invoked to explain how cells coordinate changes in shape, motility FRET-Based Trilateration Applied to the Mapping of CaM within the and vesicle fusion, but the underlying propagation has never been observed Ryanodine Receptor directly. Here, we present a simple biophysical assay to measure the propaga- Bengt Svensson, Robyn T. Rebbeck, David D. Thomas, Razvan L. Cornea. tion of membrane tension in mammalian cells. In cell-attached membrane Biochemistry, Molecular Biology and Biophysics, Univ. of Minnesota, blebs, tension equilibrated within 1 s, but in intact cells we observed no Minneapolis, MN, USA. sign of tension propagation in measurements that lasted up to 500 s. The fail- We have carried out trilaterations, based on simulated annealing calculations and time-resolved FRET measurements, to map the location of calmodulin ure of tension to propagate is explained by a simple fluid dynamical model 2þ that incorporates the flow resistance due to cytoskeleton-bound transmem- (CaM) within the ryanodine receptor (RyR) Ca channel. Fluorescence life- brane proteins. This model predicts that the membrane behaves as a two- time (FLT) detection of FRET allows resolving multiple distances for each dimensional gel, through which molecule-sized tracers can readily diffuse, FRET pair, which are represented by Gaussian distance distributions fitted to but where bulk flow is largely suppressed. Under physiological conditions, the FRET data. Our improved general trilateration approach takes into account cellular membrane tension is therefore heterogeneous and far from equilib- the distance distributions from multiple FRET pairs, and shows the location of rium. Our results show that membrane tension is not a global mediator of the acceptor probe as a probability distribution in the format of an MRC/CCP4- long-range signaling, but that local changes in membrane tension may format density map. This map can be displayed using common molecular mediate distinct processes in different sub-cellular domains. graphics software. A more specialized approach takes into account the four- fold symmetry of the RyR channels in calculating the predicted FRET from 584-Pos Board B354 a donor with known location to symmetrically located FRET acceptors. Several Cell Type Specific Splicing of Piezo2 Regulates Mechanotransduction single-cysteine variants of the 12-kDa FK506-binding protein (FKBP) were Marcin Szczot1, Leah Pogorzala2, Hans Jurgen€ Solinski1, Mark Hoon2, labeled with FRET donor (Alexa Fluor 488), and targeted to RyR1 and Alexander T. Chesler1. RyR2. Effective average positions of the donors were calculated from simu- 1NCCIH, NIH, Bethesda, MD, USA, 2NIDCR, NIH, Bethesda, MD, USA. lated annealing, constrained by the FKBP atomic structure and by the RyR1 Piezo2 is a mechanically activated ion-channel required for touch discrimina- and RyR2 cryo-EM maps. Four single-cysteine variants of CaM, two N-lobe tion, vibration detection and proprioception. Here we discovered that Piezo2 is and two C-lobe sites, were labelled with fluorescence acceptor (Alexa Fluor extensively spliced, producing different Piezo2 isoforms with distinct proper- 568). FRET from the FKBP donors to the CaM acceptor was measured via ties. Sensory neurons from both mice and humans express a large repertoire FLT detection. The FRET-based trilateration has allowed us to resolve the of Piezo2 variants whose expression is cell-type specific. Biophysical charac- location of the N- and C-lobes of CaM within the RyR1 and RyR2 cryo-EM terization revealed substantial differences among Piezo2 splice variants. maps. A shift in the location of CaM dependent on resting or contracting

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[Ca2þ] is observed. The upgraded methodology is setup for determining the loading. Taken together, we found that the N-terminal RyR2R420Q mutation structure-function relationship of other RyR modulators. This work was sup- results in unique cellular Ca2þ signaling alterations. Supported by SNF and ported by NIH grants R01 HL92097 (RLC) and R37 AG26160 (DDT), and ANR. by American Heart Association Postdoctoral Fellowship 16POST31010019 (RTR). 589-Pos Board B359 Dual Ablation of the RYR2-S2808 and RYR2-S2814 Sites Increases Pro- 587-Pos Board B357 pensity for Pro-arrhythmic Spontaneous Calcium Releases Generation and Characterization of CPVT1 Cardiomyocytes using Duilio Michele Potenza1, Radoslav Janicek1, Miguel Fernandez-Tenorio1, Human Induced Pluripotent Stem Cells and CRISPR/Cas9 Gene Editing Hector H. Valdivia2, Ernst Niggli1. Naohiro Yamaguchi1,2, Xiao-Hua Zhang1,2, Hua Wei1,2, Martin Morad1,2. 1Department of Physiology, University of Bern, Bern, Switzerland, 2Center 1Regenerative Medicine and Cell Biology, Medical University of South for Arrhythmia Research, University of Michigan, Ann Arbor, MI, USA. Carolina, Charleston, SC, USA, 2Cardiac Signaling Center of University of During physical exercise or stress, the sympathetic system stimulates cardiac South Carolina, Medical University of South Carolina and Clemson contractility via b-adrenergic receptor (b-AR) activation, resulting in PKA- University, Charleston, SC, USA. mediated phosphorylation of the cardiac ryanodine receptor (RyR2). So far Over 150 missense mutations in human type2 ryanodine receptor (RYR2) three RyR2 phosphorylation sites have been analyzed. S2808 and S2030 are gene have been found to be associated with type1 catecholaminergic polymor- described as PKA specific sites and S2814 is a CaMKII specific target. To phic ventricular tachycardia (CPVT1). Although recent advance in stem cell examine the contribution of these sites to calcium signaling, we performed technology enables us to study CPVT1 pathology using skin biopsies of hu- confocal Ca2þ imaging in a double knock-in (DKI) mouse model in which man patients, many of CPVT1 mutations identified had lethal consequences, S2808 and S2814 phosphorylation sites have been replaced by alanine and patient’s tissue is not readily available, making characterizations of (RyR2-S2808A/S2814A). Isoproterenol was used to stimulate b-ARs. In con- certain mutation sites difficult if not impossible. As an alternative approach trol conditions we observed a higher Ca2þ spark frequency, resulting in we introduce the CPVT1 associated RYR2 mutation in healthy wild type reduced SR Ca2þ content and smaller Ca2þ transients in myocytes from human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 gene- DKI compared to wild type (WT) animals. It has been shown that the refrac- editing. This approach enables us to: (1) generate human CPVT1 model toriness of Ca2þ spark triggering depends on the Ca2þ sensitivity of the RyRs. cardiomyocytes without patients’ tissue; (2) carry out comprehensive charac- Moreover, it was demonstrated that b-AR stimulation shortens RyRs refracto- terization of Ca2þ signaling aberrancies and their pharmacological sensitiv- riness. We measured Ca2þ spark restitution using the ‘‘low-dose ryanodine ities by drug screening; (3) validate whether the mutation is causative for method’’ in which repetitive sparks originating from the same cluster of CPVT1, and (4) establish homozygote cell-lines carrying the mutation. Since RyRs are recorded. We found that DKI RyRs were more Ca2þ sensitive in most of the CPVT1 mutations in patients are heterozygous, this approach will control conditions, compared to WT. Isoproterenol further shortened RyR provide us with deeper insights in mutation-related structure/function changes refractoriness in DKI cardiomyocytes. Measurements of latencies to the 1st in RYR2. As a first step, we introduced F2483I RYR2 mutation in wild type wave after a train of depolarizations revealed increased propensity for spon- hiPSCs, because we had previously characterized the calcium signaling pa- taneous Ca2þ releases in DKI animals, both in control and during b-AR thology in hiPSC-derived cardiomyocytes from CPVT1 patient carrying the stimulation. F2483I mutation, making it possible to compare the results from gene- Together, our results suggest that ablation of both the RyR2-S2808 and RyR2- edited cells with those of the patient-derived cells. Our preliminary studies S2814 sites increases propensity for pro-arrhythmic spontaneous Ca2þ releases, show Ca2þ sparks with significantly longer durations compared to the wild as previously suggested for hypo-phosphorylated RyRs. Moreover, since the type, increased level of diastolic Ca2þ and smaller sarcoplasmic reticulum DKI cells present a full response to isoproterenol, it seems that in these cells store size in gene-edited F2483I hiPSC-derived cardiomyocytes, consistent phosphorylation of S2030 is sufficient for b-AR mediated sensitization of the with the data from patient-derived F2483I cardiomyocytes. Supported by RyRs. Supported by SNF. NIH. 590-Pos Board B360 588-Pos Board B358 Search for Type 2 Ryanodine Receptor Inhibitor by Monitoring Endo- The RyR2R420Q Mutation Triggers Catecholaminergic Polymorphic Ven- plasmic Reticulum Ca2D tricular Tachycardia in Mouse Cardiomyocytes via SR Calcium Loading Mai Tamura1, Nagomi Kurebayashi1, Takashi Murayama1, Shuichi Mori2, Miguel Fernandez-Tenorio1, Radoslav Janicek1, Riccardo Rizzetto2, Mari Ishigami-Yuasa2, Hiroyuki Kagechika2, Junji Suzuki3,4, Alexandra Zahradnikova Jr.2, Yue Yi Wang2, Jean-Pierre Benitah2, Kazunori Kanemaru5, Masamitsu Iino3,5, Takashi Sakurai1. Esther Zorio3, Ana M. Gomez2, Ernst Niggli1. 1Jutendo University, Tokyo, Japan, 2Tokyo Med. Dent. University, Tokyo, 1Department of Physiolgy, University of Bern, Bern, Switzerland, 2Inserm, Japan, 3The Univ Tokyo, Tokyo, Japan, 4University of California, San UMR-S 1180, Chaˆtenay-Malabry, France, 3Hospital La Fe, Valencia, Spain. Francisco, CA, USA, 5Nihon Univ. Sch. Med., Tokyo, Japan. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is a The type 2 ryanodine receptor (RyR2) is a Ca2þ release channel on the endo- genetic disease associated with mutations in the RyR2, characterized by syn- plasmic reticulum (ER) and plays a central role in the excitation-contraction cope and/or sudden death during exercise or emotional stress. For this study coupling in the heart. Amino acid mutations in RyR2 are known to cause we used a CPVT mouse model bearing the RyR2R420Q mutation in the N-ter- many types of fatal arrhythmic diseases including catecholamine-induced minal region. These knock-in (KI) mice recapitulate the human disease polymorphic ventricular tachycardia (CPVT). Because spontaneous Ca2þ phenotype on the organ and cellular level, with bidirectional ventricular release from ER Ca2þ store, which induce triggered activity, is likely to occur tachycardia after epinephrine/caffeine injection in KI animals and delayed in cardiac cells in patients with CPVT, drugs that suppress RyR2 are expected afterdepolarizations (DADs) and triggered activity in KI myocytes during to have antiarrhythmic effects. We have studied properties of RyR2 using b-adrenergic receptor (b-AR) stimulation. Intact cardiomyocytes showed HEK293 expression system and indicated that the ER Ca2þ signal by the higher Ca2þ spark frequency with longer duration and small differences in ER Ca2þ indicator, R-CEPIA1er, well reflects the Ca2þ releasing activity of amplitude. Unlike WT myocytes, the median of the Ca2þ spark recovery dis- RyR1 and RyR2 (Murayama et al, Human Mut., 2016, Uehara et al. J. Gen. tribution in low-dose ryanodine did show a leftward shift, suggesting an un- Physiol., 2017, Fujii et al. Heart Rhythm, 2017). In this study, we aimed to expected absence of RyR2R420Q sensitization during b-AR stimulation. KI search for RyR2 inhibitors by ER Ca2þ monitoring. HEK293 cells stably ex- cells also exhibited a lower SR-Ca2þ threshold for the initiation of Ca2þ pressing wild-type or mutant RyR2 and R-CEPIA1er were generated and the waves (recorded with Fluo-5N entrapped in the SR of permeabilized cells), R-CEPIA1er signal was measured in a 96-well plate by a FlexStation II spec- suggesting an enhanced RyR Ca2þ sensitivity. The SR-Ca2þ depletion during trophotometer. Approximately 1,600 of well-characterized compounds were Ca2þ waves was similar in RyR2R420Q and WT animals, however the frac- screened, and compounds that alter the R-CEPIA1er signal exceeded the set tional SR Ca2þ release was increased in RyR2R420Q cells. When b-AR stim- reference value (mean value þ/- 4SD) were selected. For the selected hit com- ulation was mimicked with cAMP, the wave threshold in RyR2R420Q cells pounds, single cell Ca2þ imaging of HEK 293 cells and cardiac muscle reached values similar to that in WT. These findings indicate that the SR derived cell lines were carried out to investigate the reversibility of the drugs Ca2þ leak mediated by the hypersensitive RyR2R420Q does not increase dur- and the effect on action potential. Furthermore, cytoplasmic Ca2þ-dependent ing b-AR stimulation and thus cannot prevent substantial SR Ca2þ loading. Ca2þ release activity of RyR2 was determined by [3H]ryanodine binding Therefore, the SERCA stimulation may initially add an element of negative assay. Several promising compounds were found. This screening method feed-back to the wave generation (resulting in elevation of the threshold), but with ER Ca2þ monitoring was shown to be useful for searching RyR2 sup- finally contribute to triggering of arrhythmogenic Ca2þ waves by SR (over)- pressors.

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591-Pos Board B361 593-Pos Board B363 Regulation of Store Ca2D Level in HEK293 Cells Expressing Type 2 Rya- Novel Inhibitors of Leaky Skeletal Ryanodine Receptor Calcium Channels nodine Receptor (RyR2) with CPVT Mutations Discovered via FRET-Based High-throughput Screening Nagomi Kurebayashi1, Takashi Murayama1, Ryosaku Ohta2, Claire E. Haskin1, Robyn T. Rebbeck1, Gregory D. Gillispie2, Fumiyoshi Yamashita3, Junji Suzuki4, Kazunori Kanemaru5, David D. Thomas1, Razvan L. Cornea1. Masamitsu Iino4, Takashi Sakurai1. 1Biochemistry, Molecular Biology, and Biophysics, Univeristy of Minnesota, 1Dept. of Pharmacology, Juntendo University, Faculty of Medicine, Tokyo, Minneapolis, MN, USA, 2Fluorescence Innovations Inc., Minneapolis, MN, Japan, 2Dept. of Drug Delivery Research, Kyoto University, Kyoto, Japan, USA. 3Kyoto University, Kyoto, Japan, 4The University of Tokyo, Tokyo, Japan, Using fluorescence lifetime (FLT) detection of FRET, we carried out high- 5Dept. of Pharmacology, The University of Tokyo, Japan. throughput screening (HTS) of a 50,000-compound library for modulators of RyR2 is the major Ca2þ release channel in ER Ca2þ store in heart. Many the skeletal-muscle intracellular Ca2þ release channel, the ryanodine receptor mutations in RyR2 are linked to catecholaminergic polymorphic ventricular (RyR1), for therapeutic applications. Under resting Ca2þ, increased propensity tachycardia (CPVT). The CPVT-linked RyR2s expressed in HEK293 cells of channel openings due to RyR dysregulation is associated with severe skeletal 2þ 2þ are reported to decrease threshold ER Ca ([Ca ]ER) for spontaneous and cardiac myopathies, diabetes and neurological disorders. This is the leaky Ca2þ release compared to WT. Two types of Ca2þ dependent regulation state of the RyR that has become an attractive target for pharmacological agents 2þ of RyR2 are likely explanations for the effects on the [Ca ]ER.Oneisan to treat such pathologies. Our FRET-based HTS assay specifically detects RyR increase in luminal Ca2þ sensitivity of RyR2 known as store-overload binding of accessory proteins, calmodulin or FKBP12.6, that may mitigate the induced Ca2þ release (SOICR), and the other is an increase in cytosolic leak state of RyR1. We have previously demonstrated an inverse correlation be- Ca2þ sensitivity of RyR2. We aimed to examine the contribution of cyto- tween a compound’s effect on the FRET readout and RyR1 activity; com- 2þ 2þ 2þ solic Ca regulation to the threshold [Ca ]ER. WT and 8 mutant RyR2s pounds that increase FRET tend to inhibit RyR1 in resting [Ca ]. Under 2þ were inducibly expressed in HEK293 cells. [Ca ]ER was monitored with conditions that mimic a pathological state, we carried out a screen of a R-CEPIA1er. Protein level of WT and mutant RyR2s similarly increased af- 50,000-compound chemical library with extensive pharmacophore coverage, 2þ ter induction until 24 hours. With this increase the [Ca ]ER gradually to identify modulators of RyR1 in skeletal muscle SR membrane preparations. 2þ 0 decreased, and at 24 hours, [Ca ]ER was lower in the CPVT mutants With an excellent HTS assay quality factor (z = 0.9650.01) for every 1536- than WT. To know cytoplasmic dependent RyR2 activity, bell-shaped well plate, our screen produced a 0.8% hit rate at the 5SD threshold. Ongoing Ca2þ dependent [3H]ryanodine binding was measured and the three param- studies are testing the functional effect of 43 compounds that increased FRET. 2þ eters for the activity, affinities for activating (KACa) and inactivating Ca This work was supported by American Heart Association Postdoctoral Fellow- (KICa) and gain (Amax), were determined by curve fitting. The CPVT muta- ship 16POST31010019 (RTR) and Grant-in-Aid 15GRNT25610022 (RLC), tions increased 1/KACa and/or Amax, and as a consequence, all CPVT mu- and NIH grants R01HL092097 (RLC), R37AG26160 (to DDT), and 2þ tants showed higher RyR2 activity at physiological [Ca ]cyto (pCa6-7) R42DA037622 (to GDG/DDT). compared to WT. At the same expression level, there was a good inverse 2þ 594-Pos Board B364 correlation between the threshold [Ca ]ER and RyR2 activity at pCa7. Furthermore, at different expression level, similar correlation was observed A Functional Significance of Amino-Terminal Cysteines of RyR2 2þ Roman Nikolaienko, Elisa Bovo, Daniel Kahn, Aleksey Zima. between threshold [Ca ]ER and global release rate, which is product of the expression level and RyR2 activity of individual mutants. The threshold The Department of Cellular and Molecular Physiology, Stritch School of 2þ 2þ Medicine, Loyola University Chicago, Maywood, IL, USA. [Ca ]ER appears to be regulated by cytosolic Ca dependent RyR2 activity in HEK293 cells. The type 2 ryanodine receptor (RyR2) is the major sarcoplasmic reticulum (SR) Ca release channel in cardiomyocytes. Mutation mappings have revealed a functional importance of the RyR2 N-terminus. Recent studies have shown 592-Pos Board B362 that this region forms a tetramer stabilized by a disulfide bond. Given a substan- The Mechanism of Flecainide Action in CPVT Involves a Direct Effect on tial number of cysteines in the N-terminus of RyR2, we investigated the RyR2 functional significance of these residues and their involvement in intersubunit 1 1 1 Dmytro O. Kryshtal , Daniel J. Blackwell , Nieves Gomez-Hurtado , cross-linking during oxidative stress. To test a possible redox sensitivity of 2 2 1 Suzanne M. Batiste , Jeffrey N. Johnston , Bjo¨rn C. Knollmann . RyR2 N-terminus, we mutated the first nine cysteines of RyR2. We treated 1Medicine, Vanderbilt University Medical Center, Nashville, TN, USA, 2 HEK293 cells expressing wild type (WT) RyR2 or its N-terminus cysteine Chemistry, Vanderbilt University, Nashville, TN, USA. mutant (NT) by increasing concentrations of an oxidizing agent (diamide) The class Ic antiarrhythmic drug flecainide has now become the world-wide and visualized RyR2 cross-linking by western blotting. Similar to WT RyR2, standard of care for patients with catecholaminergic polymorphic ventricular the mutations in NT RyR2 did not prevent cross-linking at lower diamide con- tachycardia (CPVT), however there is a controversy regarding its mecha- centrations. However, we observed a partial protection at higher diamide con- nism of action. Several reports have indicated that sodium channel block centrations. Next, we studied the functional properties of WT and NT RyR2 by alone is responsible for its efficacy, whereas data from our group point to- monitoring endoplasmic reticulum (ER) [Ca] in permeabilized HEK293 cells ward the direct blocking effect of flecainide on RyR2 as the major mecha- expressing the ER-targeted Ca sensor Cepia-1er and SERCA2a. Cells express- nism for arrhythmia suppression in CPVT. Here we utilize a calsequestrin ing WT RyR2 produced regular Ca waves, which were sensitive to caffeine and knockout (Casq2-/-) CPVT mouse model to test the effects of flecainide oxidative stress. However, cells expressing NT RyR2 neither produced Ca and fully-charged quaternary flecainide analogue (QX-FL), which is a potent waves nor showed response to caffeine. Next, we studied the impact of two sodium channel blocker but does not affect RyR2 activity, on spontaneous doses of diamide (that had the different effect on WT and NT RyR2 cross- SR calcium release in membrane-permeabilized and intact ventricular linking) on ER Ca leak. A major increase in Ca leak occurred at lower diamide myocytes. In membrane-permeabilized cardiomyocytes — lacking intact concentrations that similarly affected WT and NT RyR2 cross-linking. sarcolemma and devoid of sodium channel contribution — flecainide, but Together our data demonstrate a critical role of amino-terminal cysteine resi- not QX-FL, effectively reduced the frequency of spontaneous Ca waves dues in normal RyR2 gating. However, these residues do not seem to be in a dose-dependent manner. In voltage-clamped intact cardiomyocytes, involved in functionally significant oxidative cross-linking, suggesting the pretreatment with 30 mM tetrodotoxin (TTX) completely blocked sodium presence of other redox-sensitive sites beyond the N-terminus of RyR2. current and reduced frequency of spontaneous calcium waves (vehicle: 21.455.6 waves/min.; TTX: 12.852.9 waves/min.; p=0.3, 8 cells/group) 595-Pos Board B365 although the difference was not statistically significant. Treatment with fle- High-throughput Screening Yields Allosteric Inhibitors of Leaky RyRS for cainide (6 mM in both pipette and extracellular solutions) dramatically Therapeutic Applications reduced frequency of spontaneous calcium waves regardless of the presence Robyn T. Rebbeck1, Xiaoqiong Dong2, Kenneth S. Ginsburg2, of TTX (Flec.: 4.650.8 waves/min. (n=10); Flec.þTTX: 2.550.5 waves/ Daniel P. Singh3, Gregory D. Gillispie4, David D. Thomas1, min. (n=7); p<0.05 for both groups vs. vehicle), while QX-FL did not Bradley S. Launikonis3, Donald M. Bers2, Razvan L. Cornea1. (QX-FL: 18.753.9 waves/min. (n=8); QX-FLþTTX: 14.253.7 waves/ 1Biochemistry/Molecular Biology/Biophysics, University of Minnesota, min. (n=9); p>0.4 for both groups vs. vehicle). Importantly, the SR Ca Minneapolis, MN, USA, 2Pharmacology, University of California, Davis, content, estimated for each cell by rapid caffeine application (10 mM) at Davis, CA, USA, 3School of Biomedical Sciences, The University of the end of the experiment, was not different between all experimental Queensland, Brisbane, Australia, 4Fluorescence Innovations, Inc., groups. Our results further support the unique role of flecainide as a direct Minneapolis, MN, USA. inhibitor of RyR2-mediated Ca release activity that determines its efficacy Using fluorescence lifetime detection of FRET, we have developed and vali- in CPVT. dated high-throughput screening (HTS) methods to discover compounds that

BPJ 8570_8572 118a Sunday, February 18, 2018 modulate the ryanodine receptor (RyR) calcium release channel, for therapeu- rithm). Analysis of these receptor locations revealed that RyRs are spaced tic applications. Intracellular calcium regulation is critical for striated muscle at nearest neighbour distances of 40.1 5 0.9 nm (n = 1802 clusters), similar function, and RyR is a central player. Under resting calcium, increased pro- to recent measurements on electron tomograms but greater than the in vitro pensity of channel opening due to RyR dysregulation is associated with severe RyR packing distances observed in artificial bilayers. A new Monte Carlo cardiac and skeletal myopathies, diabetes and neurological disorders. This model of protein cluster assembly which reproduced the cluster morphologies leaky state of the RyR is an attractive target for pharmacological agents to observed in the DNA-PAINT image data suggested that RyRs commonly treat such pathologies. Our FRET-based HTS detects RyR binding of acces- cluster through a random assembly process into irregular arrays containing sory proteins calmodulin or FKBP12.6. Under conditions that mimic a path- significant gaps. These gaps were occupied by other proteins, including the ological state, we have carried out pilot screens of the 1280-compound inhibitory protein junctophilin-2 (JPH2) whose stoichiometry with RyRs, library of pharmaceutically active compounds (LOPAC), to identify modula- measured through a modified indirect target counting algorithm, varied tors of RyR in either skeletal or cardiac SR membrane preparations. These from cluster to cluster (0.5-3.5). Together, these observations reveal a new screens yielded compounds (hits) that are specific for either RyR1 (skeletal level of complexity in the regulation of RyRs which predict inter- isoform) or RyR2 (cardiac isoform), or that act on both RyR1 and RyR2. nanodomain variations of excitability and calcium release properties, even Ongoing studies indicate good correlation between the FRET (structural) within the same cellular locality. Our data underscore how improved precision readout for the hits and their effects on RyR function in isolated sarcoplasmic in localisation microscopy can reveal intimate protein organisation within reticulum, skeletal myofibers, and adult ventricular myocytes. This work is thousands of signalling nanodomains and observe molecular cluster regulation supported by NIH grants R01HL092097 (RLC/DMB) and R37AG26160 in situ. (DDT), and by American Heart Association Postdoctoral Fellowship 16POST31010019 (RTR). 598-Pos Board B368 Molecules to Membranes: The Calcium Release Unit 596-Pos Board B366 Sophia P. Hirakis1,2, Thomas M. Bartol2, Terrence J. Sejnowski2, CRISPR/Cas9 Created CPVT1 Associated RyR2 Mutations Reliably Rommie E. Amaro1. Reproduces the Calcium Signaling Aberrancy in Human iPSC-CMS 1Chemistry and Biochemistry, UC San Diego, La Jolla, CA, USA, Xiaohua Zhang1, Hua Wei1, Naohiro Yamaguchi2, Martin Morad1. 2Computational Neurobiology Laboratory, Salk Institute, La Jolla, CA, USA. 1Cell Biology & Anatomy, Cardiac Signaling Center of USC, MUSC and Calcium Induced Calcium Release (CICR) is used by multicellular organisms Clemson University, Charleston, SC, USA, 2Regenerative Medicine and Cell to translate an extracellular activation signal to an amplified intracellular Biology, Medical University of South Carolina, Charleston, SC, USA. response. In the heart, membrane depolarization leads to an increase in cyto- Human induced pluripotent stem cells (hiPSCs) generated from patients ex- solic Ca2þ concentration. In the Calcium Release Unit (CRU), huge proteins pressing catecholaminergic polymorphic ventricular tachycardia (CPVT) ar- called Ryanodine Receptors (RyR) respond to the signal by releasing up to rhythmias have been well developed for studying cellular pathology and four orders of magnitude more Ca2þ into the cytosol; an event known as a genetic dysfunction of the disease in vitro. Over 150 missense mutations, ‘‘Calcium Spark.’’ Millions of perfectly timed Ca2þ sparks produce synchro- throughout the 5000 amino acids of RyR2, are associated with CPVT1 pa- nous heart muscle contraction. In failing hearts, sparks are replaced by waves thology in patients. To overcome the limitation of generating cardiomyocytes which cause the muscle to contract asynchronously. CICR occurs in the CRU from CPVT1 patients, and to study CPVT1 pathology in a systematic way in region known as the dyad; which is composed of two membranous structures vitro, we used CRISPR/Cas9 gene editing to introduce specific RYR2 muta- and systems: 1) T-Tubules (TT)- extracellular membranes with axial and tions into hiPSCs, thus establishing human cell-lines of CPVT1. Three different transverse invaginations that increase surface area for Ca2þ influx; 2) Sarco- hiPSC-CM lines carrying the CPVT1-associated RyR2 mutations R420Q, plasmic Reticulum (SR)- intracellular calcium stores. Mitochondria surround F2483I, and Q4201R, located in the 3 hotspot mutation regions (amino-termi- dyads, providing ATP for active Ca2þ pumping into the SR by the Sarco/ nal, central and carboxyl-terminal domains) of RyR2, were created. Ca2þ Endoplasmic Reticulum Calcium/ATPase (SERCA). Harnessing computa- signaling and electrophysiological profiles of spontaneously beating cardio- tional power, CRU modelers have predicted experimentally testable hypothe- myocytes (hiPSC-CMs), carrying specific RyR2 point mutations was studied ses. Nevertheless, many simulations inaccurately represent molecular using total internal reflection fluorescence (TIRF) microscopy and patch clamp distribution by using constant concentration gradients. We present a multi- technic. All three mutant hiPSC-CMs exhibited long-lasting and spatially wan- scale spatial model of the CRU. Using MCell, we model the positions of mil- dering Ca2þ sparks as compared with wild type hiPSC-CMs. Aberrant Ca2þ re- lions of molecules in an explicit particle reacting biochemical system. leases and DADs, aggravated by exposure to isoproterenol, were observed in all Membrane structures and RyR channel positions are derived from electron three-cardiomyocyte cell-lines. The frequency of spontaneous beating also var- microscope tomograms and refined with GAMer software. Mathematical ied in the three mutant CMs lines. SR Ca2þ content, CICR gain, domain- models of SERCA reveal the importance of proper Ca2þ-clearing action in specific CPVT1 pathologies, and pharmacological sensitivities were all studied CICR termination phases. Using CellPack our molecular system is populated in detail. CRISPR/CPVT1 created hiPSC-CMs provide a valuable platform to with all-atom detail. Our model can be used to examine small-molecule and critically study CPVT1 arrhythmia in the human cell lines and identify specific protein diffusion in the dyadic junction. Our work aims to accurately model drug therapy for arrhythmias associated with different regions. This study was molecular concentration and membranous morphology changes in healthy supported by NIH grant RO1 HL16152. and diseased cardiomyocytes. 597-Pos Board B367 599-Pos Board B369 Molecular Scale Visualisation of Variable Clustering Properties of the Using Statistical Mechanics to Understand Calcium Sparks Cardiac Ryanodine Receptor Anna V. Maltsev1, Michael D. Stern2, Victor A. Maltsev2. I.D. Jayasinghe1, A.H. Clowsley2, R. Lin2, T. Lutz2, Carl Harrison2, 1Queen Mary University London, London, United Kingdom, 2Laboratory of E.M. Green2, D. Baddeley3, L. Di Michele4, C. Soeller2. Cardiovascular Science, National Institute on Aging, NIH, Baltimore, 1Faculty of Biological Sciences, University of Leeds, Leeds, United MD, USA. Kingdom, 2Living Systems Institute, University of Exeter, Exeter, United The crucial importance of local Ca signaling for regulation of cardiac muscle Kingdom, 3Bioengineering Institute, University of Auckland, Exeter, New contraction was theoretically predicted in 1992 as part of local control theory Zealand, 4Emmanuel College, University of Cambridge, Cambridge, United and validated by the experimental discovery of Ca sparks. Sparks are generated Kingdom. by release channel clusters in the form of localized Ca releases from junctional Localisation microscopy allows observation of protein organisation at previ- sarcoplasmic reticulum (SR). As release channels interact, the spark is an emer- ously unattained resolution and sensitivity. Limited brightness, and hence pre- gent behavior of the collective, rather than a property of an individual channel. cision, of switchable dyes has however made resolving and quantifying This nanoscale system of interacting molecules which generates elementary individual proteins clustered within cellular signalling nanodomains chal- biological signals is particularly interesting because this is where physics meets lenging. The excitability of intracellular calcium release nanodomains in car- biology. We have constructed an exact mapping of the release channel cluster diomyocytes formed by clusters of the cardiac ryanodine receptors (RyR) to a famous model of an interacting particle system in statistical physics, have been suggested to strongly determined by the molecular-scale receptor namely the Ising model. We demonstrated that the channel synchronization distribution. Harnessing the high localisation precision of DNA-PAINT that corresponds to termination of signals is described by the same equation (10 nm), we visualised the positions of cardiac RyRs within thousands of as the phase transition associated to the change of magnetic field in an Ising sub-plasmalemmal clusters in rat cardiac ventricular myocytes. Resolved ferromagnet. Our Ising-based theory predicts the critical level of SR [Ca] puncta of RyR labelling were confirmed as single RyRs with several imaging that is required for spark termination. If the SR depletion does not reach this and analysis tools (cluster analysis and an indirect marker counting algo- level the spark does not terminate and becomes metastable. The prediction

BPJ 8570_8572 Sunday, February 18, 2018 119a was confirmed by numerical simulations using 2013 Stern et al. spark model. In Ca2þ spark wave propagations when the spacing between Ca2þ release units an ongoing work, we explore the fine structure of calcium sparks using Peierl’s is small and SR Ca2þ load is high. These results are consistent with experi- contour analysis to investigate the famous Onsager’s phase transition to mental observations. Our study provides a more detailed mathematical describe the transition from ordered to disordered sparks as a function of b description of intracellular Ca2þ cycling in order to improve our understand- (effective inverse temperature). The disordered sparks were manifested in nu- ing of the dynamics involving Ca2þ leaks, Ca2þ waves, delayed after depolar- merical simulations by the presence of Ca embers, i.e. small clusters of opened izations, and triggered activities. channels. In summary, we describe deterministically the behavior of a system on a coarser scale (release unit) that is random on a finer scale (release chan- 602-Pos Board B372 nels), bridging the gap between scales. Molecular Contacts between MCU and its Regulatory Machinery Charles Phillips. 600-Pos Board B370 Brandeis University, Waltham, MA, USA. A Novel Computational Model of the Rabbit Atrial Myocyte Offers Insight Mitochondrial calcium uptake is critical for physiological processes such as into Calcium Wave Propagation Failure calcium signaling, energy production, and apoptosis, and is mediated by a cal- Marcia R. Vagos1, Jordi Heijman2, Hermenegild Arevalo1, cium sensitive calcium channel known as the mitochondrial calcium uniporter, Mary M. Maleckar1, Bernardo Lino de Oliveira1, Ulrich Schotten3, or MCU. MCU is actually a protein complex, including the important regula- Joakim Sundnes1. tory proteins EMRE, MICU1, and MICU2, among others. Although it is well 1Simula Research Laboratory, Lysaker, Norway, 2Cardiology, Maastricht established that EMRE, a small 10kDa protein owning a single membrane- University, Maastricht, Netherlands, 3Physiology, Maastricht University, spanning helix, is absolutely required for MCU activity, little is known about Maastricht, Netherlands. the molecular interactions between it and the pore-forming subunit of the com- Atrial cardiomyocytes have a less well-developed T-tubule system than ven- plex, ‘‘MCU’’. Here, using a Tryptophan scanning assay, we identify key res- tricular cells, resulting in intracellular calcium waves propagating from the idues within EMRE’s transmembrane helix as well as within the membrane membrane to the center via centripetal calcium diffusion. Failure of centripetal spanning TM1 of MCU that are required for MCU-EMRE binding and subse- calcium-wave propagation (‘calcium silencing’) has been observed in a rabbit quent activity. Analysis of these important residues show that EMRE likely model of rapid atrial pacing and in patients with atrial fibrillation, but the un- forms a well characterized GXXXG motif to facilitate helix/helix packing derlying mechanisms remain incompletely understood. The goal of this study against TM1 of MCU. Residue swapping along with co-immunoprecipitation was to develop a novel computational model of the rabbit atrial cardiomyocyte show that the interaction between EMRE and MCU occurs by direct binding that incorporates detailed compartmentalization of intracellular calcium dy- and is not facilitated by another complex member(s). This work is an important namics, which can be used to investigate the mechanisms underpinning calcium first step toward understanding how EMRE regulates MCU and helps explain silencing. why animals have evolved a requirement for EMRE to facilitate mitochondrial We incorporated ion-current formulations reflecting rabbit electrophysiology calcium uptake. into a previously published human atrial cardiomyocyte model. The model was validated with published experimental data and the effects of altered 603-Pos Board B373 rate of calcium diffusion between the calcium-release-unit space and The Function of MICU2 in Mitochondrial Calcium Uniport the cytosol (tdiff) were investigated. tdiff modulates local calcium levels Kai-Ting Huang1, Melaine Paillard1, Peter Varnai2, Gyorgy Hajnoczky1. available to activate neighboring calcium-release sites, affecting wave 1Department of Pathology, MitoCare, Thomas Jefferson University, propagation. Philadelphia, PA, USA, 2Department of Physiology, Semmelweis University, Simulation results showed that calcium-wave propagation was highly sensitive Budapest, Hungary. to tdiff during normal pacing at 2 Hz. We observed impaired calcium-wave MICU2 has been established as a component of Mitochondrial Calcium Uni- propagation for a range of values of tdiff, with full calcium-wave propagation porter (mtCU), which dimerizes with MICU1 to support the closed state of þ þ for values of tdiff exceeding 12.7 ms, and calcium silencing for values of tdiff mtCU at low [Ca2 ]. However, MICU2’s exact role in Ca2 sensing remains below 10.6 ms due to insufficient local positive feedback for calcium-induced contentious and its possible direct interaction with the mtCU’s pore has not calcium release to maintain centripetal wave propagation. We also observed been tested. To address these problems, we used MICU2- and MICU1- calcium alternans between propagating and non-propagating waves for inter- deficient HEK cells (2014, embr.201337946) and mouse embryonic fibroblasts þ mediate values of tdiff. rescued with MICU2 point mutations. Mitochondrial Ca2 handling was eval- þ This study provided new insight into the mechanisms of calcium-wave propa- uated by measuring either the Ruthenium Red sensitive Ca2 clearance in per- gation failure in rabbit atrial cardiomyocytes and motivates further investiga- meabilized cells or simultaneous measurements of both cytoplasmic and þ þ tion of the effects of altered calcium diffusion on wave-propagation mitochondrial matrix [Ca2 ] in intact cells during store operated Ca2 entry. abnormalities and calcium-dependent arrhythmogenesis. Moreover, the newly Our results confirm that MICU2 promotes the closure of mtCU but MICU1 þ developed model will be a useful tool for studying conditions which permit can alone also suppress opening at low cytoplasmic [Ca2 ]. Furthermore, our þ restoration of normal calcium wave propagation. data support that the putative Ca2 binding motifs, the EF hands of MICU2 are required for Ca2þ-dependent activation in mtCU. Ongoing studies evaluate 601-Pos Board B371 the relevance of the EF hand sequence differences between MICU2 and The Modeling of Calcium Dynamics within the Dyadic Space using MICU1 and possible effects of MICU2 independent of MICU1-MICU2 heter- Random Walks odimer formation. Jessica Au1, Zana Coulibaly2, Leighton Chen2, Daisuke Sato2. 1University of California-Davis, Davis, CA, USA, 2Department of 604-Pos Board B374 Pharmacology, University of California-Davis, Davis, CA, USA. MICU2 Restricts Spatial Crosstalk between InsP3R and MCU Channels by Calcium (Ca2þ) sparks play an important role in regulating the uniform Regulating Threshold and Gain of MICU1-Mediated Inhibition and Acti- contraction and relaxation of cardiac myocytes. Ca2þ sparks occur due to a vation of MCU positive feedback process known as Ca2þ induced Ca2þ release. Tens to hun- Riley Payne, Henry Hoff, Anne Roskowski, J. Kevin Foskett. dreds of ryanodine receptors (RyRs) form a cluster within the dyadic space University of Pennsylvania, Philadelphia, PA, USA. þ þ between the cell membrane and the sarcoplasmic reticulum (SR) membrane. Ca2 entry into mitochondria is mediated by the Ca2 uniporter channel þ Often, Ca2þ diffusion is modeled by using a partial differential equation. complex containing MCU, the Ca2 -selective pore, and associated regulatory However, since the dyadic space is extremely small and cytosolic Ca2þ con- proteins. The roles of MICU proteins are controversial. MICU1 was proposed centration is relatively low during diastole, the continuum assumption may not to be necessary for MCU activity whereas subsequent studies suggested it 2þ 2þ 2þ provide a sufficiently accurate basis for describing Ca leaks and sparks. In inhibits the channel in the low-cytoplasmic Ca ([Ca ]c) regime, a 2þ this study, we used random walks, which are the fundamental processes that mechanism referred to as ‘‘gatekeeping’’, that imposed a [Ca ]c threshold underlie diffusion, to model Ca2þ dynamics within the dyadic space. We for channel activation at 1-3 mM. Here we quantitated MCU activity over 2þ began by modeling a single RyR. We found that the opening rate of the single a wide range of quantitatively-controlled and recorded [Ca ]c. MICU1 alone RyR obeys the function k[Ca2þ]2 for some constant k without its actual can mediate gatekeeping as well as highly-cooperative activation of MCU embedding in our model. Afterwards, by using that single RyR model, we activity, whereas the fundamental role of MICU2 is to regulate the threshold modeled a single Ca2þ release unit (CRU, RyR cluster) and then, multiple and gain of MICU1-mediated inhibition and activation of MCU. Our results CRUs. The model of the single CRU shows the transition from non-spark provide a unifying model for the roles of the MICU1/2 hetero-dimer in leaks to Ca2þ sparks as SR Ca2þ load becomes high. We also observed MCU-channel regulation and suggest an evolutionary role for MICU2

BPJ 8570_8572 120a Sunday, February 18, 2018

2þ in spatially-restricting Ca crosstalk between single InsP3R and MCU 607-Pos Board B377 channels. Molecular Interactions that Contribute to the Regulation of HCN Chan- nels by KCNE2 Yoann Lussier, Karen Callahan, Rikard Blunck, Nazzareno D’Avanzo. Posters: Voltage-gated K Channels and Physiology, University of Montreal, Montreal, QC, Canada. Mechanisms of Voltage Sensing and Gating I Hyperpolarization activated cyclic-nucleotide gated (HCN) channels are the molecular correlate of neuronal and cardiac Ih. The four mammalian isoforms 605-Pos Board B375 that have been identified (HCN1-4) are widely expressed in the central and pe- Investigating Cyclic Dinucleotide Binding to HCN Channels by Surface ripheral nervous systems as well as cardiac conduction tissue. KCNE2 is the only member of the KCNE protein family known to regulate HCN expression, Plasmon Resonance and Isothermal Calorimetry -/- Sebastien Hayoz1, Purushottam B. Tiwari2, Grzegorz Piszczek3, Aykut U¨ ren2, voltage-sensitivity, and gating kinetics. KCNE2 mice have diminished Ih in Tinatin I. Brelidze1. cortical and hippocampal neurons and increased excitability, consistent with 1Pharmacology and Physiology, Georgetown University School of Medicine, experiments in over-expression systems. Here we examine the molecular de- Washington, DC, USA, 2Oncology, Georgetown University School of tails of HCN-KCNE2 complex formation, including subunit stoichiometry, Medicine, Washington, DC, USA, 3Biophysics Core, National Heart, Lung and sites of protein-protein interactions. Single-molecule fluorescence experi- and Blood Institute, National Institutes of Health, Bethesda, MD, USA. ments indicate that HCN1 channels interact with WT-KCNE2 with a 4:2 stoi- Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels chiometry in a concentration independent manner. Some KCNE2 gene variants regulate the rhythmic firing of neurons and cardiac myocytes. Cyclic nucleo- can alter this stoichiometry and alter HCN1 function, while others have no tides facilitate the opening of HCN channels by binding to the intracellular cy- discernible impact. Using computational approaches, we have identified puta- clic nucleotide-binding domain (CNBD). Recently, cyclic dinucleotides were tive sites of interaction in the HCN1-KCNE2 complex that are being validated shown to antagonize the effect of cyclic nucleotides in HCN4 channels. Based experimentally. Further biophysical evidence suggest that KCNE2 alters the on the structural analysis and mutational studies it has been proposed that cyclic regulation of HCN1 channels by other modulators including cAMP, which dinucleotides affect HCN4 channels by binding to the C-linker that connects could have potential implications of its role in neuronal function. the CNBD to the pore-forming transmembrane domain. However, a direct ev- 608-Pos Board B378 idence of cyclic dinucleotide binding to the C-linker was missing. Here, we Towards Revealing a Cooperative Mechanism of Camp Binding to HCN2 used surface plasmon resonance (SPR) and isothermal calorimetry (ITC) to Cyclic Nucleotide Binding Domains at the Single-Molecule Level probe the direct binding of cyclic dinucleotides to isolated C-linker/CNBDs David S. White1,2, Marcel P. Goldschen-Ohm1,3, Ruohan Zhang1,2, of HCN channels. As a positive control we first determined binding affinities Vadim A. Klenchin1, Randall H. Goldsmith2, Baron Chanda1. of cyclic nucleotides (cAMP and cGMP) to HCN4 C-linker/CNBDs with 1Neuroscience, University of Wisconsin-Madsion, Madison, WI, USA, SPR and ITC. The binding affinities were in agreement with cyclic nucleotide 2Chemistry, University of Wisconsin-Madison, Madison, WI, USA, binding affinities determined previously. We then used SPR and ITC to inves- 3Neuroscience, University of Texas at Austin, Austin, TX, USA. tigate cyclic dinucleotide binding. To our surprise, we detected no binding of Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are cyclic dinucleotides (c-di-GMP, c-di-AMP and cGAMP) to immobilized critical for oscillatory neuronal activity in the brain and pacemaking in the monomeric C-linker/CNBDs with SPR and to non-immobilized monomeric heart. Although activation of these tetrameric channels can be driven solely C-linker/CNBDs with ITC. Since the cyclic dinucleotide effect on the currents by hyperpolarizing voltages, the binding of cyclic nucleotides to their intracel- from HCN4 channels was only detected in the presence of cyclic nucleotides, lular cyclic nucleotide binding domains (CNBDs) enhances their voltage- we examined cyclic dinucleotide binding to the monomeric C-linker/CNBD in dependence of activation through a mechanism that remains unclear. Previous the presence of cyclic nucleotides and still did not detect binding with SPR and studies dissecting the cooperative nature of subunit activation upon cAMP ITC. Finally, we examined cyclic dinucleotide binding under the conditions binding have relied on macroscopic methods, such as ensemble channel cur- inducing tetramerization of the C-linker/CNBDs and also did not detect binding rents or fluorescence. Although these studies provide pivotal information in with ITC. Taken together, our results suggest that the interaction of the determining the gating-mechanism of HCN channels, they both afford only C-linker/CNBD with other parts of the channel and/or additional membrane- ensemble-averaged data, which obscures the resolution of individual binding associated factors are necessary for cyclic dinucleotide binding in HCN4 steps. Herein, we aim to circumvent this limitation and determine the cooper- channels. ative mechanism of cAMP binding to HCN2 CNBDs at the single-molecule level. By tethering an artificially-linked tetrameric CNBD construct into nano- 606-Pos Board B376 fabricated devices called zero-mode waveguides, we resolve the stepwise bind- Examining Drug Binding in HCN Channels ing of fluorescent cAMP molecules to all four binding sites of our complex. Jeremie Tanguay, Nazzareno D’Avanzo. Using hidden Markov modeling of our idealized single-molecule fluorescence University of Montreal, University of Montreal, Montreal, QC, Canada. time series, we begin to reveal a cooperative mechanism of cAMP binding to Neuronal HCN channels are open when neurons are not firing action- HCN2 CNBDs. potentials (sub-threshold potentials), and reduce the input resistance of the cell membrane making it less responsive to incoming inputs. These combined 609-Pos Board B379 properties make them excellent targets for fine-tuning of intrinsic neuronal Role of Interactions between Transmembrane and C-Terminal Regions in excitability. HCN channels are also expressed in cardiac conduction tissue Voltage-Dependent Activation of HCN4 Channels where they establish the pace of the heartbeat, and enable sensitivity to auto- Dana A. Page, Kaylee E. Magee, Jessica Li, Edgar C. Young. nomic simulation. Moreover, since HCN channels are not found in vascular Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, tissue, targeted inhibition of HCN channels has strong therapeutic potential BC, Canada. as bradycardic agents, anti-convulsants, and analgesics without adverse effects The HCN4 subtype of Hyperpolarization- and Cyclic Nucleotide-activated on pulmonary and vascular smooth muscle tone. While several molecules that (HCN) channels is noted for slower kinetics and a hyperpolarized activation target HCN channels have been identified, their low affinity and lack of iso- midpoint voltage (V1/2) compared to the HCN2 subtype. To test whether the form specificity prevents wide-spread use of these current HCN inhibitors. To HCN4 transmembrane (TM) region requires interactions with the C-terminal advance the development of isoform specific inhibitors, we are using compu- region to determine V1/2, we substituted the HCN4 TM region into previously tational and electrophysiological methods to characterize the binding sites of characterized HCN2 channels with various truncations of the C-terminal re- known inhibitors and identify key interactions. Using computational docking gion. Substituting the HCN4 TM region into HCN2 channels - either full- approaches, we have examined the binding of 13 known inhibitors to the length or truncated after the cyclic nucleotide-binding (CNB) fold - resulted atomic structure of human HCN1 (PDB: 5U6O), and a homology model of in a significant (more than 8 mV) negative shift of V1/2. Strikingly, in HCN2 the open state derived from the closely related eukaryotic CNG channel channels lacking the CNB fold, or channels where the majority of the CNB (PDB: 5H3O). Our results indicate that ‘‘bradine’’ inhibitors bind in the fold was present except for the final C-helix, the HCN4 TM substitution failed pore cavity with no preferred orientation, providing possible insight into their to shift V1/2 significantly (less than 2 mV difference). This suggests that the hy- low affinity. Other results indicate that clonidine and alinidine are likely pore perpolarized V1/2 of HCN4 channels relative to other HCN subtypes relies on a blockers as well, but with greater conformational restraint. Detailed character- functional interaction of the TM and C-terminal regions, and that the specific ization of the mode of binding and residues lining the binding pocket may conformation supporting this inhibitory interaction requires an intact CNB enable the development of isoform specific inhibitors that can be used as fold. To test whether this inhibitory interaction coincides with any inhibition chemical probes or therapeutic agents. caused by eliminating CNB fold-mediated cAMP potentiation, we repeated

BPJ 8570_8572 Sunday, February 18, 2018 121a the HCN4 TM substitution in a full length HCN2 channel whose CNB fold is 612-Pos Board B382 mutated (R591E) so it cannot bind cAMP. HCN2 R591E exhibits V1/2 20 mV USE- and State-Dependent Binding of KCNQ Channel Openers more negative than cAMP-potentiated HCN2 channels, yet the HCN4 TM sub- Caroline K. Wang1, Harley T. Kurata1, Alice W. Wang2. 1 2 stitution in HCN2 R591E produced a further 10 mV negative V1/2 shift, match- Pharmacology, University of Alberta, Edmonton, AB, Canada, University ing that produced by HCN4 TM substitution in cAMP-potentiated channels. of British Columbia, Vancouver, BC, Canada. This suggests a new role for the CNB fold in supporting an inhibitory interac- Retigabine is the first approved anticonvulsant that acts via opening of voltage- tion between the C-terminal region and the HCN4 TM region, independent of gated potassium channels formed by KCNQ2-5 subunits. An important unex- and additive with the well-known autoinhibition caused by eliminating cAMP plored feature of retigabine and its derivatives is their state- and use- potentiation. dependent properties. Drugs that exhibit use-dependence may exhibit enhanced effectiveness with more frequent channel stimulation, making them especially 610-Pos Board B380 useful for selectively targeting hyperactive cells that trigger seizures in epi- Ethanol Increases Neuronal Firing by Regulating PI(4,5)P2 Sensitivity of D lepsy. We aimed to generate a detailed understanding of the mechanism of ac- M-Type K Channels tion of KCNQ channel openers retigabine and ICA069673 (ICA73) on WT Kwon-Woo Kim, Byung-Chang Suh. KCNQ2 channels. We assessed drug binding to pre-open states by applying DGIST, Daegu, Republic of Korea. compounds at different holding potentials, and subjected cells to frequent repet- Ethanol affects the physiological functions of the central nerve systems by itive stimulations to assess use-dependent drug interactions. WTQ2 channels changing signaling pathways through the plasma membrane or by altering exhibited marked state- and use- dependent binding of ICA73, but an attenu- the membrane electrical properties. It has been reported that ethanol regu- ated state-dependence of retigabine binding. Rapid application of compounds lates neuronal firing by changing the gating of ion channels, including at different voltages demonstrated close overlap between the voltage- GIRK, BK, CaV,NaV and M-type potassium channels. It has been known dependence of channel activation and the rate of ICA73 binding. In contrast, that the M-type KCNQ2/3 channels require the membrane phosphoinositide retigabine could access both open and closed channel states and was less phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for their activation. affected by voltage. We also compared the mechanism of ICA73 binding to Although various studies on the physiological effects of ethanol has been re- KCNQ2[A181P] mutant channels, previously shown to have a weaker satu- ported, molecular mechanisms of the ethanol regulation on KCNQ2/3 chan- rating response to ICA73. Unlike WTQ2, A181P mutants did not show use- nel gating and neuronal activity have not been studied well. Here, we have dependent activation. Rapid perfusion assays demonstrated that ICA73 examined the molecular mechanisms for ethanol regulation of M-type unbound from A181P channels much faster than from WTQ2, despite a similar KCNQ2/3 current and membrane excitability in SCG neurons. First, our rate of drug binding to both channels, suggesting that ICA73 has a weaker af- data show that 100 mM and 400 mM concentrations of ethanol inhibits finity for binding A181P. Taken together, these findings explore the mecha- M-current in SCG neurons by 20% and 65%, respectively. Similar re- nisms that underlie state-dependent interactions of KCNQ openers and sponses are found in tsA201 cells expressing the KCNQ2/3 channels. We highlight pronounced occlusion of certain retigabine analogs from resting chan- also found that the ethanol inhibition is decreased when the cells are co- nel states. transfected with the PI(4,5)P2 synthesizing kinase PIPKIg (8% inhibition in 400 mM ethanol-treated cells). In addition, KCNQ2 or KCNQ3 homo- 613-Pos Board B383 meric channels show different ethanol sensitivity, where KCNQ2 and Effect of Camp When Varying the Number of KCNE1 Subunits in the IKs KCNQ3 homomeric currents were inhibited by 40% and 10%, respec- Complex tively, upon 200 mM ethanol application. Those results suggest that mem- Emely Thompson, Jodene Eldstrom, Maartje Westhoff, Donald McAfee, brane PI(4,5)P2 plays an important regulator in the ethanol suppression of David Fedida. M-channels. In consistent, ethanol application increased neuronal firing in Anesthesiology, Pharmacology and Therapeutics, University of British all three classes of SCG neurons sorted by their firing patterns (phasic-1, Columbia, Vancouver, BC, Canada. phasic-2 and tonic neuron). Taken together, our results suggest that ethanol The slow delayed rectifier potassium current, IKs, is composed of two types of elevates neuronal firing in the sympathetic SCG neurons by suppressing the subunit; KCNQ1, which comprises the tetrameric channel, and the accessory M-channel activity via the suppression of PI(4,5)P2 sensitivity of KCNQ subunit, KCNE1. The number of KCNE1 subunits present is variable with up channel proteins. to four associating with the channel complex. During beta-adrenergic stimula- tion KCNQ1 becomes phosphorylated, which increases the current and hyper- 611-Pos Board B381 polarizes the voltage dependence of activation. Previous studies have shown Molecular Basis of Voltage Activation of an Epilepsy-Causing Mutation in that KCNQ1 alone does not elicit a functional response to phosphorylation, the S4 of KCNQ3 Channel as KCNE1 appears to be required for the channels’ response. The application Rene Barro-Soria. of a membrane-permeable cAMP analog, 8-(4-chlorophenylthio)-cAMP (8- Department of Medicine, University of Miami, Miami, FL, USA. CPT-cAMP) to a fusion construct with a fixed stoichiometry of 4:4 Epilepsy is a clinically devastating disease affecting more than 50 million (KCNE1:KCNQ1, EQ), or separately-expressed KCNQ1 þ KCNE1 in mouse people worldwide. Epilepsy is characterized by abnormal neuronal activity ltk- cells, hyperpolarized the V1/2 of activation by 18 mV in whole cell re- in the brain that can cause delayed psychomotor development, mental retar- cordings and by 25 mV in macropatches, and shortened the first latency to dation or even death. At present, more than 30% of patients suffering from opening of single channels in the presence of 8-CPT-cAMP. Using other fusion epilepsy do not benefit from available anti-epileptic drugs. Therefore, there constructs with either 2 (EQQ) or 1 KCNE1 subunits (EQQQQ) present, the de- is an ongoing need to understand the molecular basis of the disease to design pendency of the IKs channel complex response to 8-CPT-cAMP upon the num- treatments. One of the major potassium currents in neurons is the muscarine- ber of KCNE1 subunits present was investigated. In both EQQ and EQQQQ, regulated M-current (IKM). The IKM, formed by the heteromerization of there was reduced hyperpolarisation of the V1/2 of activation upon exposure KCNQ2 and KCNQ3 channels, activates in the time frame of action potential to cAMP of 13 mV at the whole cell level, and 18 mV in macropatches. initiation, thereby controlling neuronal excitability. We here study the molec- There was also a reduction in the first latency to opening for both EQQ and ular mechanisms by which the epileptic-inducing mutation R230C in KCNQ3 EQQQQ at the single channel level. These results suggest that only one causes channel malfunction. For instance, KCNQ3-R230C is assumed to lock KCNE1 subunit is required in the IKs channel complex to facilitate a response the voltage sensor (S4) of KCNQ3 channel in the activated state, resulting in to cAMP. a constitutively open channel. We use voltage clamp fluorometry to under- stand how R230C affects the voltage sensor and the gate of KCNQ3 channel. 614-Pos Board B384 We show that R230C, contrary to what was previously assumed, allows S4 Molecular Mechanism of Polyunsaturated Fatty Acid Analogues as movement in KCNQ3-R230C channels. R230C shifts the closing and S4 KV7.1-Channel Modulators movement of KCNQ3 to extreme negative potentials, such that at physiolog- Sara I. Liin1,2, Rosamary Ramentol2, Rene Barro-Soria2, H. Peter Larsson2. ical voltage range (80 mV to þ40 mV), the channel is always open. By 1Clinical and Experimental Medicine, Linko¨ping University, Linko¨ping, comparing the functional properties of substituting R230 by alanine, cysteine Sweden, 2University of Miami, Miami, FL, USA. or histidine, we found that the main functional effect of this mutation seems Negatively charged analogues of polyunsaturated fatty acids (PUFAs) have to be the loss of the positive charge. Thus, either by external application of been shown to activate the human KV7.1þKCNE1 channel, which generates MTSEA that converts R230C to a charged, lysine-like residue or by proton- the repolarizing cardiac IKs current. Specific PUFA analogues activate both ation of a substituted lysine at position R230 the voltage dependence of chan- wild type KV7.1þKCNE1 and KV7.1þKCNE1 containing Long QT nel activation and the equilibrium of the S4 movement are shifted to positive syndrome-associated mutations. Therefore, PUFA analogues represent a new values. class of KV7.1þKCNE1 channel activators that may aid in development of

BPJ 8570_8572 122a Sunday, February 18, 2018 novel anti-arrhythmic drugs. To determine the molecular mechanism of how expression and function. On the other hand, 9 mutants that traffic with nearly PUFA analogues activate KV7.1þKCNE1, we here combine electrophysiology WT-like efficiency are nevertheless electrophysiologically dysfunctional. recordings and site-directed mutagenesis with a panel of PUFA analogues These results illuminate the exact defects induced by each mutation associated with distinct properties. We identify two main effects of PUFA analogues on with LQTS, potentially providing information that can be used to inform KV7.1 that are dependent on discrete channel motifs. The first effect is a shift personalized treatment of LQTS subjects harboring KCNQ1 mutations. This in the voltage dependence of channel opening that is critically dependent on work was supported by NIH Grant RO1 HL122010. specific S4 arginines in the voltage sensor domain of KV7.1. Intriguingly, 617-Pos Board B387 different S4 arginines are important in KV7.1 and KV7.1þKCNE1. The second effect is alteration in the maximum conductance that is critically dependent A Non-canonical VSD-Pore Coupling is Responsible for the AO State of KCNQ1 Channels on the S6 residue K326 in the pore domain of KV7.1. Both effects appear to be primarily electrostatic. Our findings provide novel insights into how Panpan Hou. Biomedical Engineering, Washington University in St Louis, St. Louis, MO, KV7.1þKCNE1 channel activators, such as PUFA analogues, simultaneously may interact with the voltage sensor domain and the pore domain to alter chan- USA. nel activity. Our findings also contribute pharmacological support to the hy- In voltage dependent activation of Kv channels, membrane depolarization ac- tivates the voltage sensor(VSD), which then opens the pore via the VSD-pore pothesis that KV7.1 and KV7.1þKCNE1 open from different conformational states. coupling. The interaction between the S4-S5 linker and the cytosolic end of S6 has been proposed to mediate the VSD-pore coupling. KCNQ1 (Kv7.1) in as- 615-Pos Board B385 sociation with the auxiliary subunit KCNE1 forms the IKs channel in the heart that is critical for controlling heart rhythm. It has been shown that KCNQ1 Modulation of KV7.1 by NaVb1 Subunit Elisa Carrillo-Flores, Carlos A. Villalba-Galea. opens to two open states when the voltage sensor is activated to the intermedi- Physiology and Pharmacology, University of the Pacific, Stockton, CA, USA. ate state (termed IO) and the fully activated state (AO), respectively. The asso- Several small proteins, referred to as ‘‘b-subunits’’, have been shown to ciation of KCNE1 suppresses the IO state and enhances the AO state. Therefore, the IKschannel only opens to the AO state, which is more important modulate the activity of potassium-selective, voltage-gated (KV)channels, rendering Kþ-conductance with different voltage dependence and kinetics. to the cardiac physiology. We have previously shown that the F351A mutation The physiological role of these interactions has been identified in some selectively suppressed the IO state by disrupting the VSD-pore coupling. Since F351 is a conserved residue important for the above mentioned VSD-pore case. For instance, the interaction of KV7.1 and KCNE1 produces the car- diac Kþ-current known as I , which has a slower kinetics and negatively coupling mechanism, this result suggests that the canonical coupling mecha- KS nism is important for the IO state, In this study we find that three mutations shifted voltage dependence for activation with respect to KV7.1 alone. In contrast, KCNE3 renders a channel that is open at physiological potentials. of KCNQ1 that are associated with Long QT syndrome, S338F, F339S and L342A disrupt the VSD-pore coupling to selectively eliminate the AO state. This characteristic of KV7.1 makes it into a versatile ‘‘a-subunit’’ with ac- tivity that can be physiologically customized by b-subunits. Recent studies These residues are located in the middle of S6 and cannot interact with the S4-S5 linker. These results indicate that different VSD-pore coupling mecha- have shown that NaVb1, which is regarded as a b-subunit of sodium- selective, voltage-gated (Na ) channels, can modulate the activity of several nisms are responsible for the IO and AO states, and mutations in different struc- V tural motifs can selectively disrupt either of the mechanisms. Furthermore, the KV channels. Since both KV7.1 and NaVb1 are expressed in the hippocam- pus and cerebellum of mice, we wonder whether these two proteins could middle of S6 is involved in a non-canonical mechanism that specifically cou- functionally interact. Thus, we recorded Kþ-currents from Xenopus oocytes ples VSD activation to the AO state. co-expressing KV7.1 and NaVb1 using the Cut-open Oocyte Voltage Clamp 618-Pos Board B388 technique. We found that the expression of NaVb1 caused KV7.1 to display a þ Properties of Polyunsaturated Fatty Acid Tail Influence Affinity for IKs slow inactivating-like behavior, decreasing the K -conductance over 20% Channel after depolarization to 10 mV and above. Further, the deactivation rate 1 2 1 þ Briana Watkins , Sara Liin , Peter Larsson . of the K -current decreased, displaying at least three phases, namely, a par- 1Physiology and Biophysics, University of Miami, Miami, FL, USA, tial recovery from inactivation, a fast deactivating phase, and a slower deac- 2Linkoping University, Linkoping, Sweden. tivating phase. The rate of the initial fast deactivation was similar to the The activity of voltage-gated ion channels underlies the generation and propa- deactivating rate of the KV7.1 expressed alone. In contrast, the rate of the gation of action potentials in the brain and heart. The cardiac IKs channel is a þ slow deactivation was 2-fold slower than the rate of deactivation of KV7.1 voltage-gated K channel responsible, in part, for the repolarization and termi- alone. Furthermore, our results indicate that NaVb1 was able to modulate nation of the ventricular action potential. Disruption of the IKs channel’s activ- the voltage dependence for activation KV7.1 showing an apparent displace- ity can lead to a particular type of cardiac arrhythmia known as Long QT ment of 20 mV. The physiological significance of this interaction is yet to Syndrome (LQTS), and can predispose individuals to ventricular fibrillation be determined. and sudden cardiac death. Previous work in our lab has shown that the activity of the IKs channel can be modified by polyunsaturated fatty acid (PUFA) ana- 616-Pos Board B386 logues such as N-arachidonoyl taurine (N-AT). We have demonstrated the Comprehensive Assessment of Disease Mutant Forms of the Human importance of a negatively charged head group for participating in an electro- KCNQ1 Potassium Channel static interaction between the PUFA head and the voltage-sensor, however little 1 2 1 1 Hui Huang , Georg Kuenze , Jarrod Smith , Keenan Taylor , is known about the importance of the PUFA tail. By conducting two-electrode 2 2 3 3 Amanda Duran , Jens Meiler , Carlos Vanoye , Alfred George , voltage-clamp experiments while perfusing different concentrations of PUFA 1 Charles Sanders . compounds, we have shown that the tail group of a PUFA molecule is important 1Biochemistry, Vanderbilt University, Nashville, TN, USA, 2Biochemistry for the PUFA’s binding affinity to the IKs channel. There is little correlation be- and Pharmacology, Vanderbilt University, Nashville, TN, USA, tween the length of the PUFA’s carbon tail and binding affinity or the PUFA’s 3Pharmacology, Northwestern University, Chicago, IL, USA. effects in shifting the voltage-dependence of channel activation (V0.5). Simi- The voltage-gated potassium channel KCNQ1 is critical for the cardiac action larly, the number of double bonds found in the tail does not seem to alter bind- potential. Mutations in KCNQ1 and its accessory protein KCNE1 are the most ing affinity or effect. However, our results suggest that the position of the first common cause of congenital long-QT syndrome (LQTS). There are a variety of double bond in proximity to the head group influences the affinity and effect of mechanisms by which a given mutation may cause KCNQ1 channel dysfunc- several PUFAs for the I channel. PUFAs with their first double bond closer to tion and prolonged activation potentials. The ideal treatment of patients Ks the head group display higher affinity to the IKs channel compared to PUFAs in harboring a KCNQ1 mutation is dependent on which specific mechanisms which the first double bond occurs at a more distal position along the carbon cause loss of function. In the present study, we have employed a multidisci- tail. plinary approach to systematically investigate the specific effects of 51 KCNQ1 mutations on the channel structure, stability, trafficking, and electro- 619-Pos Board B389 physiological properties. The 51 mutations, located in the voltage sensor Probing the Molecular Mechanism of Polyunsaturated Fatty Acid Modu- domain (VSD), are disease causing, benign, or of unknown significance. Our lation of the IKs Channel results show nearly all of the benign mutants were efficiently expressed and Sammy Yazdi, Sara Liin. functional, with variable changes in biophysical properties. About 70% of Linko¨ping University, Linko¨ping, Sweden. loss of function mutants exhibited impaired expression and channel function. The current generated by the cardiac slow-delayed rectifier potassium (IKs) For many of these the NMR spectra were significantly-broadened, indicating channel is an important determinant in the repolarization phase of the cardiac that such mutations severely destabilize the channel, resulting in reduced action potential. The IKs channel is composed of the voltage-gated potassium

BPJ 8570_8572 Sunday, February 18, 2018 123a

channel KCNQ1 (KV7.1) and its auxiliary subunit KCNE1. Mutations in the IKs thought to contribute to M-current heterogeneity. Here we show the func- channel have been implicated in a range of cardiac diseases, in particular tional consequences of de novo missense Kv7.5 mutations identified by congenital long QT syndrome, which may cause ventricular arrhythmias, ven- whole exome sequencing in four children affected by intellectual disability tricular fibrillation and even sudden death. Analogues of polyunsaturated fatty and/or epileptic encephalopathy. We also mapped the position of the mutants acids have previously been shown to facilitate the opening of the IKs channel, in a homology model of Kv7.5 based on the cryo-EM structure of Kv7.1 restore rhythmic firing of embryonic rat cardiac myocytes, and correct a pro- channels. Electrophysiological characterization showed that 3 of the muta- longed QT interval in isolated guinea pig hearts. In order to understand the tions (V145G in S1, L341I in S6, and S448I in the unstructured C-terminus) mechanism of the modulation of the IKs channel by polyunsaturated fatty resulted in loss-of-function, causing a depolarizing shift of the voltage- acid analogues, we have in this study generated models of KCNQ1 in complex dependence of activation and slowing of activation kinetics. The positions with KCNE1 in both its open and closed states based on the recent cryo-EM of V145 and L341 in the Kv7.5 protein lead us to hypothesize that mutations structure of the KCNQ1/CaM complex. Furthermore, through applying molec- at these sites influence voltage sensor movement or its coupling to the ular dynamics simulations, we study how lipid bilayers enriched with polyun- pore, in the case of V145, and the stability of the open state of the pore, in saturated fatty acid analogues interact with an integral KCNQ1/KCNE1 the case of L341. On the other hand, P369R (in helix-A of the C-terminus) complex. These simulations aim to provide insights into how the enrichment causes a hyperpolarizing shift of the voltage-dependence of activation, accel- of polyunsaturated fatty acid analogues on particular regions on the IKs channel eration of activation kinetics and slowing of deactivation kinetics. These find- enhances channel activity. ings suggest that P369R is a gain-of-function mutation, increasing the stability of the open state of Kv7.5 channels. In summary, these data charac- 620-Pos Board B390 terize the first mutations in Kv7.5 channels to be associated with human Combining Population Whole Exome Sequencing and Functional Analysis neurological disease, and the findings suggest that Kv7.5 channels present to Detect LQT1 a potential therapeutic target for the treatment of neuronal excitability 1 1 1 Cassandra M. Hartle , Jonathan Z. Luo , Ann N. Stepanchick , disorders. Uyenlinh L. Mirshahi1, Dustin N. Hartzel2, Kandamurugu Manickam3, Michael F. Murray3, Tooraj Mirshahi1. 1 622-Pos Board B392 Department of Molecular and Functional Genomics, Geisinger, Danville, Activation of N-Methyl-D-Aspartate Receptor Inhibits Enkephalinergic PA, USA, 2Biomedical and Translational Informatics, Geisinger, Danville, Neurons in the Superficial Dorsal Horn of Mouse Spinal Cord PA, USA, 3Genomic Medicine Institute, Geisinger, Danville, PA, USA. Eiko Kato, Yuuichi Hori. Long QT Syndrome (LQTS) is a rare genetic disorder the can lead to life threat- Physiology and Biological Information, Dokkyo Medical University, ening arrhythmias. Mutations in KCNQ1, the pore forming subunit of the slow Tochigi, Japan. rectifying potassium current IKs, are responsible for 35% of all genetically iden- Enkephalin (Enk)-containing neurons are distributed in high concentrations in tifiable cases of LQTS. Impaired IKs prolongs repolarization of the cardiac ac- the superficial dorsal horn (SDH) of the spinal cord, where they play an impor- tion potential leading to prolonged QT interval. We have completed whole tant role in the transmission and modulation of nociceptive information. In the exome sequencing on more than 90,000 Geisinger patients and have identified present experiments, we investigated the involvement of the N-methyl-D- a large number of rare missense and putative loss of function variants in aspartate receptor (NMDAR) and the large conductance calcium-activated po- KCNQ1. A previous report using in silico and genetic lab reports could not tassium (BK) channels in the regulation of Enk-containing neurons activity in establish a link between a LQTS diagnosis and variants of unknown signifi- the SDH. cance (VUS) in LQT2 and LQT3 genes (KCNH2 and SCN5A). However, those Tight-seal whole-cell recordings were obtained from neurons localized in the data did not include functional analysis. We have combined genetic and clinical SDH of the lumbar spinal cord in mice. L-glutamate (Glu) was applied by pres- data with functional analysis on variants of unknown significance to help sure ejection through a glass pipette placed near the recorded neurons. After the establish pathogenicity of rare KCNQ1 variants. In doing so, we found a het- electrophysiological experiments, the recorded neurons were collected, and erozygous carrier of KCNQ1(G314V), a mutation in the highly conserved single-cell real-time RT-PCR was performed to analyze the expression profile selectivity filter region of KCNQ1. Expressing KCNQ1(G314V) with of genes of interest in each SDH neuron. KCNE1 in HEK293 cells, we find that the mutant channel is expressed at The puff application of Glu evoked an inward current at a holding potential of similar levels to the wild-type channel. Using whole cell patch clamp 70 mV. Depolarizing the holding potential to 0 mV, an outward current last- recording, we find no I current in HEK293 cells expressing KCNQ1 Ks ing a long duration appeared after the initial inward current. The NMDAR (G314V)/KCNE1. Examining Electronic Health Records (EHR), we found antagonist AP-V abolished the outward current. The outward current was that the G314V carrier was a 48-year-old female with a median QTc of also abolished by the selective BK channels antagonist iberiotoxin. The 493ms. The patient is deceased and, while she was at risk for an arrhythmia, single-cell real-time RT-PCR analysis revealed that single SDH neurons ex- the cause of death is unknown. Our data suggest in vitro functional analysis pressing the preproenkephalin mRNA also expressed the BK channel a -sub- should be included in any population based genetic studies that aim to identify unit, NR1, and NR2B subunit mRNAs. Furthermore, the neurons generating pathogenic variants in LQT1. the outward current showed a significant tendency to express the BK channel 621-Pos Board B391 a-subunit mRNA, which is consistent with the pharmacological results of a Mutations in Kv7.5 Channels Associated with Intellectual Disability or BK channels antagonist. 2þ Epileptic Encephalopathy Our results suggest that Ca influx through NMDAR may activate BK chan- Anna Lehman1, Samrat Thouta2, Grazia M.S. Mancini3, nels, thereby inhibiting Enk-containing neurons in the SDH. The NMDAR- Marjon van Slegtenhorst3, Sakkubai Naidu4, Sonal Desai4, Kirsty McWalter5, mediated inhibition of Enk-containing neurons is speculated to be one of the Richard Person5, Jill Mwenifumbo1, Ramona Salvarinova6, Ilaria Guella8, possible cellular mechanisms of chronic pain induced by peripheral nerve Marna B. McKenzie8, Matthew J. Farrer8, Anita Datta6, Mary B. Connolly7, injury. Michelle Demos6, Somayeh Mojard Kalkhoran2, Damon Poburko2, Jan M. Friedman1, Thomas Claydon2. 623-Pos Board B393 1Medical Genetics, University of British Columbia, Vancouver, BC, Canada, Exotic Properties of a Voltage Gated Proton Channel in the Snail Helisoma 2Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, trivolvis BC, Canada, 3Clinical Genetics, Erasmus University Medical Center, Vladimir V. Cherny1, Sarah Thomas2, Deri Morgan1, Susan M.E. Smith2, Rotterdam, Netherlands, 4Neurogenetics, Kennedy Krieger Institute, Thomas E. DeCoursey1. Baltimore, MD, USA, 5GeneDx, Gaithersburg, MD, USA, 6Pediatrics, 1Physiology & Biophysics, Rush University, Chicago, IL, USA, 2Molecular University of British Columbia, Vancouver, BC, Canada, 7University of and Cellular Biology, Kennesaw State University, Kennesaw, GA, USA. 8 British Columbia, Vancouver, BC, Canada, Centre for Applied Voltage gated proton currents, HV1, were first reported in snail neurons (Helix þ Neurogenetics, University of British Columbia, Vancouver, BC, Canada. aspersa and Lymnaea stagnalis). These H channels open very rapidly, 2-3 þ The M-current is a slowly activating and deactivating K current that plays a orders of magnitude faster than mammalian HV1. We identified an HV1 crucial role in regulating neuronal excitability by impeding repetitive action gene in the snail Helisoma trivolvis and expressed it in mammalian cells. potential firing during sustained depolarizing inputs. Inhibition of the M-cur- The resulting HtHV1 currents in most respects resembled those described in rent leads to enhanced neuronal excitability associated with neurological dis- other snails, including rapid activation. In contrast with most HV1, activation orders. Although their role is poorly understood, Kv7.5 channels, which are of HtHV1 was exponential, suggesting first-order kinetics. Also consistent highly expressed in the brain, co-assemble with Kv7.3 subunits and are with first order kinetics, tact and ttail overlapped at intermediate voltages

BPJ 8570_8572 124a Sunday, February 18, 2018 and the voltage at which the gating time constants were slowest occurred at of the S3 transmembrane helix that appeared to account for this difference in the midpoint of the gH-V relationship. However, the large gating charge of kinetics. Replacing His164 in mHV1 with Ser, the corresponding amino acid 5.5 e0 and the existence of extensive predicted coiled-coil regions in the in SpHV1, accelerated mHV1 gating by an order of magnitude. We focused C terminus both suggest that HtHV1 functions as a dimer, but evidently on this position, and replaced the corresponding His168 in human HV1 with with more highly cooperative gating than exists in other species. In stark Gln, the corresponding residue in HtHV1. The mutant did activate 10 times contrast with mammalian HV1, HtHV1 opening is extremely sensitive to faster, but remained two orders of magnitude slower than HtHV1. We conclude pHo whereas closing is nearly independent of pHo. All known HV1 exhibit that this position does influence kinetics, but does not account for the rapid DpH dependent gating that in results in a 40 mV shift of the gH-V relationship gating of snail HV1. for a 1 unit change in either pHo or pHi. This property, called DpH dependent Supported by NIH grant GM-102336 and NSF grant MCB-1242985. gating, is crucial to all of the functions of HV1 in many species and in numerous human cells. The HtHV1 channel exhibits normal or supernormal 626-Pos Board B396 pHo dependence, but anomalously weak pHi dependence. The average slope Insight into the Role of Hv1 C-Terminal Domain in Dimer Stabilization of the pHi dependence is 15 mV/unit between pHi 5 and 9, with pHo 7. Panisak Boonamnaj, Pornthep Sompornpisut. Evidently, HtHV1 has distinct internal and external pH sensors. The anoma- Chemistry, Chulalongkorn University, Bangkok, Thailand. lous DpH dependent gating of HtHV1 channels provides clues to the structural The voltage-gated proton-selective channel (Hv1) regulates the proton conduc- basis for this important property. tion process in response to electrical membrane voltage. The Hv1 channel is a Supported by NIH grant GM-102336 and NSF grant MCB-1242985. dimer in membrane. Crystal structures of Hv1 channels have revealed the pri- mary contacts between the two monomers are located at C-terminal domain 624-Pos Board B394 (CTD) which forms a coiled-coil structure. The role of Hv1-CTD is not fully Gating Currents in Hv1 Proton Channels understood. Here molecular dynamics (MD) simulations of full-length and Victor De La Rosa, I. Scott Ramsey. truncated CTD models of human and mouse Hv1 channels illustrated an impact Physiology and Biophysics, Virginia Commonwealth University, Richmond, of the CTD on a packing between dimeric transmembrane domains. MD results VA, USA. of the only Hv1-CTD models highlighted four fundamental interactions of the Hv1 proton channels are evidently unique among voltage sensor (VS) do- key residues contributing to the stability of Hv1 dimer. These include salt- þ mains in mediating an intrinsic H -selective conductance (GAQ) gated by bridges, hydrophobic interactions, hydrogen bond and disulfide bond across changes in both the transmembrane voltage and pH (DpH) gradients. In order the dimer interface. At neutral pH, salt-bridge interactions increase dimer sta- to investigate how changes in DpH are coupled to shifts in the GAQ-V relation, bility but the dimer becomes less stable at acidic pH. Hydrophobic core packing we sought to measure gating charge movements in Hv1. Combining mutations of the heptad pattern is important for the stability, as shown by favorable non- that a) activate more rapidly than WT Hv1 (W207A), and b) confer voltage- polar binding free energies rather than by electrostatic components. Moreover, þ dependent block of outward H current carried by GAQ (N214R) permits mea- free energy calculations indicate that a better uniformed hydrophobic core in surement of whole-cell Hv1 gating currents in tetracycline-inducible HEK- the coiled-coil structure of the Hv1-NIN mutant leads to an increase in dimer 293 expression system (Randolph, et al., 2017). We calculate ‘ON’ gating stability with respect to the wide-type in which the hydrophobic packing is dis- charge (QON) from the integrals of transient outward currents measured at rupted by a hydrogen bond between hydrophilic Asn. A Cys disulfide bond has þ100 mV after voltage prepulses (VPP) to a range of potentials. The midpoint a strong impact on dimer stability, by holding the dimer together and facili- of the steady-state QON-V relation (measured at long VPP durations) is 10 tating the interactions described above. These results are consistent with disso- mV more negative than the GAQ-V relation, indicating that Hv1 exhibits a ciative temperatures and energy barriers of dimer dissociation obtained from form of electromechanical coupling that is phenomenologically similar to the temperature-accelerated MD. þ the archetypal Shaker K channel. Correlating maximal QON and GAQ-medi- ated ITAIL permits estimates of channel number and GAQ unitary conductance 627-Pos Board B397 2D (gAQ). Consistent with the effect of N214R to block GAQ, we find that gAQ Zn Modulates Hv1 Proton Channel Gating via Conformational (0.8 fS in W207A-N214R) is approximately 40-fold smaller than a previous Coupling to an Intracellular Coulombic Network þ estimate of gAQ based on H current variance analyses in human eosinophils Ashley L. Bennett, Victor De, La Rosa, I. Scott Ramsey. that natively express Hv1 (Cherny, et al., 2003). Assuming that each dimeric Physiology and Biophysics, Virginia Commonwealth University, Richmond, Hv1 channel complex moves 6 e0 during VS activation (Gonzalez, et al., VA, USA. þ 2013), we calculate that accurate measurement of gating currents in Gating of the intrinsic ‘aqueous’ H conductance (GAQ) in the Hv1 proton W207A-N214R requires very high channel density in the plasma membrane channel is sensitive to extracellular Zn2þ and other divalent cations in the (> 3x106 channel complexes per cell). C-terminal truncation (W207A- micromolar range. Previous studies showed that the extracellularly- N214R-DC), which is reported to monomerize Hv1 channels and decrease accessible residues H140 and H193 are required for Zn2þ to positively shift the effective gating valence (Gonzalez, et al., 2013), also decreases the the GAQ-V relation. However, the molecular mechanism by which extracel- 2þ QON/ITAIL ratio by 50%. lular Zn occupancy is coupled to voltage-dependent GAQ gating remains known. We used site-directed mutagenesis and electrophysiology to identify 625-Pos Board B395 Zn2þ-interacting residues and used the experimental data to build new models RAPID Gating Kinetics of a Voltage Gated Proton Channel in the Snail of the Hv1 voltage sensor (VS) domain in Zn2þ-liganded (Hv1F$Zn2þ) and Helisoma trivolvis unliganded (Hv1F þ Zn2þ) conformations. Although imidazole nitrogen 1 2 2 3 Sarah A. Thomas , Vladimir V. Cherny , Deri Morgan , Liana Artinian , atoms (H140-Nd1 and H193-Nd1) are close enough to simultaneously coordi- þ þ Vincent L. Rehder3, Thomas E. DeCoursey2, Susan M.E. Smith1. nate a single Zn2 ion, we did not observe a stable Zn2 coordination sphere 1Molecular and Cellular Biology, Kennesaw State University, Kennesaw, during all-atom molecular dynamics (MD) simulations using the GA, USA, 2Department of Physiology & Biophysics, Rush University, CHARMM36 force field. To simulate electronic interactions between Zn2þ 3 Chicago, IL, USA, Biology, Georgia State University, Atlanta, GA, USA. and H140-Nd1 and H193-Nd1, we imposed harmonic constraints (5 kcal/ ˚ 2þ Voltage gated proton currents, HV1, were first reported in snail neurons (Helix mol/A) and equilibrated the Hv1F$Zn model during a 50 ns MD simulation. aspersa and Lymnaea stagnalis). The most obvious difference between proton 20 ns after releasing constraints, ZN13 spontaneously dissociates from H140- þ currents in snails and in other species is that snail H channels open very Nd1 and H193-Nd1 but remains associated with the carboxyl group of E119. rapidly, 2-3 orders of magnitude faster than HV1 in mammals, dinoflagellates, The VS also undergoes backbone reorganization involving the S1, S3 and coccolithophores, fish, and insects. Preliminary analysis of RNA-seq data from S4 helices after ZN13 dissociation that suggest Zn2þ-dependent conforma- the snail Helisoma trivolvis suggests that HV1 is differentially expressed in two tional changes. Concomitant changes in the organization of an electrostatic neurons. We cloned a HV1 gene from H. trivolvis brains, confirmed its protein network involving intracellular acidic residues (E153 and D174) and S4 expression in H. trivolvis brain tissue, and expressed it in mammalian cells. The ‘gating charge’ Arg side chains may reveal how voltage-dependent gating 2þ resulting HtHV1 currents in most respects resembled those described in other is altered by Zn . In support of this hypothesis, we find that E153 neutrali- snails, including rapid activation. Another species with rapidly activating pro- zation also alters Zn2þ potency. In summary, extracellular Zn2þ occupancy is ton currents is the sea urchin, Strongylocentrotus purpuratus (SpHV1). Sakata coupled to reorganization of an intracellular Coulombic network that is et al (2016, BBA 1858:2972-2983) localized a single amino acid at the inner end required for voltage-dependent gating in Hv1.

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Posters: Ligand-gated Channels I spontaneous jumps between patterns of gating characterized by distinctive open probabilities and rate constants. It is thought to control the decay time of the 628-Pos Board B398 NMDA receptor excitatory postsynaptic current. Quantitative investigations of Activation and Desensitization Mechanism of AMPA Receptor-TARP modal gating have remained elusive due to difficulties in identifying modal tran- Complex by Cryo-EM sitions with sufficient resolution. Here we present a novel computational method Shanshuang Chen1, Yan Zhao1, Yuhang Wang2, Mrinal Shekhar2, based on change-point detection and k-means clustering to identify modal tran- Emad Tajkhorshid2, Eric Gouaux3. sitions within a time-continuous single-channel recording. With this method we 1Vollum Institute, OHSU, Portland, OR, USA, 2University of Illinois at analyzed one-channel electrical recordings from recombinant GluN1-1a/ Urbana-Champaign, Urbana, IL, USA, 3Vollum institute, Howard Hughes GluN2A and GluN1-1a/GluN2B receptors to estimate for the first time rate con- Medical Institute, OHSU, Portland, OR, USA. stants for NMDA receptor modal transitions We found that mode dwell-times The majority of fast excitatory neurotransmission in mammalian synapses is initi- followed exponential distributions for all three modes and that the rate constants a for High to Medium and Medium to Low modes are faster than previously ated by -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) recep- 1 1 assumed for both GluN2A (kHM, 1.53 s ; kML, 1.03 s ) and GluN2B (kHM, tors, receptors which transduce presynaptic neurotransmitter release to 1 1 1.94 s ; kML, 0.71 s ) channels. In contrast, Low to High transitions were depolarization of postsynaptic membrane by coupling glutamate binding to the 1 1 slow for both GluN2A (kLH, 0.07 s ; kHL, 0.23 s ) and GluN2B (kLH, 0.004 opening of a cation channel. In the post synaptic densities, AMPA receptors 1 1 co-assemble with a variety of auxiliary proteins to yield signaling complexes s ; kHL, 0.005 s ) channels. These results also show that GluN2B- with modulated gating kinetics, ion channel properties and pharmacology. Trans- containing channels exhibit High and Low mode transition rates several orders membrane AMPA receptor regulatory proteins (TARPs) represent the principle of magnitude slower than GluN2A-containing channels. Simulations of macro- family of auxiliary proteins, from which the prototypical member TARP g2, scopic responses to 1 ms glutamate pulses using the derived rate constants pre- also known as stargazin, potentiates AMPA receptors by decelerating deactiva- dict biphasic decay times consistent with experimental observations. The data tion and desensitization, facilitating resensitization, boosting the efficacy of presented here fill an important gap in modeling the NMDA receptor reaction partial agonists such as kainate, and attenuating polyamines from blocking mechanism. In addition, our computational workflow may provide a novel strat- calcium-permeable AMPA receptors. Here, we report structures of the GluA2 egy to probe structural determinants of modal gating. AMPA receptor -TARP g2 complex in the presence of the partial agonist kainate 631-Pos Board B401 or the full agonist quisqualate together with a positive allosteric modulator or with Effects of T686A Mutation on the Structural Stability of the AMPA Recep- quisqualate alone, to illustrate the activation and desensitization mechanism of tor Ligand-Binding Domain the complex. These structures show how TARPs buttress the receptor ligand bind- Hiraku Oshima1, Suyong Re1, Masayoshi Sakakura2, Hideo Takahashi2, ing domain to enhance the coupling between ligand binding and channel opening, Yuji Sugita1. to promote kainate efficacy, and to reduce the ensemble of desensitized states. In 1Riken Quantitative Biology Center, Kobe, Japan, 2Graduate School of particular, structural and computational analysis of the full-agonist and modulator Medical Life Science, Yokohama City University, Yokohama, Japan. bound complex indicate spontaneous ion permeation through the channel gate, AMPA ionotropic glutamate receptors mediate fast excitatory neurotrans- thus offering structural insights of AMPA receptor in an active state. mission in the central nervous system and their dysfunction is associated 629-Pos Board B399 with neurological diseases. The cleft of the ligand-binding domain (LBD) Single Molecule FRET Studies into the Effects of Sodium on Kainate closes upon glutamate binding, which induces the channel opening in the Receptor Dynamics transmembrane domain. T686A mutation reduces the efficacy of glutamate Douglas Litwin, Sana Shaikh, Vladimir Berka, Vasanthi Jayaraman. (Glu), while that of quisqualate (Quis) is not affected by the mutation. Biochemistry and Molecular Biology, UT Health at Houston, Houston, The X-ray crystallography shows that wild type (WT) and T686A mutant TX, USA. structures are quite similar to each other. However, our recent NMR exper- Ionotropic glutamate receptors (iGluRs) are the primary neurotransmitter re- iment suggests that the mutation changes the chemical shift of residues ceptors involved in excitatory synaptic transmission. Amongst the iGluR fam- around the ligand-binding site. This indicates that T686A has intermediate ily, kainate receptors (KARs) are the most diverse in function but also the least or hidden states that cannot be observed by X-ray. We here investigate understood. KARs contribute to postsynaptic responses and can also control the the effect of the mutation on the LBD structure for two ligands, Glu and presynaptic release of both excitatory and inhibitory neurotransmitters. KARs Quis, using molecular dynamics simulations. The free energy landscapes consists of an N-terminal domain (NTD), ligand binding domain (LBD), trans- governing the opening/closing of the LBD are calculated for WT and membrane domain (TMD) and a C-terminal intracellular tail. Moreover, the T686A by the replica-exchange umbrella sampling method. We found that LBD-LBD/NTD-NTD contacts are influential sites in shaping the KAR confor- only closed state similar to the crystal structures is stable for Glu-bound mational landscape. Studies have also shown that KARs have a downregulation WT, Quis-bound WT, and Quis-bound T686A, whereas both the closed state in function in the absence of sodium and an affinity for sodium in the range of and a semi-open state are stable for Glu-bound T686A. The semi-open state physiological conditions. This would make sodium a negative regulator during cannot open the channel wider than the closed state. These results suggest times of high synaptic activity. However, no data is available on the effect of that Glu behaves as a partial agonist for the T686A mutant. We also discuss sodium on KAR structural dynamics. In this study we investigated the struc- the effect of flip/unflip of the peptide bond of D651 and S652 on the stability tural dynamics of KARs when occupied and lacking sodium. We initially of the LBD. compared measurements made in full length protein and in whole cells to cur- rent structural models using luminescence energy resonance transfer (LRET). 632-Pos Board B402 We then characterized the structural dynamics of KARs in the presence and Subunit Independence in AMPA Type Glutamate Receptors 1,2 1,2 absence of sodium using single molecule FRET (smFRET). The smFRET Jelena Baranovic , Andrew J.R. Plested . 1Molecular Physiology and Cell Biology, Leibniz-Forschungsinstitut fur€ and LRET data matched well for corresponding sites. Current smFRET data 2 shows high similarly between the unliganded sodium-bound state as seen in Molekulare Pharmakologie (FMP), Berlin, Germany, Institute of Biology, EM models and LRET measurements. The glutamate bound state smFRET Cellular Biophysics, Humboldt University, Berlin, Germany. data shows a state unique from EM models. Cesium replacement experiments Glutamate is the primary excitatory neurotransmitter in the vertebrate brain. are underway. These are the first smFRET studies into the structural dynamics Once released into the synaptic cleft, it is swiftly bound by AMPA recep- of KARs and there are similarities and differences between current structural tors (AMPARs), opening their ion channels and initiating depolarization of models and smFRET measurements. Additional measurements are being the postsynaptic membrane. Synapses undergo activity dependent changes made and it appears likely that there are unique unliganded states for KARs in strength that may underlie cognitive processes such as memory and occupied or lacking sodium. learning. These changes are linked to ion flux and membrane depolariza- tion. Therefore alterations of the AMPAR kinetics could change signal 630-Pos Board B400 transduction between excitatory neurons. AMPARs are tetrameric proteins. Quantifying Kinetic Transitions between NMDA Receptor Gating Modes Their ion channels activate rapidly upon binding of glutamate, but also shut Gary J. Iacobucci, Gabriela K. Popescu. down quickly even with glutamate still bound, a property known as desen- Biochemistry, State University of New York at Buffalo, Buffalo, NY, USA. sitization. Recently, it has been found that a mutation near the transmem- NMDA receptors are glutamate- and glycine-gated cation channels whose activ- brane segment of AMPARs, A622T, known as Lurcher from earlier ity is critical to normal brain development and function. They activate with com- studies on mice, recurs in patients with neurodevelopmental disorders. plex gating kinetics, which includes modal transitions. These transitions reflect Here, we characterized the mutation by recording currents from single

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A622T-carrying AMPARs. The most striking property of the A622T mutant 635-Pos Board B405 was greater conductance, which revealed the full subunit independence in Assembly of Kainate and AMPA Receptors AMPA receptor gating. Each subunit bound glutamate and activated Mark L. Mayer1, Huaying Zhao2, Suvendu Lomash1, Sagar Chittori3, independently, leading to a step-wise increase in ion channel conductance. Carla Glasser4, Peter Schuck2. Using a classical antagonist unbinding assay, all four sublevels of AMPA 1NINDS, NIH, Bethesda, MD, USA, 2NIBIB, NIH, Bethesda, MD, USA, receptor activation were resolved. Accordingly, deactivation of the receptor 3NCI, Bethesda, MD, USA, 4NIMH, NIH, Bethesda, MD, USA. followed the same 4-step process. In addition, freely desensitizing recep- The gating characteristics and permeability of tetrameric glutamate receptor tors, exhibited dimer independence, with each dimer desensitizing sepa- ion channels are determined by subunit composition. Competitive homo- rately. Desensitization of individual dimers was a more frequent event dimerization and hetero-dimerization of their amino-terminal domains is a than simultaneous desensitization of both dimers, leading us to identify key step controlling iGluR assembly. Using fluorescence-detected sedimenta- sublevels associated with ‘‘partial desensitization’’. Pharmacological block tion velocity analytical ultracentrifugation we systematically measured the of desensitization effectively removed this partial desensitization. The thermodynamic stabilities of all kainate and AMPA receptor ATD homodimer Lurcher results demonstrate a hierarchy of subunit independence in and heterodimer combinations. The measured affinities span many orders of GluA2 across different states of activity. They also confirm that the magnitude, and the complexes show large differences in kinetic stabilities. Kai- A622T channel can conduct at very low glutamate concentrations, a prop- nate receptor ATD dimers are generally lower affinity than AMPA receptor erty that could easily modify signal transduction between excitatory ATD dimers, but both show a general pattern of increased heterodimer stabil- neurons. ities as compared to the homodimers of their constituents, matching well phys- iologically observed receptor combinations. Free energy maps of AMPA and 633-Pos Board B403 kainate receptor ATD dimers provides a framework for the interpretation of Single-Channel Study of AMPA Receptors Restrained by an Intersubunit observed receptor subtype combinations and possible assembly pathways. Zinc Bridge Sebastian Opfermann1,2, Jelena Baranovic1,2, Andrew J.R. Plested1,2. 636-Pos Board B406 1 Molecular Phyisiology and Cell Biology, Leibniz-Forschungsinstitut fuer Membrane Lipid Compositions Control Dye-Permeable Pore of the P2X7 2 Molekulare Pharmakologie, Berlin, Germany, Institute of Biology, Cellular Receptor Biophysics, Humboldt University, Berlin, Germany. Akira Karasawa, Kevin Michalski, Polina Mikhelzon, Toshimitsu Kawate. AMPARs (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors) belong Cornell University, Ithaca, NY, USA. to the ionotropic glutamate receptor family and mediate fast excitatory transmis- The P2X7 receptor is an extracellular ATP-gated cation channel. Activation of sion at synapses in the vertebrate brain. They have a tetrameric structure with an P2X7 leads to the opening of a dye permeable pore, which causes cell lysis and amino terminal domain, ligand binding (LBD), transmembrane and carboxy- eventually results in various health problems including chronic pain, neurodegen- terminal domain. Glutamate binding at the LBDs results in a conformational rear- eration, and tumor growth. However, it remains controversial what constitutes rangement that triggers opening of the integral ion channel in an iris-like fashion. this dye-permeable pore and how it opens. Two prevailing hypotheses are: 1) However, the LBD layer has multiple degrees of freedom including the domain P2X7 dilates its transmembrane channel to form a larger pore and 2) P2X7 acti- closure of individual clamshells, the number of LBDs bound by agonist, and their vation recruits other large conductance channels. To clarify whether P2X7 itself relative arrangements within the dimer and the tetramer. Here, we investigated one constitutes a dye permeable pore, we purified and reconstituted P2X7 into lipo- aspect of the LBD layer dynamics at the single channel level, using a previously somes and measured pore activity. We observed strong dye uptake from P2X7 re- studied interdimer metal bridge. This bridge holds the receptor in a compact constituted liposomes, suggesting that P2X7 can form a dye permeable pore in the arrangement and concomitantly reduces macroscopic currents activated by satu- absence of other cellular components. To our surprise, dye uptake was indepen- rating glutamate. We recorded glutamate-activated currents from single AMPAR dent of the C-terminal domain, which has been considered to be absolutely neces- channels with restrained (zinc-bridged) and ‘freely moving’ LBD layers, and sary for pore formation. Furthermore, dye uptake was immediately observed after compared them to wild-type channels. We wanted to establish whether the inhibi- ATP application, indicating that pore dilation is unlikely. We also discovered that tion by the zinc bridge is because the channels open less frequently, or because the the P2X7-dependent pore was facilitated by phosphatidylglycerol and sphingo- channels open to a lower conductance. The results show that AMPARs with a myelin but strongly inhibited by cholesterol through direct interactions with the compact LBD layer can reach similar conductance levels to non-bridged receptors. transmembrane domain of P2X7. In combination with a fatty acid incorporation However, once the LBD layer is immobilized, the open probability of the channel is assay, our results suggest that palmitoylation at the C-terminal domain counter- reduced, and latency to opening increases. These results suggest that the correct acts the inhibitory effect of cholesterol by facilitating P2X7 channel opening. angle and register of the two LBD dimers is essential for full AMPA receptor activation. 637-Pos Board B407 Characterization of Hearing Loss-Related Mutations in ATP-Activated 634-Pos Board B404 Ion Channels Functional Validation of Heteromeric Kainate Receptor Models Benjamin I. George, Mufeng Li, Kenton J. Swartz. 1 2 1 3 Teresa Paramo , Patricia M.G.E. Brown , Maria Musgaard , Derek Bowie , NINDS, National Institutes of Health, Bethesda, MD, USA. Philip Biggin1. 1 The P2X family of receptors are trimeric ion channels that open in response to Department of Biochemistry, Oxford University, Oxford, United Kingdom, binding of ATP to the extracellular surface of the protein. There are seven 2Integrated Program in Neurosciences, McGill University, Montreal, QC, 3 genes that encode P2X1 to 7 subunits and when expressed alone, all form func- Canada, Department of Pharmacology and Therapeutics, McGill University, tional receptors except P2X6. Structurally, each subunit is comprised of intra- Montreal, QC, Canada. cellular N and C termini, a large extracellular domain containing the ATP It is been shown that homomeric kainate receptors require external ions for binding site and two a-helical transmembrane (TM) domains that form a gating. However, in vivo, kainate receptors are most likely arranged as het- pore permeable to cations such as Kþ,Naþ and Ca2þ. These ligand-gated erotetramers and the extent to which external ions are required for gating is cation channels are expressed throughout the body, but are localized in the cen- less clear. A large amount of structural and biochemical work over the years tral nervous system, peripheral nervous system, skeletal, smooth, cardiac mus- has indicated that agonists bind to the ligand-binding domain (LBD), which cle, immune cells and the auditory system. P2X2 receptors are hypothesized to is arranged as a dimer of dimers as exemplified in homomeric structures. play a role in cochlear adaptation to elevated sound levels and protection from However, no high-resolution structure currently exists of heteromeric overstimulation. Whole-exome sequencing of individuals with dominantly kainate receptors and in a full-length heterotetramer, the LBDs could poten- inherited progressive sensorineural hearing loss (DFNA41) revealed three tially be arranged either as a GluK2 homomer alongside a GluK5 homomer different mutations of P2X2 receptors: V60L, D273Y and G353R. We are or as two GluK2/K5 heterodimers. To investigate this further we built studying how these mutations influence the functional properties of P2X2 re- models of the LBD dimers based on the GluK2 LBD crystal structures ceptors, and we will present results showing that two of these produce distinct and investigated their stability with molecular dynamics simulations. The alterations in how these channels gate. models were then used to make predictions about the functional behavior of the full-length GluK2/K5 receptor including the anticipation that lithium 638-Pos Board B408 ions would bind to the dimer interface of GluK2/K5 heteromers and Magnesium Modulation of P2X Receptor Channels slow their desensitization. These predictions were confirmed via electro- Mufeng Li, Shai D. Silberberg, Kenton J. Swartz. physiological recordings giving high confidence in the models suggesting NINDS, NIH, Bethesda, MD, USA. they find future use in the absence of high-resolution crystallographic or P2X receptors are cation channels that are activated by extracellular ATP. NMR data. Seven subtypes of P2X receptors are widely expressed in virtually every tissue,

BPJ 8573_8577 Sunday, February 18, 2018 127a and they play important roles in sensory signaling and inflammation. It is well rescent rim. There is a rim of bright green fluorescence around the outer known that physiologically abundant divalent cations like Mg2þ can regulate surface of the alveolus in some views. There is also localization of green ion channels through metal-protein interaction. In the case of P2X receptors, fluorescence at the basal surface of some receptor cells (the presynaptic re- Mg2þ also modulates channel function by forming a complex with ATP, which gion). With optimal imaging, the membrane near the receptor cell apex is is ionized in physiological solutions. Previously, we have demonstrated that visible and appears yellow, which suggests staining for both BK and both MgATP2- and ATP4- can bind to the slow-desensitizing P2X2 receptors, Cav1.3. Refinement of these methods should lead to additional insights con- but only ATP4- can activate these channels. Therefore, Mg2þ behaves as a cerning electroreception. competitive antagonist for P2X2 receptors through chelating ATP4-.In contrast, both free and Mg2þ bound ATP can bind and effectively activate 641-Pos Board B411 fast-desensitizing P2X3 receptors. The mechanism of this subtype specific Crystal Structure of a Mycobacterial RCK Domain 1 1 1 MgATP2- activation is unknown. Recently published crystal structures of Alexandre G. Vouga , Katia K. Matychak , Michael E. Rockman , 2 2 1 hP2X3 receptors with Mg2þ (or Mn2þ) bound revealed a cation binding site Sebastian Garcia , Sebastian Brauchi , Brad S. Rothberg . 1Department of Medical Genetics and Molecular Biochemistry, Temple near the ATP binding pocket. We are currently investigating the functional con- 2 sequences of Mg2þ binding to this site, and whether it is responsible for the University School of Medicine, Philadelphia, PA, USA, Department of subtype specific MgATP2- activation. Physiology, Universidad Austral de Chile, Valdivia, Chile. Potassium (Kþ) channels play a critical role in bacterial electrolyte homeostasis 639-Pos Board B409 and electrical signaling. In mycobacterial pathogens (including M. tubercu- Role of the Left Flipper Domain in the Homotrimeric Assembly and Func- losis), Kþ homeostasis is regulated in part by KefC, a multimeric Kþ tion of P2X Receptors channel-like protein, with each subunit consisting of a transmembrane pore Angela Hein1, Achim Kless2, Ralf Hausmann1, Fritz Markwardt3, tethered to an RCK domain. Understanding the structure and function of these Gunther€ Schmalzing1. channels may lead to novel therapeutics to combat the spread of mycobacterial 1Molecular Pharmacology, RWTH Aachen University, Aachen, Germany, infection in humans. Here we present the X-ray crystal structure of the Myco- 2Grunenthal€ Innovation, Drug Discovery Technologies, Grunenthal€ GmbH, bacterium intracellulare KefC (MycK) RCK domain in complex with ATP at Aachen, Germany, 3Julius Bernstein Institute for Physiology, Martin Luther 2.8 A˚ resolution. The crystal structure shows that ATP is coordinated by a University, Halle/Saale, Germany. hydrogen bond between N6 (on the adenine base) and the side chain of Asp- P2X receptors (P2XRs) are trimeric ATP-gated channels (Nicke et al. EMBO J. 200, a pair of hydrogen bonds between O2’ and O3’ (on the ribose sugar) 17, 3016-3028, 1998) that assemble from a repertoire of seven subunits, P2X1- and the two oxygens on side chain of Asp-180. In addition, an intersubunit P2X7. ATP binding triggers the opening of an intrinsic ion channel that allows salt-bridge is formed between the gamma phosphate of ATP and the side chain for the non-selective passage of cations along their electrochemical gradients. of Arg-249 in the neighboring subunit, which may be key to stabilizing the acti- P2X6 subunits co-assemble with other P2XR subunits to functional heterotri- vated conformation. Electrophysiological recordings from purified MycK meric channels, but are incapable of forming functional homotrimers. In Xen- incorporated in planar lipid bilayers demonstrate that ATP can activate opus laevis oocytes, we have previously found that singly expressed P2X6 MycK with an EC50 of 6 mM. We have begun to estimate affinity of ATP subunits form random homooligomers, mostly tetramers, rather than defined to the purified MycK RCK domain using the fluorogenic ATP derivative homotrimers (Aschrafi et al. JMB 342, 333-343, 2004). This suggested to us TNP-ATP; these measurements yielded an apparent Kd of 30 mM. TNP-ATP that P2X6 subunits have a genuine assembly defect. A sequence alignment fluorescence is inhibited in the presence of ATP, ADP, and AMP, yielding based on the X-ray structure of the zebrafish (zf) zfP2X4R disclosed that IC50 values of 10, 21, and 20 mM, respectively, whereas cAMP and NAD P2X6 subunits lack nine residues forming the so-called left flipper domain; did not significantly inhibit TNP-ATP fluorescence at concentrations up to 1 this may compromise subunit-subunit contacts and thus explain the inability mM, suggesting that the binding site is selective for non-cyclic purine mono- of forming functional homotrimers (Kawate et al. Nature 460, 592-599, nucleotide phosphates. Together these results are consistent with MycK acting 2009). Here, we analyzed the role of the left flipper domain in the homotrimeric to regulate bacterial Kþ homeostasis or membrane potential as a function of en- assembly and ATP-gated channel function by site-directed mutagenesis, blue ergy metabolism. native polyacrylamide gel electrophoresis (BN-PAGE) and two-electrode voltage-clamp electrophysiology (TEVC). We found that the insertion of resi- 642-Pos Board B412 The Role and Determinants of Biphasic Regulation of SK Channels by dues 281-287 or 290-297 of the rP2X1 or hP2X2 subunit, respectively, into the 2D rP2X6 subunit neither enabled efficient homotrimerization nor ATP-gated Ca in Hypertrophic Rat Ventricular Cardiomyocytes 1 1 1 1 channel function. A deletion of the appropriate left flipper residues from Radmila Terentyeva , Iuliia Polina , Shanna Hamilton , Kevin R. Murphy , 1 2 rP2X1, hP2X2, hP2X3 or rP2X7 did not prevent homotrimeric assembly, but Gideon Koren , Dmitry Terentyev . 1Medicine, Brown University and Rhode Island Hospital, Providence, RI, interfered with the function of the P2XRs as ATP-gated ion channels. The 2 respective left flipper deletions decelerated the desensitization of the rP2X1R USA, Medicine, Rhode Island Hospital, Providence, RI, USA. Small conductance calcium (Ca2þ)-activated Kþ channels (SK) have been es- and the hP2X3R, while they abolished the functions of the hP2X2R and the 2þ rP2X7R. These data will be discussed in terms of the known P2XR X-ray tablished as mediating the feedback between intracellular Ca dynamics and structures. sarcolemma repolarization in ventricular myocytes (VMs) from heart failure patients. Constitutively bound calmodulin mediates Ca2þ-dependent activa- 640-Pos Board B410 tion of SK channels (EC50 500 nM), while channels are also inhibited by 2þ 2þ Confocal Microscopy of Skate Electroreceptors: Fluorescent Antibodies Ca (IC50 20 mM) in a voltage-dependent manner. Given that [Ca ]i Used to Localize Cav1.3 and BK Channels changes dramatically during cardiac contraction and relaxation in specific sub- William T. Clusin. cellular compartments, we sought to determine the exact location and identity Medicine, Stanford Medical Center, Stanford, CA, USA. of Ca2þ sources for SK channel activation and inhibition using the rat model Skate electroreceptors are exquisitely sensitive to small voltage changes in of hypertrophy induced by thoracic aortic banding (TAB). Simultaneous the environment. This sensitivity is due to co-localization of voltage- confocal Ca2þ imaging and whole-cell voltage-clamp experiments showed sensitive calcium channels (Cav1.3) and large calcium-activated Kþ chan- that the component of integral current sensitive to specific SK inhibitor Apa- nels (BK) in the apical membranes of the receptor cells. Regenerative re- min (APA, 100 nM), ISK, demonstrates strong rectification with IV peak at sponses depolarize the basal membranes, which form synapses with 20 mV. Importantly, at 0 mV where Ca2þ transient amplitude reaches 2þ afferent nerve fibers and have presynaptic calcium channels. We character- maximum, ISK was undetectable indicative of Ca /voltage-dependent block. ized this localization using fluorescent antibodies against conserved Accordingly, APA application affected action potential waveform only in 2þ Cav1.3(a)andBK(a) sequences. Single ampullae were dissected and fixed phase 3 at voltages below 0 mV in current-clamped VMs when Ca transient with paraformaldehyde and treated with detergent. Images were initially was in decline. Under b-stimulation with isoproterenol APA induced arrhyth- obtained with a stereomicroscope. Both antibodies caused bright staining mogenic early-afterdepolarizations. In Blue-Native Page experiments, anti-SK of the receptor epithelium. For confocal microscopy, both primary anti- antibody recognized a protein band of similar molecular weight as a1c sub- bodies were applied together. Stacks of 1mm merged and single color im- unit of L-type Ca2þ channel (LTCC) antibody suggesting complex formation, ages were obtained at 10x and 40x. One alveolus filled the field at 40x. further confirmed by proximity ligation assay. Immunolocalization study re- Staining of Cav1.3 produced green images, while staining of BK produced vealed a low level of colocalization between SK/LTCC complexes and red images. Receptor cells were most clearly identified as a monolayer of RyR2 channels (<20%). Taken together, these data suggests that a large in- reddish disks, presumably nuclei, which harbor BK (Li et al 2014). Some crease in [Ca2þ] in the sub-membrane space during RyR2-mediated Ca2þ cell images do not show the nucleus but appear as a black void with a fluo- release is sufficient to effectively inhibit SK channels at Vms>0mV.

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LTCC-associated SKs are activated by a small LTCC-mediated Ca2þ influx in myocytes from both sexes and, the shortening was significantly greater in phase 3 of AP to counter its depolarizing force, thereby reducing early exercise-trained compared to sedentary groups. The KATP inhibitor glibencla- afterdepolarizations. mide (2 mM) prolonged the 10Hz APD more in exercise-trained than control rats. Regular exercise enhanced KATP outward current density in apex myo- 643-Pos Board B413 cytes from both sexes; in base, trained females exhibited larger KATP inward Molecular Basis of the Nucleotide-Dependent Conformational Change in current than controls. In both sexes, Kir6.2 expression was elevated by an RCK Domain wheel-running in apex but not base region. Exercise-training increased Celso M. Teixeira-Duarte,Fa´tima Fonseca, Joa˜o H. Morais-Cabral. SUR2A density in base region of both sexes, and eliminated its reginal dif- i3S/IBMC, Porto, Portugal. þ ference in females. In conclusion, exercise-training induced APD shortening Potassium (K ) is the major inorganic intracellular cation in cells, playing a under energy demanding conditions (e.g. high HR) is caused at least in part fundamental role in essential physiological processes. Regulation of Kþ trans- by KATP up-regulation, an adaptation that reduces energy requirements for port is therefore a tightly regulated process in all organisms, mediated mainly ion homeostasis and maintains a diastolic interval adequate for myocardial by channels and transporters. In several of these proteins, potassium flux is regu- relaxation. lated through a cytoplasmic RCK (regulator of Kþ conduction) domain. Ligand- binding to this domain results in a conformational change that propagates to the 646-Pos Board B416 membrane subunit of the system and controls its activation state. The KtrAB po- Gating Mechanism Investigation in Homotetramer CNGA1 Ion Channel tassium transporter is composed by a homodimeric membrane protein, KtrB, and by Coarse-Grained Molecular Dynamics Simulation an octameric cytosolic RCK ring, KtrA. KtrA adopts different conformations Mangesh V. Damre1, Alejandro Giorgetti2, Vincent Torre1. upon binding of ATP or ADP, resulting in a high or low-activity state of KtrAB, 1Neurobiology, International School for Advanced Studies (SISSA), Trieste respectively. Comparison of the X-ray structures of KtrA-ADP and KtrA-ATP (Italy), Trieste, Italy, 2Department of Biotechnology, University of Verona, shows a symmetric contraction of the RCK ring in the presence of the activating Verona, Italy. ligand (ATP). The ATP induced conformation appears to be mediated by inter- Cyclic nucleotide-gated (CNG) ion channels are activated by cyclic nucleo- subunit ATP g-phosphate interactions but the nature of these interactions is un- tides. Crystal structure of TAX4 (PDB id: 5H3O) gives the insight about the clear. In this work we strived to understand how the conformational change in the open state conformation of the CNGA1 ion channel in presence of cyclic gua- RCK domain occurs. We have explored the role of binding site residues and nosine monophosphate (cGMP) molecule. Although, Structural studies have of divalent and monovalent cations in the stabilization of the activated state. identified the channel pore and selectivity filter, conformation changes associ- We present a novel conformational regulatory mechanism for nucleotide- ated with gating remain poorly understood. Here we use coarse-grained dependent RCK domains, which depends on the interplay between nucleotide molecular dynamics simulation by applying martini forcefield parameters on and cation binding to control ion flux. homotetrameric homology model of bovin CNGA1 channel. We studied 644-Pos Board B414 conformational changes from the open - in the presence of cGMP - to the closed A Mutation in the Intrinsivally Disordered Fragment of SK2 Channel state - in the absence of cGMP -conformation of CNGA1 channel embedded in Confers Ca2D Hypersensitivity the membrane environment. Despite of the low specificity of the martini Young Woo Nam1, Saba N. Baskoylu2, Meng Cui3, Razan Orfali1, coarse-grained forcefield, the open and closed state conformations of Anne C. Hart2, Miao Zhang1. CNGA1 channels are in agreement with several electrophysiological experi- 1School of Pharmacy, Chapman University, Irvine, CA, USA, 2Department ments with mutant CNGA1 channels. These results suggest that upward of Neuroscience, Brown University, Providence, RI, USA, 3School of movement of cyclic nucleotide binding domain (CNBD) following binding Pharmacy, Northeastern University, Boston, MA, USA. of cGMP induces conformational changes in the S4, S5, p-helix and selectivity Small-conductance Ca2þ-activated potassium (SK) channels play an essential filter leading to the opening of CNGA1 channels. role in the regulation of neuronal excitability. There are three subtypes of SK channels expressed in the neurons, with SK2 and SK3 predominant in the motor 647-Pos Board B417 neurons. Elevated intracellular Ca2þ levels exclusively activate the SK chan- Functional and Spectroscopic Studies of Prokaryotic Cyclic Nucleotide- nels. The Ca2þ-binding protein calmodulin (CaM) is constitutively associated Gated Ion Channels 1,2 1 1 1,2 with SK channels at the CaM binding domain and serves as the Ca2þ sensor Eric G.B. Evans , Jacob L.W. Morgan , Zachary M. James , Stefan Stoll , 1 of the channels. An intrinsically disordered fragment connects the CaM binding William N. Zagotta . 1Physiology and Biophysics, University of Washington, Seattle, WA, USA, domain and the transmembrane domain S6. Here, we report that a phenylala- 2 nine mutation in the intrinsically disordered fragment caused a 6-fold Chemistry, University of Washington, Seattle, WA, USA. increase in the Ca2þ sensitivity of SK2-a channels. Also, equivalent phenylal- Cyclic nucleotide-gated (CNG) channels and the related hyperpolarization- anine mutations in SK1 or SK3 channels conferred Ca2þ hypersensitivity. activated cyclic nucleotide-gated (HCN) channels belong to the Kv superfam- Additionally, an equivalent phenylalanine substitution in the Caenorhabditis ily of voltage-gated ion channels and are activated by cyclic nucleotide bind- elegans SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing ing to an intracellular cyclic nucleotide-binding domain (CNBD). These C. elegans ALS model, in which human SOD1G85R is expressed at high levels channels play fundamental roles in physiological processes including in neurons. Combined, these results provide the most direct functional and mammalian visual and olfactory signal transduction as well as cardiac pace- behavioral evidence that activation of SK channels ameliorates defects in making and neuronal rhythmicity. However, the molecular mechanism by ALS models. which ligand binding is coupled to channel activation is currently unknown. In the past year, several high-resolution structures of intact CNG/HCN chan- 645-Pos Board B415 nels have been reported. These structures reveal key features of channel archi- Role of ATP Sensitive Potassium Channel in Exercise-Training Mediated tecture but have contributed little to our understanding of cyclic nucleotide Adaptations in Ventricular Repolarization regulation, in part because they are static pictures that are difficult to associate Xinrui Wang, Robert H. Fitts. with functional states. To better probe the conformational changes and ener- Marquette, Milwaukee, WI, USA. getics that accompany cyclic nucleotide regulation in CNG/HCN channels, we Regular exercise-training is known to reduce the incidences and severity of have turned to a recently-discovered family of prokaryotic CNG orthologs. ischemic heart disease, but the mechanistic nature is unknown. We showed We expressed different prokaryotic channel constructs in giant E. coli sphero- that regular exercise shortened the left ventricular action potential duration plasts and used patch-clamp electrophysiology to screen for constructs with (APD) at high heart rate (HR), which would prevent Ca2þ overload and/or properties suitable for biophysical characterization. In order to correlate func- inadequate filling under conditions of metabolic stress. Here we test the hy- tional states - as observed with electrophysiology - with the conformational pothesis that exercise-training induced APD shortening at high HR is due to ensemble of channel structures, we generated cysteine-free constructs and the up-regulation of ATP sensitive potassium channel (KATP). Female and introduced nitroxide spin labels for double electron-electron resonance male Sprague-Dawley rats were randomly assigned to voluntary wheel (DEER) spectroscopy. Intersubunit distance distributions obtained by DEER running or control groups. After 6-8 weeks training, cardiac myocytes in detergent solution reveal conformational changes in the presence of acti- were isolated from the apex and base regions of the left ventricle with colla- vating cyclic nucleotide. These distributions allow us to characterize the struc- genase–protease dispersion technique. APD was measured with glass micro- tural rearrangements that occur during cyclic nucleotide-dependent activation electrode, current-voltage relationship was recorded with discontinuous sin- of CNG/HCN channels. The combination of DEER, electrophysiological mea- gle electrode voltage clamp, and the expression level of KATP channel pore- surements in spheroplasts, and mutational analysis, are now enabling a more forming subunit Kir6.2 and regulatory subunits SUR2A were determined by detailed understanding of the structures and energetics of this important class Western blots. At 1Hz, KATP activator pinacidil (100 mM) shortened APD in of ion channels.

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648-Pos Board B418 the amplifier is reduced to less than 4 pF. This measurement configuration Functional Characterization of the Cyclic Nucleotide-Gated Channel SthK allows us to measure the type-1 ryanodine receptor (RyR1) at bandwidths Xiaolong Gao, Philipp A.M. Schmidpeter, Crina M. Nimigean. up to 500 kHz. We are able to observe channel gating events at temporal res- Weill Cornell Medicine, New York, NY, USA. olutions as short as 2 ms. Further, single channel recordings at these band- Cyclic nucleotide-modulated (CNG and HCN) ion channels play essential widths for different calcium concentrations provide heretofore unavailable roles in various physiological processes, such as, photon detection in pho- details about the kinetics of activation and inactivation processes in RyR1. toreceptors, odorant sensation by olfactory neurons and pace making activ- Specifically, we are able to separate multiple closed and open states that ity in the heart and brain. Previous studies provided many insights into the are indistinguishable at bandwidths less than 10 kHz. Additional data anal- function and the regulation of these channels, but little is known about the ysis with extended beta distributions allows us to report time constants asso- function of purified channel proteins in a defined environment. Here we pre- ciated with channel gating events as short as 35 ns. These time constants sent the expression, purification and functional characterization under are on the order of single-file transition events and have the potential to defined conditions of a bacterial cyclic nucleotide-gated potassium channel, link single-channel recordings with state-of-the-art molecular dynamics SthK, from Spirochaeta thermophila. Using lipid bilayer electrophysiology simulations. and stopped-flow Tlþ flux assay measurements of purified channels recon- stituted in liposomes, we show that cAMP alone can activate SthK, with an 651-Pos Board B421 activation time constant of about one second in saturating cAMP. In the Comparing Ion Transport between a Chloride Channel and a Phospho- absence of cAMP, the channel is always closed, while application of lipid Scramblase in the TMEM16 Family cGMP alone leads to very low but non-zero SthK channel activity, suggest- Dung M. Nguyen, Louisa S. Chen, Wei-Ping Yu, Tsung-Yu Chen. ing that cGMP can also activate the channel albeit less efficiently than University of California, Davis, Davis, CA, USA. cAMP. Furthermore, in the presence of saturating cAMP, increasing con- The TMEM16 family of transmembrane proteins consists of dimeric mole- cules acting as ion channels and phospholipid scramblases. High-resolution centrations of cGMP lead to a decrease in SthK channel activity, indicating 2þ that the two ligands compete for the same binding sites, as previously structures of mouse TMEM16A, a Ca -activated chloride channel, and shown. In addition, single channel recordings in planar lipid bilayers with nhTMEM16, a phospholipid scramblase, suggest that the hypothesized saturating cAMP show that SthK is inwardly-rectifying and opens only ion-transport pathways of these two molecules may be similar. Electrophys- briefly and infrequently with hyperpolarized membrane potentials while iological experiments showed that TMEM16A conducts mostly anions longer and more frequent openings are observed with increasingly depolar- while TMEM16F, also a phospholipid scramblase like nhTMEM16, ized membrane potentials, suggesting channel activity is also modulated by conducts both anions and cations. The structural basis of different ion voltage, in addition to cAMP. Our initial functional characterization shows selectivity between a mammalian chloride channel and a phospholipid that SthK is a good model system to understand gating and ligand modula- scramblase is not known. Here, we compare the ion transport between tion in cyclic nucleotide-modulated channels. TMEM16A and TMEM16F using excised inside-out patch clamp record- ings. We found the side-chain charge of the amino acid placed at position 649-Pos Board B419 584 of TMEM16A appears to exert an electrostatic effect on the rectifica- Quantitative Proteomic Analysis of the Putative NAADP-TPC Signaling tion of channel’s I-V curve. On the other hand, the charge of the corre- Complex sponding residue at position 559 in TMEM16F has very little effect on Jiyuan Zhang, Xin Guan, Qin Li, Jiusheng Yan. the current rectification. However, mutating Q559 of TMEM16F into aro- Anesthesiology and Perioperative Medicine, MD Anderson Cancer Center, matic amino acids significantly reduces the degree of outward current recti- Houston, TX, USA. fication of this phospholipid scramblase. Furthermore, the Q559K mutation Nicotinic acid adenine dinucleotide phosphate (NAADP)isthemost of TMEM16F slightly increases the permeability ratio of Cl- versus Naþ 2þ potent known Ca -mobilizing second messenger which uniquely mobilizes (PCl/PNa). These results show that K584 in TMEM16A and the correspond- þ Ca2 from acidic endolysosomal organelles. Despite the importance ing residue, Q559, in TMEM16F may be located at a comparable position in þ of NAADP-evoked Ca2 signaling, the molecular identities of the NAADP their respective ion-transport pathways. The observation that aromatic side- þ receptors and Ca2 -release channels involved in this process have yet to chain at position 559 significantly decreases current rectification of be unequivocally defined. Accumulated evidence indicates that the TMEM16F may suggest a participation of lipid molecules in the ion trans- poorly understood two-pore channels (TPCs) participate in NAADP- port across this phospholipid scramblase. evoked Ca2þ release. However, how TPCs are involved in NAADP- 652-Pos Board B422 signaling remains largely unknown or debatable. A more unified hypothesis D is that TPCs are not directly involved in NAADP binding but are part of the Towards the Identification of the Neuropeptide Binding Site of Hydra Na NAADP receptor/Ca2þ-release channel complex, in which the molecular Channels (HyNaCs) identities of other key proteins remain unknown. To dissect the molecular Axel Schmidt, Katrin Augustinowski, Marc Christopher Assmann, € components of the putative NAADP-TPC signaling complex, we performed Stefan Grunder. Institute of Physiology, RWTH Aachen University, Aachen, Germany. affinity purification using both TPCs and NAADP as baits to purify the þ signaling complex in HEK-293 and SKBR3 cells. We employed stable The DEG/ENaC (Degenerin/Epithelial Na Channel) ion channel family is isotope labeling with amino acids in cell culture (SILAC)-based quantitative comprised of channels that are activated by diverse stimuli such as mechanical proteomic approach to specifically identify the TPC and NAADP interacting stress, peptides or other ligands. The best known ligand-activated family mem- bers are the Acid-Sensing Ion Channels (ASICs), which are activated by pro- proteins in these cells. Upon systematic investigation, we have identified þ large and comprehensive datasets of TPC-interactome and NAADP- tons. Interestingly, the closely related Hydra Na channels (HyNaCs) from interactome. the freshwater polyp Hydra magnipapillata are activated by much more com- plex ligands, namely the neuropeptides Hydra RFamides I and II. The bona fide 650-Pos Board B420 ligand binding sites of both ASICs and HyNaCs are unknown. In this study, we CMOS-Integrated Electrophysiology and Data Analysis by Extended aimed to identify the neuropeptide binding site on heteromeric HyNaC2/3/5 to BETA Distributions Reveal Nanosecond Closed State Flickering of the explore how ligands activate this DEG/ENaC ion channel. To identify the bind- TYPE-1 Ryanodine Receptor ing site, we combined molecular modelling with site-directed mutagenesis. Peijie Ong1, Andreas J. Hartel1, Indra Schro¨der2, M Hunter Giese1, Mutant ion channels were subsequently expressed in Xenopus laevis oocytes Siddharth Shekar1, Oliver Clarke1, Andrew Marks1, Wayne Hendrickson1, and Hydra RFamide I affinity was determined by two-electrode voltage clamp. Kenneth L. Shepard1. Molecular modelling suggested a Hydra RFamide I binding site in a region on 1Columbia Unviersity, New York City, NY, USA, 2Technical University of subunit 3 of a HyNaC2/3/5 homology model that corresponds to the acidic Darmstadt, Darmstadt, Germany. pocket described in the crystal structure of ASIC1. When four residues of Hy- The temporal resolution of single ion channel measurements is typically NaC3 surrounding the suggested binding site were mutated individually, the af- limited due to signal-to-noise constraints imposed by the measurement elec- finity of Hydra RFamide I was strongly (3.5 to >100-fold for each mutant) tronics. In particular, the parasitic capacitance of the membrane system com- reduced. In contrast, less conservative mutations of the corresponding bined with the input capacitance of the amplifier reduces achievable residues in HyNaC2 or HyNaC5 left the peptide affinity relatively unaffected bandwidths to less than 100 kHz (often to as little as 10 kHz). Here, by using (<3.5-fold reduced for all mutants). In summary, our results suggest that Hydra suspended membranes with areas less than 700 mm2 fabricated directly on RFamide I binds to a single binding site on subunit 3 of HyNaC2/3/5 in a region top of a custom preamplifier chip built in complementary metal-oxide- homologous to the acidic pocket of ASIC1, highlighting the importance of this semiconductor (CMOS) technology, the total capacitance at the input of region for ligand binding in DEG/ENaC ion channels.

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653-Pos Board B423 of new and improved ASIC-modulating compounds. In this study, we aimed to Inhibitor-Induced Conformational Changes in ASIC1A decipher the molecular basis of peptide modulation of ASICs by employing a Camilla Lund, Christian B. Borg, Timothy Lynagh, Stephan A. Pless. combination of molecular biology, electrophysiology, and synthetic peptide Department of Drug Design and Pharmacology, University of Copenhagen, chemistry. Synthetic peptides and peptide analogs were produced by solid- København Ø, Denmark. phase peptide synthesis and their effects on ASICs were measured by two- Acid-sensing ion channels (ASICs) are involved in acid-induced neuronal electrode voltage clamp electrophysiology. In addition, a series of ASIC conca- injury during pathological conditions in the central nervous system, such as sei- temers were constructed to allow for the introduction of mutation(s) into one or zures and ischemic brain injury. These cation-permeable membrane proteins more specific ASIC subunits. By evaluating the effects of modified ASIC- are also involved in several pain-related mechanisms in the periphery. modulating peptides on both wild-type, mutated and concatenated ASICs, we Understanding their function and pharmacology is therefore of great interest. have gained detailed insights into the molecular requirements for peptide modu- ASICs are activated by protons and inhibited by both small molecule com- lation of ASIC function. Information gained from this study will allow the rational pounds, such as ibuprofen, and peptides like the tarantula toxin psalmotoxin design of novel peptides with improved potency and selectivity in the future. 1 (PcTx1). In this project, Voltage-Clamp Fluorometry (VCF) is used to track the conformational changes of mASIC1a in response to exposure to both Posters: Ion Channel Regulatory Mechanisms I ibuprofen and PcTx1. We show that ibuprofen induces global, concentration- dependent conformational changes in the extracellular domain of ASIC1a. 656-Pos Board B426 These ibuprofen-induced conformational changes appear to be distinct from Intragenic Rescue of the Function of Long QT Syndrome-Causing hERG those induced by channel opening and/or desensitization and are therefore Mutant Channels likely to be compound-specific conformational changes. Furthermore, mutation Jordan H. Davis, Jun Guo, Shetuan Zhang. of a residue critical to functional ibuprofen inhibition, K422, rendered the chan- Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, nel less sensitive to ibuprofen, but did not affect the concentration-dependence Canada. The human-ether-a-go-go-related gene (hERG) encodes the pore forming sub- of the ibuprofen-induced conformational changes. These observations might þ help decipher the recently proposed allosteric inhibition mechanism of unit of the rapidly activating delayed rectifier K current (IKr) which is impor- ibuprofen. The peptide toxin PcTx1 inhibits ASICs by inducing steady-state tant for the repolarization phase of cardiac action potentials. A reduction in IKr desensitization in ASIC1a. Here we show that it induces global conformational due to a loss of hERG function can lead to long QT syndrome (LQTS). There changes in the extracellular domain that are modulated by the proton concen- are a variety of loss-of-function hERG mutations known to cause LQTS, the tration in the extracellular solution. Mutations of F350 have previously been majority of which are thought to be trafficking deficient, including G601S. It shown to abolish PcTx1 inhibition, but our VCF work shows that the PcTx1- has been shown that the plasma membrane (PM) expression of the G601S induced conformational changes persist despite severely reduced PcTx1 inhibi- hERG mutant can be rescued by reduced temperature culture. We have shown that wild-type (WT) hERG channels require extracellular Kþ for their stability tion of the mutant channel. Similar to our observations with ibuprofen, this þ strongly suggests that Pctx1 still binds to and interacts with ASIC1a, despite in the PM. They undergo internalization under 0 mM K culture conditions, which can be prevented by reduced temperature culture. We also identified the mutation-induced reduction of channel inhibition. Together, these findings þ provide new insight on inhibitor-induced conformational changes in ASIC1a. that the S624T mutation makes hERG insensitive to extracellular K , thereby preventing hERG channel internalization under 0 mM Kþ culture conditions. 654-Pos Board B424 We hypothesize that certain hERG mutants, including G601S, may not be Evolution of Acid-Sensing Ion Channels able to sense extracellular Kþ, making them unstable in the PM, leading to a Timothy Lynagh, Janne M. Colding, Stephan A. Pless. loss of PM expression. In this study, we added a secondary mutation, S624T, University of Copenhagen, Copenhagen, Denmark. which does not depend on extracellular Kþ for its PM expression, to the Each major lineage in the animal kingdom possesses genes from the ENaC/ G601S mutant hERG channel. The addition of S624T suppressed the loss-of- DEG channel family, a broad family of functionally diverse ion channels. A function G601S phenotype, and rescued its current to a level similar to WT small sub-family is that of the acid-sensing ion channels (ASICs), proton- channels. The addition of S624T also rescued LQTS-causing hERG mutants gated sodium channels that contribute to the neuronal signals underlying, for T474I and P596R. Our data reveals the most effective intragenic rescue of example, learning and nociception in mammals. Although ASICs have only LQTS-causing hERG mutants to date and lends insight into the mechanisms been described in vertebrates, it seems peculiar that such a signaling protein through which these mutations confer the loss-of-function phenotype. should be absent from lower organisms that also express such fundamental behavioral phenotypes. The identification of primitive ASICs has perhaps 657-Pos Board B427 been hampered by our poor understanding of the molecular mechanism of Regulation of hERG C-terminal Isoform Expression by KCNH2 Intronic proton-sensing. Here, we use molecular phylogenetics, conventional mutagen- Elements esis, unnatural amino acid incorporation and electrophysiological recordings to Matthew R. Stump1, Sequoyah N. Tate1, Rachel T. Nguyen1, explore proton-sensing in a broader sample of ASICs than previously possible, Anastasiya V. Goldys-Olson2, Qiuming Gong2, Zhengfeng Zhou2. 1Department of Biology, George Fox University, Newberg, OR, USA, in light of recent genomic data. We find that ASICs are not confined to the 2 vertebrate lineage, and a comparison of this extended ASIC family with other Knight Cardiovascular Institute, Oregon Health & Science University, ENaC/DEG channels points towards somewhat surprising determinants of Portland, OR, USA. proton-sensing. Using unnatural amino acids, we were able to dissect some The KCNH2 gene encodes the hERG potassium channel that conducts the rapidly of the chemical interactions behind the molecular mechanism of proton- activating delayed rectifier current in the heart. Two C-terminal hERG isoforms sensing. Finally, we provide an explanation for the loss of proton sensitivity are expressed in the heart. Splicing of KCNH2 intron 9 leads to the formation of a in ASIC4 of the mammalian lineage. Together, this combined evolutionary full-length, functional hERGa isoform and polyadenylation of intron 9 results in and biochemical approach throws new light on the mechanism of proton- the production of a non-functional, C-terminally truncated hERGa-USO isoform. sensing and the distribution of ASICs within the ENaC/DEG family. In the heart, only one-third of KCNH2 pre-mRNA is processed to hERGa due to inefficient intron 9 splicing. A unique feature of KCNH2 intron 9 is the presence of 655-Pos Board B425 a 25 nt adenosine stretch immediately downstream of the weak, noncanonical, Peptide Modulation of Acid-Sensing Ion Channels polyadenylation site, AGTAAA. Interestingly, non-human primate species Christian B. Borg, Linda M. Haugaard-Kedstro¨m, Timothy Lynagh, contain the AGTAAA poly(A) site, but differ in the length of the downstream Kristian Strømgaard, Stephan A. Pless. poly(A) stretch. It is not known how the length of the adenosine stretch effects Department of Drug Design and Pharmacology, University of Copenhagen, the alternative processing of KCNH2 and the generation of C-terminal hERG iso- Copenhagen, Denmark. forms. To test the effect of adenosine stretch length on alternative processing, the Acid-sensing ion channels (ASICs) are homo- or heterotrimeric proton-gated KCNH2 intron 9 poly(A) stretch was changed from 25 nt to 13 nt and 33 nt, cor- cation channels which are involved in numerous biological processes, including responding to the sequences of the marmoset and baboon. In the presence of a 13 initiation of pain and neuronal death associated with ischemic stroke. A number of nt adenosine stretch, intron 9 splicing outcompeted polyadenylation and resulted neuropeptides and peptide toxins have been shown capable of modulating ASIC in two-thirds of KCNH2 pre-mRNA being processed to hERGa. Introducing the function and activity. Thus, these peptides have great potential as therapeutics for 33 nt adenosine stretch into intron 9 also resulted in increased hERGa expression the treatment of these disorders, as well as molecular tools for the understanding and concomitantly decreased expression of the hERGa-USO isoform. Whole-cell and control of ASIC function. Despite the recent advances in structure determina- patch clamp analyses showed increased hERG current in HEK-293 cells trans- tion of ASIC-peptide complexes, the precise molecular mechanisms underlying fected with full-length KCNH2 gene constructs containing 13 nt and 33 nt aden- peptide modulation of ASICs remain elusive, which hampers the development osine stretches. Although the expression of hERGa-USO has not been previously

BPJ 8573_8577 Sunday, February 18, 2018 131a reported in other species, our present findings suggest that length of the poly(A) both transgenic animal models and heterologous cell lines. These interactions stretch within KCNH2 contributes to the species-specific regulation of hERG are abrogated by cAMP, although cAMP does not directly bind either protein; C-terminal isoform expression. rather, PKA-mediated phosphorylation is hypothesized to be responsible for regulation of these interactions. Nonetheless, the precise mechanisms regu- 658-Pos Board B428 lating hERG-KVLQT1 interactions, as well as their subcellular localization, Differential Regulation of HERG Current and Expression by Activation of remain to be elucidated. As such, there is considerable interest in assessing Protein Kinase C the role of the cAMP/PKA pathway in the trafficking and membrane localiza- Morgan Sutherland-Deveen, Shetuan Zhang. tion of hERG and KVLQT1. In implementing a biotinylation-based surface Queen’s University, Kingston, ON, Canada. protein assay for purification of membrane-localized proteins, we semi- The human ether-a`-go-go-related gene (hERG) encodes the pore-forming alpha quantified surface expression levels of hERG and KVLQT1 and thus deter- subunits of the channel responsible for the rapidly activating delayed rectifier mined the roles played by hERG-KVLQT1 interactions and cAMP in plasma potassium current (IKr) in the heart. Reductions in IKr cause long QT syndrome, membrane expression of these alpha subunit proteins. Characterizing the which predisposes individuals to potentially fatal arrhythmias that can be trig- endogenous mechanisms regulating hERG and KVLQT1 trafficking has broad gered by stress. One link between stress and hERG function is protein kinase C clinical implications and may lead to improved therapies. (PKC) activation. Up to date, PKC activation has been shown to have conflict- ing effects on hERG function. This study investigates how activating PKC with 661-Pos Board B431 phorbol 12-myristate 13-acetate (PMA) affects hERG channels expressed in Regulation of BK Channel Auxiliary Gamma Subunits by N-Glycosylation human embryonic kidney (HEK) 293 cells. We show that chronic (24 hour) Qin Li, Jamie Smith, Karen Yen, Jiusheng Yan. PKC activation increases hERG protein expression intracellularly and in the The University of Texas MD Anderson Cancer Center, Houston, TX, USA. plasma membrane. However, the increased channel abundance is not accompa- BK channels consist of pore-forming, voltage- and Ca2þ-sensing a subunits nied by an increase in hERG current (IhERG). Our data reveal that acute (30 min- and auxiliary b and g subunits. The auxiliary g1-3 subunits potently modulate ute) PKC activation inhibits IhERG, and this effect is dependent on the the BK channel’s function by shifting voltage-dependence of channel activa- N-terminus of the channel. Upon truncation of hERG’s N-terminus, chronic tion by 50 - 150 mV toward the negative voltage direction. All BK channel activation of PKC increases both hERG protein expression and current. The g subunits contain single or multiple consensus N-linked glycosylation sites PKC-mediated increase in hERG expression is partially due to inhibition of in their extracellular LRR domains. We found that inhibition of protein the E3 ubiquitin ligase Nedd4-2, which mediates degradation of hERG chan- N-glycosylation in HEK-293 cells by tunicamycin caused a great reduction nels. Our findings demonstrate that PKC activation regulates hERG in a (g1) or full loss (g2 and g3) of the g subunit’s effectiveness in modulating balanced manner, altering both hERG current and expression. BK channels. We identified the glycosylation sites of N147 in the g1 subunit, N112, N148 and N211 in the g2 subunit, and N82, N111 and N137 in the g3 659-Pos Board B429 subunit to be all necessary for the g subunits’ channel-modulation function. The Role of Glycosylation in hERG Channel Stability and Susceptibility to Blockade of glycosylation at single or multiple sites by mutation (N/Q) Extracellular Proteases caused a decrease in protein size and a conversion of either all or a significant Shawn M. Lamothe, Maggie Hulbert, Jun Guo, Wentao Li, Tonghua Yang, portion of BK channels to be unmodulated by the auxiliary g subunit. Overex- Shetuan Zhang. pression of the g1-N147Q mutant can partially compensate its deficiency in BK Biomedical and Molecular Sciences, Queen’s University, Kingson, ON, channel modulation. We conclude that N-glycosylation plays a critical role in Canada. maintaining the modulatory function of the BK channel auxiliary g subunits, The human ether-a-go-go related gene (hERG) encodes the alpha subunit of the presumably through facilitating the protein’s surface expression. rapidly activating delayed rectifier potassium channel (IKr). A reduction in hERG current delays cardiac repolarization and predisposes affected individ- 662-Pos Board B432 uals to cardiac arrhythmias and sudden death. hERG possesses an unusually Hydrogen Sulfide Regulates the Activity of Mitochondrial Large Conduc- long S5-pore linker compared to other voltage-gated potassium channels, and tance Calcium Activated Potassium Channel (MitoBKCa) undergoes N-linked glycosylation at position 598 which is located in the S5- Agnieszka Walewska, Piotr Koprowski, Adam Szewczyk. pore linker. It has been shown that the non-glycosylated hERG channels are Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of less stable and undergo accelerated degradation compared to glycosylated Sciences, Warsaw, Poland. hERG channels. Additionally, we and others have previously shown that Hydrogen sulfide (H2S) is recognized as an important gaseous signaling mole- hERG/I in hERG-HEK cells and isolated cardiomyocytes are uniquely sus- Kr cule. Mechanism of H2S action is complicated and includes redox reactions, ceptible to proteolytic cleavage in the S5-pore linker by extracellular proteinase persulfide formation with -SH groups of cysteines and sulfide-metal interac- K (PK) and calpain. In the present study, using biochemical, mutagenesis and tions in heme proteins. MitoBKCa is formed by so called DEC splice variant biophysical techniques, we sought to understand the role of N-linked glycosyl- of KCNMA1 gene, which encodes also plasmalemmal BKCa channel. Even ation on hERG channel susceptibility to extracellular proteases. We found that though mitoBKCa and BKCa share common features including large conduc- cell-surface localized non-glycosylated hERG channels were significantly tance, synergetic gating by voltage and Ca2þ, inhibition by heme, activation more sensitive to protease cleavage compared to their glycosylated counter- by carbon monoxide or free fatty acids it seems that in some cases channels parts. The S620T mutation, which is completely resistant to extracellularly differ in their response to regulators. It was previously shown that H2S applied PK, became susceptible to PK cleavage when glycosylation was in- increased plasmalemmal BKCa channel activity in rat pituitary tumor cells hibited. We previously showed that the scorpion toxin BeKm-1 prevents PK and this effect was linked to its reducing action on sulfhydryl groups of the cleavage of hERG channels. In the present study, we found that the BeKm- channel protein. In this study we recorded by patch-clamp the activity of single 1-mediated protection against PK cleavage was no longer present when glyco- mitoBKCa channels derived from mitochondria of astrocytoma U-87MG cells. sylation was prevented. We conclude thatN-linked glycosylation protects We applied NaHS as a hydrogen sulfide donor in various conditions and hERG against protease-mediated degradation, thereby enhancing hERG stabil- observed complex effects of this compound indicating that H2S could have ity in the plasma membrane. more than one site of action in this channel. This work was supported by Polish National Science Center, grant no. 2015/ 660-Pos Board B430 17/B/ NZ1/02496. Investigating Camp-Mediated Protein-Protein Interactions as Modulators of hERG and KVLQT1 Plasma Membrane Expression 663-Pos Board B433 Laurel F. Kinman1, Louise E.O. Darling2. b2 is Not Required for Daytime Activation of BK Currents by L-Type 1Biological Sciences, Wellesley College, Wellesley, MA, USA, 2Biological Calcium Channels Sciences and Biochemistry, Wellesley College, Wellesley, MA, USA. Joshua Whitt, Jenna Harvey, Andrea Meredith. Regulation of the heart’s rhythm is achieved by the propagation of currents Dept. of Physiology, Univ. of Maryland School of Medicine, Baltimore, through membrane-localized cardiac ion channels, dysfunction of which can MD, USA. lead to potentially deadly arrhythmias or cardiac arrest. Indeed, mutations in BK channel accessory b subunits are important for functional coupling to some the genes encoding potassium channel alpha subunit proteins hERG and Ca2þ sources. In the suprachiasmatic nucleus (SCN), two b subunits are ex- KVLQT1, responsible for carrying delayed rectifier currents IKr and IKs respec- pressed; b2 and b4. No differences in BK current properties were observed in tively, have been found to underlie the majority of Long QT syndrome cases. the absence of the b4 subunit, suggesting this modulation is not critical for regu- hERG and KVLQT1 have been previously shown to undergo dynamic physical lating BK current properties. On the other hand, the majority of BK currents re- interactions, and the two currents exhibit mutual functional downregulation in corded from SCN neurons during the day exhibit macroscopic inactivation,

BPJ 8573_8577 132a Sunday, February 18, 2018 demonstrating that the b2 subunit modulates BK current properties. Because residue, such as glycine, valine, and leucine failed to restore significant daytime SCN BK channels depend on multiple Ca2þ channel subtypes for cur- Ca2þ influx after store depletion either as monomers or heterodimers with rent activation, we examined the relationship between BK inactivation and wildtype Orai1.Using different ammonium ion derivatives of increasing Ca2þ sources in the SCN. Inhibiting L-type Ca2þ channels (LTCCs) eliminated size, we found all the nonselective heterodimers showed enlarged pore macroscopic inactivation of BK currents, but inhibition of N/P/Q-type and RyR size compared with the wildtype dimer. Therefore, we conclude that the Ca2þ channels had little effect. The LTCC agonist BayK increased the number of E106Q, E106N and E106D mutations in the heterodimer alter the geometry neurons with inactivating BK currents, suggesting that inactivating BK channels of the channel pore. Both the charge and size of the Glu106 residue are crit- may be preferentially coupled to LTCCs. To test whether b2 was required for BK ical to function of the tightly regulated and highly Ca2þ selective Orai1 channel activation by LTCCs, BK currents were recorded from b2 KO SCNs in channel. Nimodipine. Inhibition of LTCCs reduced the BK current, demonstrating that the b2 subunit is not required for BK channel activation by LTCCs. However, 666-Pos Board B436 compared to WT, the relative inhibition with Nimodipine was changed in b2 Plasma Membrane ORAI1 and Septin Organization during Calcium KO neurons, suggesting b2 may modify the functional coupling. Moreover, Signaling 1,2 1 1 2 application of a cocktail of inhibitors (Nimodipine, Dantrolene, and contoxin- Zachary Katz , Chen Zhang , Ariel Quintana , Bjorn Lillemeier , Patrick G. Hogan1. MVIIC) that completely abolished the BK current in WT neurons, significantly 1 2 attenuated, but did not completely abolish, the activation of BK currents in b2 La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA, Salk KO neurons. These data demonstrate that although LTCC-mediated Ca2þ cur- Institute for Biological Studies, La Jolla, CA, USA. rent is important for inactivation of daytime BK current, the b2 subunit is not The STIM-ORAI-mediated calcium release-activated calcium channel, or required for overall LTCC-dependent BK channel activation in SCN. CRAC channel, is a key source of calcium influx that sustains intracellular Ca2þ signaling and effector function in T lymphocytes. STIM1 in the 664-Pos Board B434 ER membrane senses depletion of ER calcium stores and moves to BK Channels are Activated by Distinct Calcium Sources during Day and ER-plasmamembranejunctions,then recruits plasma membrane ORAI Night in the Circadian Clock channels to the junctions and directly gates the channels. We have shown Joshua Whitt, Beth McNally, Jenna Harvey, Andrea Meredith. previously that septins are necessary for efficient STIM1-ORAI1 cluster Dept. of Physiology, Univ. of Maryland School of Medicine, Baltimore, formation following store depletion. Septins are known to specify diffusive MD, USA. barriers in the plasma membrane and to serve as scaffolds to recruit BK Kþ channels are regulated by depolarization and increases in intracellular signaling proteins, but their detailed role in calcium signaling remains to Ca2þ. Across neurons and muscle, BK channels have been shown to differentially be characterized. Here we utilize live-cell super-resolution microscopy couple to voltage-gated Ca2þ channels L-, P-, Q-, and N-type, as well as to rya- and single-molecule tracking to map ORAI1 relative to STIM1 and nodine receptor (RyR)-mediated release from intracellular Ca2þ stores. In the su- membrane-localized septins. We further investigate the trajectories and prachiasmatic nucleus (SCN), both intracellular free Ca2þ and voltage gated Ca2þ mobility of ORAI1 molecules in the context of septin-deficient cells or channel (VGCC) currents are regulated in a circadian manner, with greater intra- when the interaction between ORAI1 and STIM1 is perturbed by mutagen- cellular Ca2þ found during the day. We used a pharmacological approach to iden- esis. These analyses at high spatial and temporal resolution are providing tify the Ca2þ sources for BK current activation in day and night SCN neurons. We new insight into the modulation of STIM-ORAI signaling by septins and found a 77% reduction in BK current with nimodipine during the day, with less the local plasma membrane environment. effect during the night (10% reduction), suggesting that L-type VGCCs (LTCCs) are the primary Ca2þ source for BK activation during the day. Consistent with 667-Pos Board B437 this, under the same conditions used to record BK current, the LTCC current A Molecular Mechanism for Orai1 Channel Activation by STIM1 was larger during the day than at night (1753 pA/pF and 1052pA/pF, Raz Palty. respectively). Conversely at night, when VGCC currents are reduced, we found Technion, Haifa, Israel. a significant decrease in BK current using dantrolene, blocking Ca2þ release The calcium release activated calcium (CRAC) channel plays a key role in from RyRs (64%). Thapsigargin, which depletes Ca2þ from intracellular stores, diverse cellular functions. Assembly of endoplasmic reticulum-plasma caused a similar reduction in BK current, demonstrating that nighttime BK acti- membrane (ER-PM) bridges between ER-resident STIM1 proteins and vation is primarily driven by intracellular Ca2þ release through RyRs. BK cur- PM-resident Orai1 proteins translate calcium depletion in ER into Orai1 rents were reduced by conotoxin-MVIIC to a similar extent between day and channel opening and replenishment of cellular calcium. Gating of Orai1 night (21% and 25%, respectively), suggesting that a fraction of BK channels by STIM1 occurs in a graded and non-linear manner which is poorly under- maintain stable Ca2þ channel coupling over the circadian cycle. These data stood. Using electrophysiological, optical and biochemical techniques, we demonstrate diurnal regulation of the activation of BK channels by their Ca2þ examined the functional roles of regions of the STIM1-Orai1 activating re- source and suggest circadian changes in BK-Ca2þ coupling could contribute to gion (SOAR) of STIM1. We find that SOAR mutants that are deficient in BK current regulation in the SCN and its role in circadian rhythmicity. binding and activation of resting Orai1 channels are able to bind to and boost activation of partially activated Orai1 channels. We further show 665-Pos Board B435 that the STIM1 binding regions on Orai1 undergo structural rearrangement Ion Conductance of Orai1 Channel Selectivity-Filter Heterodimers during channel activation. The results suggest that activation of Orai1 by Xiangyu Cai, Yandong Zhou, Robert M. Nwokonko, Natalia A. Loktionova, SOAR occurs in multiple steps. A first step in which SOAR binding to Donald L. Gill. Orai1 partially activates the channel and induces a rearrangement in the Penn State University, Hershey, PA, USA. SOAR binding site of Orai1. That rearrangement of Orai1 then permits Store Operated Ca2þ Entry (SOCE) controls a wide spectrum of phys- sequential steps of SOAR binding, via distinct molecular interactions, to iological functions. Recent studies have identified the ER Ca2þ sensor fully activate the channel. STIM1 and channel forming unit Orai1 as two main players in this process. However, the mechanism by which the Orai1 channel is gated Posters: Other Channels I has remained elusive. In this study, we focused on the selectivity filter of the Orai1 channel, the Glu 106 residue. Previous studies reported 668-Pos Board B438 that the single E106D point mutation diminishes Ca2þ selectivity, and Inferior Olivary TMEM16B Mediates Cerebellar Motor Learning E106Q or E106A mutations result in non-conductive channels. Here, using Yang Zhang1, Zhushan Zhang1, Shaohua Xiao2, Trieu Le1, Son Le1, Lily Jan2, a concatemer strategy, we utilized heterodimers of Orai1 consisting of a Jason Tien2, Huanghe Yang1. wildtype Orai1 (O) and a mutated Orai1E106Q (Q) moiety. We observed 1Duke University Medical Center, Durham, NC, USA, 2University of that such dimers form nonselective channels with increased Csþ perme- California, San Francisco, San Francisco, CA, USA. ability when transiently expressed in HEK Orai1 knockout cells stably Ca2þ-activated ion channels shape membrane excitability and Ca2þ dynamics expressing STIM1-YFP. These results were also repeated using the in response to cytoplasmic Ca2þ elevation. Compared to the Ca2þ-activated Kþ concatenated hexamers of Orai1, OQOQOQ and QOQOQO. Similar to the channels, known as BK and SK channels, the physiological importance of Orai1 E106D monomer, the aspartate-substituted OD or DO heterodimers Ca2þ-activated Cl channels (CaCCs) in neurons has been largely overlooked. still function as non-selective channels. Based on the side chain size of Here we report that CaCCs coexist with BK and SK channels in inferior olivary different amino acids, we tested the Orai1 E106N monomer, which results (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for in a non-conductive channel. However, the ON and NO heterodimers of the control of motor learning and timing. Ca2þ influx through the dendritic Orai1 can form non-selective channels. Other substitutions of the E106 high-threshold voltage-gated Ca2þ channels activates CaCCs, which contribute

BPJ 8573_8577 Sunday, February 18, 2018 133a

2þ to membrane repolarization of IO neurons. Loss of TMEM16B expression re- ties, and intracellular magnesium ([Mg ]i) modulation. The underlying molec- sulted in the absence of CaCCs in IO neurons, leading to markedly diminished ular determinants are not fully clear. Previous experimental evidence indicates action potential firing of IO neurons in TMEM16B knockout mice. Moreover, that residues in the amino terminal (NT) and the first extracellular domain these mutant mice exhibited severe cerebellar motor learning deficits. Our find- (E1) influence gj and Vj-gating of Cx50 GJs. It is also known that Cx37 GJs ings thus advance the understanding of the neurophysiology of CaCCs and the showed the largest gj among all characterized GJs. We hypothesize that residue ionic basis of IO neuron excitability. differences in the NT and E1 domains between Cx50 and Cx37 are partially responsible for the differences in their gjs. Sequence alignment of Cx50 and 669-Pos Board B439 Cx37 in these domains revealed that in Cx50 G8 and V53 positions are nega- Nonequilibrium Molecular Dynamics (MD) Simulations of Multi-ion tively charged residues in Cx37 (E8 and E53, respectively). To evaluate these Permeation in KcsA residue differences and a previously identified variant on the 46th residue 1 1 1 1 Robert A. Farley , Sarah Holzmann , Cameron Kopp , Yi Shi , (G46E), which significantly increased the gj in Cx50 GJ, we generated a triple 2 3 3 Yibo Wang , Sergei Noskov , Van A. Ngo . variant in Cx50, G8E, G46E, and V53E to study the gj,Vj-gating properties, and 1 2 2þ University of Southern California, Los Angeles, CA, USA, Mount Sinai its modulation by [Mg ]i. Our data indicate that the triple variant and individual 3 Medical Center, New York, NY, USA, University of Calgary, Calgary, AB, variants G8E and G46E, but not V53E, significantly increased Cx50 GJ gj 2þ Canada. without much change in the Vj-gating. In addition, elevated [Mg ]i reduced Non-equilibrium MD simulations were used to generate a two-dimensional po- g in Cx50 and all the variant GJs. These results and our homology structural þ j tential of mean force (2D PMF) profile for K ions in the KcsA channel. With models indicate that these residues are likely pore-lining and the variants symmetrical 140 mM KCl concentrations on both sides of a POPC lipid bilayer increased negative electrostatic potentials along the GJ pore to facilitate cation and ions occupying sites S1 and S3, a minimum free-energy path for steady- permeation and an elevated magnesium binding. Supported by NSERC. state ion permeation was identified for two Kþ ions pulled through the channel at constant velocity. Our non-equilibrium pulling technique enables assessment 672-Pos Board B442 of energetics costs for different multi-ion/water configurations, thus ranking Investigation of Anion Selectivity of CLC-K Channels ion-permeation pathways and shedding more insight into the debate of what Laura Lagostena1,2, Michael Pusch2, Alessandra Picollo1,2. 1 2 would be the most efficient ion permeation mechanism. The global free- Dulbecco Telethon Institute, Genova, Italy, Istituto di Biofisica, Genova, energy minimum configuration has Kþ ions bound at S0, S2, and S4. The high- Italy. est free-energy barrier for Kþ permeation is associated with displacement of an The CLC protein family is divided into two sub-classes: chloride channels and - þ ion from S4 into site S2. For outward permeation, electrostatic repulsion be- Cl /H exchangers. CLC channels localize to the plasma membrane, where tween an ion approaching the selectivity filter from the cytoplasm and the they are involved in the regulation of muscle and neuronal excitability as ion bound at site S4 destabilizes the system and provides sufficient energy well as transepithelial salt transport. Exchangers are expressed in the membrane for the ion in site S4 to move to site S2, displacing that ion to S0, and forcing of intracellular compartments where they contribute to modulate endolysoso- the ion in S0 to the extracellular side. At an average distance of 3.75 5 0.5 A˚ mal pH and/or chloride concentration. The comparison of the recently solved the free energy barriers between binding sites for Kþ in the selectivity filter are three-dimensional structure of the bovine CLC-K channel with structures of at a minimum, and the net DG for outward Kþ permeation is negative. Under bacterial and algal CLC exchangers indicates that the most significant differ- the same conditions, the net DG for inward Kþ permeation is positive, indi- ence between the CLC-K channel and the transporters is the orientation of res- cating that the KcsA channel structure favors outward permeation. During idues forming the aC-D loop, including a highly conserved serine residue ion permeation, the diameter of the selectivity filter increases to accommodate (SerC) known to be important for the anion selectivity of transporters. In þ þ - K ions and water. We found that permeant K ions were often, but not always, CLC transporters the side chain of SerC is located within the Cl permeation separated by a water molecule, contributing the most to the overall energetics pathway, while in the CLC-K structure it is orientated in the opposite direction of efficient permeation pathways. pointing towards the cytosolic side. This orientation suggests a limited role of SerC in the modulation of the anion selectivity in CLC-K channels. To test this 670-Pos Board B440 hypothesis, we investigated the selectivity of several SerC mutants in human Permeation of Antibiotics across Porins CLC-Ka using two electrode voltage clamp and patch clamp techniques. Pre- Mathias Winterhalter, Satya Prathysha Bhamidimarri, Ishan Ghai, liminary analysis of the permeability ratio of several anions versus chloride in- Jiajun Wang. dicates that exchanging SerC with threonine is sufficient to alter the selectivity Jacobs University, Bremen, Germany. properties of CLC-Ka, suggesting that despite the indication emerging from the - Antimicrobial resistance (AMR) is recognized as a major global public health CLC-K structure, SerC may directly interact with permeating Cl ions. threat and there is an urgent need for discovery and development of new drugs to combat the multi-resistant organisms. Within the Innovative Medicine Initia- 673-Pos Board B443 tive platform ‘‘New Drugs Against Bad Bugs’’ Translocation is devoted to un- Ionic Permeation and the Nature of Ion Selectivity in Claudin Paracellular derstand the low permeability of antibiotics in Gram negative bacteria. Here we Channels focus on the development of assays that will enable rapid and reliable quanti- Priyanka Samanta1, Yitang Wang2, Shadi Fuladi3, Jinjing Zou4, Le Shen5, Christopher Weber2, Fatemeh Khalili-Araghi3. fication of the flux of small, water-soluble molecules across the cell envelope. 1 2 Membrane channels in the outer cell wall of Gram-negative bacteria control the Chemistry, University of Illinois, Chicago, IL, USA, Pathology, University of Chicago, Chicago, IL, USA, 3Physics, University of Illinois, Chicago, IL, entry of antibiotics or nutrients. To quantify the permeation we reconstitute sin- 4 5 gle channel into planar lipid bilayer and record the ion current noise in presence USA, Pulmonary Medicine, Whuan University, Wuhan, China, Surgery, of the permeating molecules. However this simplified approach does not allow University of Chicago, Chicago, IL, USA. to distinguish permeation from binding. Accounting for electroosmosis or Tight junctions are macromolecular structures that seal the paracellular space application of concentration gradients of charged molecules may break the and provide a barrier for the passage of ions and small molecules. Claudins symmetry and provide approximate numbers.Ghai I, et al. J Phys Chem Lett. are one of the major components of tight junctions that control their selectivity 8 (2017) 1295-1301.Soundararajan G. et al. ACS Chemical Biology 12 and ion permeation properties. Claudin-15 and 2 have been shown to estab- (2017) 1656-64. lish paracellular sodium selective channels, and the latter has been shown to Ferrara G et al. J. Mol. Biol. 428 (2016) 4528-43. contribute to epithelial barrier dysfunction in intestinal diseases such as inflam- Bajaj H et al. ACS Nano 11 (2017) 5465-73. matory bowel disease and infectious colitis. Based on the available crystal structure of claudin-15 and a proposed architectural model of claudin pores, 671-Pos Board B441 we have constructed and refined atomic models of claudin-15 and claudin-2 The Residues in the Amino Terminal and First Extracellular Domains and pores in their native environment. Using all-atom molecular dynamics simula- Intracellular Magnesium Influence Cx50 Unitary Gap Junction Channel tion, we have calculated relative permeability of the channels to cations and Conductance anions of various size and shape and have compared them with in vitro mea- Mary Grace Tejada1, Swathy Sudhakar1, Hiroshi Aoyama2, Donglin Bai1. surements of channel permeability. Structural elements that interact with 1Physiol. and Pharm., The University of Western Ontario, London, ON, permeating ions have been identified from molecular dynamics simulations Canada, 2Graduate School of Pharmaceutical Sciences, Osaka University, and their role in conferring channel selectivity has been tested in vitro by Osaka, Japan. site-specific mutations. These results indicate distinct selectivity mechanisms Gap junctions (GJs) are oligomers of connexins forming channels linking neigh- in claudin-2 and claudin-15 pores that are not only controlled by electrostatic bouring cells. GJs formed by different connexins show distinct unitary channel interactions between the ions and the pore but by specific positions of charged conductance (gj), transjunctional-voltage dependent gating (Vj-gating) proper- residues in the two channels.

BPJ 8573_8577 134a Sunday, February 18, 2018

674-Pos Board B444 between barrel strand number and ion conductance. In the present work, mul- Bacterial Porins as Electrostatic Nanosieves: Exploring Transport Rules of tiple simulations of OmpX starting from different crystal structures were run to Small Polar Molecules elucidate a connection, if any, between barrel geometry and conductance. Harsha Bajaj1, Silvia Acosta Gutierrez2, Igor Bodrenko2, Giuliano Malloci2, Although water and ion permeation is not observed for any of the OmpX struc- Mariano Andrea Scorciapino3, Mathias Winterhalter1, Matteo Ceccarelli2. tures, simulations of two of them show increased water accessibility. Therefore, 1Jacobs University, Bremen, Germany, 2Physics, University of Cagliari, we also simulated alternative protonation states and mutants, which result in Monserrato, Italy, 3Biomedical Sciences, University of Cagliari, Monserrato, increased water permeability. Lastly, the barrel was expanded through the addi- Italy. tion of 2-4 duplicate strands, which leads to an increased conductance for even Transport of molecules through biological membranes is a fundamental process the wild-type OmpX, albeit still lower than that measured in experiment. in biology, facilitated by selective channels and general pores. The architecture of some outer membrane pores in Gram-negative bacteria, common to other eu- 677-Pos Board B447 karyotic pores, suggests them as prototypes of electrostatically regulated nano- A Newly Available Tool for Functional Annotation of Ion Channel Struc- sieve devices. In this study, we sensed the internal electrostatics of the two most tures Based on Molecular Dynamics Simulations abundant outer membrane channels of Escherichia coli, using norfloxacin as a Gianni Klesse1,2, Shanlin Rao2, Phillip J. Stansfeld2, Mark S.P. Sansom2, dipolar probe in single molecule electrophysiology. The voltage dependence of Stephen J. Tucker1. 1 the association rate constant of norfloxacin interacting with these nanochannels Department of Physics, University of Oxford, Oxford, United Kingdom, 2 follows an exponential trend, unexpected for neutral molecules. We combined Department of Biochemistry, University of Oxford, Oxford, United electrophysiology, channel mutagenesis, and enhanced sampling molecular dy- Kingdom. namics simulations to explain this molecular mechanism. Voltage and temper- With the discovery of novel ion channel structures continuing at an accelerated ature dependent ion current measurements allowed us to quantify the pace there is a clear need for improved tools that facilitate the functional anno- transversal electric field inside the channel as well as the distance where the tation of these structures. We have previously shown how molecular dynamics applied potential drops. Finally, we proposed a general model for transport simulations can be applied to assign a functional status to a channel structure, of polar molecules through these electrostatic nanosieves. Our model helps i.e. to determine whether it represents a conductive or non-conductive confir- to further understand the basis for permeability in Gram-negative pathogens, mation of a channel [1]. This computational approach is capable of rapidly contributing to fill in the innovation gap that has limited the discovery of effec- identifying hydrophobic gates, i.e. desolvation barriers to ion permeation that tive antibiotics in the last 20 years. result from the dewetting of hydrophobic segments within the nanometer- sized pores found in most ion channels. Such hydrophobic barriers are not 675-Pos Board B445 detectable by traditional measurements of the physical dimensions of a channel Mechanism of PH Gating in Cx26 Gap Junction Channels Revealed by pore and it is now clear that they represent an important predictor of channel cryoEM, Crosslinking and HDX permeability. The methodology and code underlying this analysis was origi- Maciej Jagielnicki1, Ali Khan1, William E. McIntire1, Michael D. Purdy1, nally developed in-house and only available to experienced users. However, Venkat Dharmarajan2, Patrick R. Griffin2, Mark Yeager1. it has recently been recompiled into a new easy to use format that is freely 1 Department of Molecular Physiology and Biological Physics, University of available to anyone wishing to gain a greater insight into the functional status 2 Virginia School of Medicine, Charlottesville, VA, USA, Department of of new ion channel structures (if interested, please contact gianni.klesse@ Molecular Medicine, The Scripps Research Institute, Scripps Florida, Jupiter, physics.ox.ac.uk). In addition, work continues to integrate this tool into the ex- FL, USA. isting MemProtMD database (http://memprotmd.bioch.ox.ac.uk/) and to make Connexins (Cx) are a family of integral membrane proteins that assemble as it accessible online. This functional annotation procedure will be illustrated hexameric hemichannels. Hemichannels from adjacent cells dock to form do- using examples of structurally unrelated classes of ion channels. decameric gap junction channels (GJCs). GJCs exhibit properties of both [1] Trick, J. L., Chelvaniththilan, S., Klesse, G., Aryal, P., Wallace, E. J., ligand- and voltage-gated channels, including channel closure at acidic pH. Tucker, S. J., & Sansom, M. S. (2016). Functional annotation of ion channel Here we used cryoEM, crosslinking and HDX to explore pH-mediated confor- structures by molecular simulation. Structure, 24(12), 2207-2216. mational changes of Cx26 GJCs. Notably, mutations of the Cx26 gene are the most common cause of sensorineural hearing loss in children. Recombinant, 678-Pos Board B448 human Cx26 was purified as dodecameric GJCs, which remained homogenous Hydrophobic Gating: Examination of Recent Ion Channel Structures and monodisperse when reconstituted in amphipol. Single-particle cryoEM Shanlin Rao1, Gianni Klesse2,1, Stephen J. Tucker2, Mark S.P. Sansom1. 1 analysis was performed on samples at pH 7.5 and 6.4. 3D classification of Department of Biochemistry, University of Oxford, Oxford, United 2 low pH images yielded two conformations, one resembling the open, neutral Kingdom, Clarendon Laboratory, Department of Physics, University of pH structure and a second that displayed an occluded state. The maps revealed Oxford, Oxford, United Kingdom. well resolved transmembrane helices and extracellular domains, whereas the Transmembrane ion channel proteins play crucial roles in membrane cytoplasmic loop (CL) and tail (CT) were less well-resolved. Modeling of biophysics and physiology. In response to specific stimuli, ion channels un- the polypeptide into the cryoEM maps suggested that the NTs undergo a dergo conformational changes that lead to opening and closing of their pores, helix-coil transition during pH-mediated gating. To elucidate the structure thereby controlling diffusion of selected ions down electrochemical gradients. and dynamics of the cytoplasmic regions we performed lysine crosslinking Driven by biophysical and pharmacological interests in these dynamic proteins, and HDX. At both neutral and acidic pH vast networks of crosslinks were de- and with advances in cryo-electron microscopy, an increasing number of struc- tected between the NT and CL, CT and CL and within the CL. At acidic pH tures are being solved for different families of ion channels, often capturing additional crosslinks were detected between residues K15 and K22 and the alternative conformations. CL, indicative of closer association compared with pH 7.5. Furthermore, Determination of the conductive state represented by a given ion channel struc- amino-tail residues 7-28 showed a reduction in hydrogen-deuterium exchange ture (i.e. whether its conformation would allow permeation of ions and water) at low pH. The kinetics of exchange suggested that NTs adopt a single confor- constitutes a key aspect of annotating channel structures to facilitate elucidation mation at acidic pH. Taken together, our results support a ‘ball-and-chain’ of functional mechanisms, and requires consideration of multiple factors, mechanism in which the NTs undergo a helix-coil transition and associate to including the hydrophobicity of the pore lining, in addition to assessing phys- occlude the channel pore during pH-mediated gating. ical dimensions. Even when sterically able to accommodate ions, sub- nanometre hydrophobic regions may present a significant energetic barrier to 676-Pos Board B446 the flow of water and ions through the pore. The Effect of Barrel Geometry on Ion Conduction through E. coli OmpX Here we apply molecular dynamics simulation-based methods to recent ion chan- Curtis Balusek1, Dirk Linke2, James C. Gumbart1. nel structures to predict the conductive states of their pores. We demonstrate that 1Physics, Georgia Inst. of Technolgy, Atlanta, GA, USA, 2University of Oslo, the crystal structures determined for TMEM175, a non-canonical Kþ channel, and Oslo, Norway. bestrophin-1, a Ca2þ-activated Cl- channel, both correspond to closed state con- OmpX is an eight-stranded, outer membrane, adhesion protein found in formations. We suggest that the hydrophobic constriction in the pore of each of Gram-negative bacteria, which has been shown to promote septicemia through these two proteins forms an energetic barrier to ion and water conduction, i.e. a host-cell interaction. Although structural data and previous simulations showed hydrophobic gate. The gate may also function as a selectivity filter upon channel OmpX to be impermeable to water and ions, electrophysiology experiments activation and widening of the region. This approach to functional annotation of exhibit a small but consistent conductance through the wild-type protein. ion channel structures is currently being applied to all known ion channel struc- Furthermore, gene duplication of OmpX displays an asymptotic relationship tures in order to assess the more general importance of hydrophobic gating.

BPJ 8573_8577 Sunday, February 18, 2018 135a

679-Pos Board B449 3San Diego State University, San Diego, CA, USA, 4Electrical and Computer Conformational Landscape of Substrate Specificity in the Fructose Trans- Engineering, University of California, San Diego, La Jolla, CA, USA, porter GLUT5 Determined via MBAR Molecular Dynamics 5Physiology and Biomedical Engineering, Northwestern University, Trevor Gokey, Jesi Lee, Anton B. Guliaev. Chicago, IL, USA. Chemistry and Biochemistry, San Francisco State University, San Francisco, Non-invasive imaging tools, such as ultrasound, magnetic resonance imaging, CA, USA. and optical coherence tomography, are blind to micrometer-scale cell organiza- GLUT5 is a member of the glucose transporter (GLUT) family and channels tion and structure that dictate cell function. In skeletal muscle, active force is fructose but not glucose through lipid membranes. Recent crystallographic determined by micrometer changes in sarcomere length (SL), which directly data indicated that GLUT5 may possess a unique transport mechanism from and predictably controls muscle force production. However, these length other GLUT members; however, the details of this mechanism are unclear. changes are too small to accurately resolve in vivo with existing tools. Inability 32ms equilibrium dynamics in combination with restrained MD were used to to measure SL in-vivo during movement is a long-standing challenge in under- generate a conformational landscape of the sugar transport mechanism in standing muscle physiology and function. To overcome this challenge, we are four independent systems containing a-fructose, b-fructose, a-glucose, or developing a new technique termed Resonant Reflection Spectroscopy (RRS) b-glucose. A PMF was generated for each substrate using the MBAR method. that is based upon principles and technology adapted from optical communica- The calculated PMF revealed two energy basins which were relatively consis- tion systems. Previosly, we demonstrated sarcomere length measurement dur- tent across the four sugars in this work. The first basin, and consequently the ing passive stretch and twitch contractions. In this report, we provide initial succeeding energy barrier, occurred at the flexible segment of the TM7 helix evidience of SL measured during tetanic contractions. at the extracellular entrance of GLUT5. This flexible region was unresolved Rabbit tibialis anterior SL during tetanic contractions was measured in vivo in recent GLUT5 crystal structures, highlighting its inherent flexibility. Here through a 250-micrometer optical probe using a combination of RRS and opti- for the first time, we report a conformational landscape of GLUT5 and details cal frequency domain interferometry (OFDI). Tetanic contractions were stim- of the required interactions of the TM7 loop with substrate. We demonstrated ulated by 0.3 millisecond pulses sent through a bipolar cuff electrode to the that initial entrance into the GLUT5 channel required overcoming an energy peroneal nerve. Even though muscle-tendon units were held at constant length, barrier associated with this loop. The second basin occurred in the GLUT5 cen- SL shortened linearly during the onset of force production due to in-series tis- tral binding pocket, where the pivotal Q166 residue that is responsible for fruc- sue viscoelasticity. Ability to study protein-level biomechanics of skeletal mus- tose specificity, made initial contact with incoming substrate. The barrier at this cle in vivo represents a critical step toward better understanding of muscle location was significantly higher for glucose compared to fructose. Moreover, disease and movement disorders. This bioengineering innovation will permit we observed several other regions in the channel which showed specific favor- new studies of muscle function and pathology, even in humans, and will permit able interactions with fructose. Such interactions could not occur with glucose efficacy testing of novel muscle treatments. The fact that the method is mini- substrates due to their minute but important steric and electrostatic incompat- mally invasive and insensitive to motion artifact exponentially expands the ibilities with GLUT5. This work improved our understanding of the GLUT5 possible measurements that can be performed in humans in realistic movement transport mechanism that is important for future development of therapies tar- settings. geting sugar metabolism. 682-Pos Board B452 Posters: Skeletal Muscle Mechanics, Structure, Residual Force Enhancement is Attenuated by Shortening in a Magnitude- and Regulation I Dependent Manner Atsuki Fukutani, Walter Herzog. 680-Pos Board B450 Kinesiology, The University of Calgary, Calgary, AB, Canada. Evidence for Actin Filament Structural Changes after Active Shortening The isometric force attained after active stretch is greater than that attained in in Skinned Muscle Bundles a purely isometric contraction at the corresponding muscle length. This prop- Venus Joumaa1, Ian Curtis Smith1, Atsuki Fakutani1, Tim Leonard1, erty is referred to as residual force enhancement (RFE). Although RFE is Weikang Ma2, Thomas Irving2, Walter Herzog1. considered to contribute to the force/work enhancement in stretch- 1University of Calgary, Calgary, AB, Canada, 2Illinois Institute of shortening cycles, it is unclear whether shortening that occurs after active Technology, Chicago, IL, USA. stretch eliminates the influence of RFE. Therefore, we evaluated the influ- Steady-state force after active shortening is smaller than purely isometric force ence of shortening on the magnitude of RFE. Skinned fibers of rabbit soleus produced at the same final muscle length without prior shortening. The mecha- (N = 45) were used for all experiments. The first test consisted of an isomet- nism responsible for this phenomenon, termed force depression, remains un- ric reference contraction at an average sarcomere length of 3.0 mm. The sec- known. It has been suggested (Marechal and Plaghki, J Gen Physiol 1979)that ond test consisted of a typical way to induce RFE. This was achieved by force depression might be associated with stress-induced conformational changes active stretching of single fibres from an average sarcomere length of 2.7 in the actin filaments after active shortening, resulting in a decrease in the propor- to 3.0 mm. The third test consisted of a stretch shortening cycle. RFE was tion of attached cross-bridges. However, no direct support has been provided for induced by active stretch from 3.0 to 3.3mm. Immediately after stretching, this hypothesis. Our aim was to determine the effect of active shortening on the fibers were shortened to 3.0 mm (12.5% of the fiber length) in 0.5 ms. Force conformation of the actin filament at the steady-state after active shortening using attained 15 s after the active stretch was used to evaluate the magnitude of small angle x-ray diffraction. Spacing changes were investigated in skinned rabbit RFE. Compared to the isometric reference condition, force was significantly greater in the normal RFE condition (108.4 5 2.5%) and in the stretch- psoas muscle bundles for three actin filament reflections: the meridional reflection 5 < at 2.7 nm which corresponds to the periodicity of axial translation of the actin sub- shortening cycle condition (RFE 103.5 2.4%) (p 0.001). This difference in the magnitude of RFE disappeared when the magnitude of shortening was unit in the thin filament, and the two actin layer-line reflections at 5.1 nm and 5.9 ¼ nm which correspond to the pitches of the right- and left-handed genetic helices of decreased from 12.5% to 1% of the fiber length (p 0.316). In conclusion, the actin filament. The spacing of the 2.7 nm actin reflection increased by 0.14% RFE is attenuated by shortening following active stretching. This attenuation during the steady-state after active shortening compared to the spacing during a effect depends on the magnitude of shortening and decreases with decreasing purely isomeric contraction. This change in spacing indicates that the thin fila- shortening magnitudes. The molecular events underlying these history- ment is likely more stretched after active shortening compared to a purely isomet- dependent force effects remain unknown. ric contraction. The spacing of the 5.1 nm actin layer-line reflection decreased by 0.88% at the steady-state after active shortening compared to the spacing during a 683-Pos Board B453 purely isomeric contraction. This change may be caused by an untwisting of the The Nonlinear Mechanical Properties of Titin Modulate Striated Muscle thin filament. These results indicate that active shortening causes the actin fila- Contraction Efficiency ment to undergo conformational changes to its structure, and these are likely to Joseph D. Powers1, C. David Williams2,3, Michael Regnier1, contribute to the force depression. Thomas L. Daniel3,1. 1Bioengineering, University of Washington, Seattle, WA, USA, 2Allen 681-Pos Board B451 Institute for Cell Science, Seattle, WA, USA, 3Biology, University of Resonant Reflection Spectroscopy and Optical Interferometry to Measure Washington, Seattle, WA, USA. Sarcomere Structure in Muscle In striated muscle, the giant protein titin spans the length of a half-sarcomere and Kevin W. Young1, Bill P.-P. Kuo2, Shawn M. O’Connor3, Stojan Radic4, extends from the thick filament backbone across the I-band, where it can revers- Richard L. Lieber5. ibly interact with the thin filament, and anchors to the z-disk. While it is known 1Independent Consultant, San Diego, CA, USA, 2Electrical and Computer that the stiffness of the I-band region of titin is modulated by various factors in Engineering, University of California, San Diego, La Jolla, CA, USA, vivo, little is known about the relationship between the basic mechanical

BPJ 8573_8577 136a Sunday, February 18, 2018 properties of titin and cross-bridge force generation. Here, we build upon our Distal muscular dystrophies are a group of inherited primary muscle disorders previous multi-filament, spatially explicit computational models of the half- showing progressive muscle atrophy and weakness preferentially in the hands sarcomere by incorporating the nonlinear mechanics of titin filaments in the and/or feet. We identified a novel frameshift mutation in the filamin C (FLNC I-band. We model the nonlinear stiffness of titin as a simple exponential c.5161delG) gene in two members of a French family affected by distal myop- spring, varying the value of the exponent in simulations of isometric contraction athy and in one healthy relative. Interestingly, the two affected members, but for a range of sarcomere lengths (SL; 1.6-3.2mm). We monitor the relationships not the healthy relative, also carry a p.Arg4444Trpvariant in the gene coding between the nonlinear stiffness of titin and isometric force and contractile for the giant protein obscurin(OBSCN). Both OBSCN and FLNC are Z-disk efficiency (force/ATP). Intermediate values of titin stiffness reproduce the associated proteins. FLNC is an actin cross-linking protein, involved with known passive force-SL relation for skeletal muscle. The active force-SL rela- Z-disk organization and muscle integrity. The FLNC c.5161delG mutation tion is unaffected by titin for SL < 2.5mm. However, for SL R 2.6mm, active here described is one nucleotide away from a previously reported FLNC muta- force significantly decreases as titin stiffness increases. At SL = 3.0 mm, tion (c.5160delC), identified in patients and in asymptomatic carriers of three the active force for a half-sarcomere without titin is 10252 pN, while it is families with distal muscular dystrophy, indicating a low penetrance of these 5951 pN with highly nonlinear (stiff) titin. Interestingly, this drop of FLNC frameshift mutations. Obscurin is a giant protein, which interacts with active force with increasing titin stiffness occurs despite an increase in the num- titin at both the M-band and the Z-disk and with muscle-specific ankyrins at ber of strongly bound cross-bridges via compliant realignment of binding sites the sarcoplasmic reticulum and the sarcolemma. The p.Arg4444Trpvariant between thick and thin filaments. Additionally, we find that at SL = 3.0mm, we identified is localized within the OBSCN Ig59 that, together with Ig58, contractile efficiency significantly decreases from 13.750.4 pN/ATP to binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional 7.050.3 pN/ATP (comparing no titin to very stiff titin, respectively). Taken studies indicated that the OBSCN p.Arg4444Trp variant results in a 10-fold together, our results point to a unique role of titin in determining muscle contrac- decrease in binding to titin. Interestingly, in vitro and in vivo models show tile efficiency. that loss of FLNC results in myofibril destabilization and that the decrease in the interaction between titin and obscurin is associated with cardiac and skel- 684-Pos Board B454 etal muscle diseases. On this line, we suggest that the combination of the Tropomyosin Translocation on F-Actin Revealed by Molecular Dynamics OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation can Simulations cooperatively affect myofibril stability and increase the penetrance of muscular 1 1 2 Farooq A. Kiani , Michael J. Rynkiewicz , Stefan Fischer , dystrophy. William Lehman1. 1Dept. Physiology & Biophysics, Boston University School Medicine, 686-Pos Board B456 Boston, MA, USA, 2IWR-Computational Biochemistry Group, Heidelberg Is Thin Filament Movement Switched On and Off by a Thermodynamic University, Heidelberg, Germany. Process Alone Tropomyosin movement across the muscle thin filament is controlled by Henry G. Zot1, Bryant Chase2, Javier E. Hasbun3, Jose R. Pinto4. troponin, calcium and myosin binding. In this process, tropomyosin alterna- 1Biology, University of West Georgia, Carrollton, GA, USA, 2Biology, tively blocks or exposes myosin-binding sites on actin, thereby regulating Florida State University, Tallahassee, FL, USA, 3Physics, University of West contraction. Indeed, fiber diffraction of intact muscles and EM-reconstruction Georgia, Carrollton, GA, USA, 4Biomedical Sciences, Florida State of isolated filaments have identified average azimuthal positions of tropomy- University, Tallahassee, FL, USA. osin on actin corresponding to off and on regulatory-states. However, these We investigated binary events at the foundation of thin filament regulation of techniques lack the spatial and temporal resolution needed to capture dynamics striated muscle contraction. With conditions held constant in a standard in vitro about these states and transitions between states. Here, computational biochem- motility assay, we recorded filaments switching between macroscopic states istry, involving molecular dynamics simulation, is a compelling choice to of pauses and runs. Individual actin-containing filaments labeled with extend understanding of tropomyosin structural transitions on actin to high res- rhodamine-phalloidin were illuminated by HeNe laser (543 nm). Magnified olution. Thus, we performed MD of tropomyosin structures that were localized fluorescence images were captured by SIT camera onto DVD at 30 fps using to multiple proposed sites of interaction over the surface of F-actin. During MD an inverted fluorescence microscope (Nikon Eclipse TE2000-U). We intro- on these conformers, tropomyosin was constrained geometrically to mimic duced fluorescent sheared F-actin or native thin filaments from skeletal muscle tropomyosin’s native end-to-end molecular length and distance to the filament into a flow cell constructed with a coverslip coated with skeletal muscle HMM surface as if part of an otherwise semi-flexible polymeric cable on F-actin. serving as the viewing surface. Motility was recorded in 2 mM Mg2þ-ATP, pH Tropomyosin showed considerable movement both in azimuthal and longitudi- 7.0 and constant free calcium concentration. Control assays demonstrated nal directions over F-actin during MD. The translocations were biased toward directed movement of actin filaments that was smooth and continuous. We blocked B-state positions, particularly when MD was initiated from the observed individual filaments change speed and run onto or off the surface. myosin-free open-state on F-actin. In contrast, little movement away from Movements of native thin filaments were indistinguishable from actin filaments the B-state locations was observed during MD. Pro333 on actin defines the of comparable length in assays containing saturating calcium (pCa 4.0). In as- boundary between blocked- and closed-states on successive actin subunits says containing low calcium (pCa 8.0), native thin filaments remained attached along F-actin. Simulations show that tropomyosin placed over Pro333 falls to the surface in stationary place. By contrast, thin filaments exposed to an in- to the B-state side of the residue during MD. Our results are completely consis- termediate free calcium concentration (pCa 7.0-5.0) exhibited spontaneous tent with predictions based on previously published potential energy landscapes events of binary switching between unbroken directed movement (run) and sta- determined by rigid-body translocation of tropomyosin over F-actin. Our re- tionary placement on the surface (pause). We expect the distribution of inter- sults support the view that blocked- and closed-state positions of tropomyosin vals of time between switching events (dwell times) to be exponential for a are energetically favored while the myosin-free open-state is not. Here, the pure thermodynamically driven process. In pCa range 6.2-5.8, assays displayed additional presence of myosin is required to pin tropomyosin in an open M- the most switching events and, hence, yielded the most run and pause dwell state configuration. times. Distributions of these dwell times will reveal if a thermodynamic process alone can, indeed, explain the mechanism by which switching is regulated. 685-Pos Board B455 Work supported by NIH grant HL128683 (JRP) and leave supported by A Novel FLNC Frameshift and an OBSCN Variant in a Family with Distal UWG (HGZ). Muscular Distrophy Daniela Rossi1, Johanna Palmio2, Anni Evil€a3, Stefania Lorenzini1, 687-Pos Board B457 Galli Lucia4, Virginia Barone1, Tracy A. Caldwell5, Rachel A. Polike5, Mapping Allosteric Pathways in the Myosin Motor Domain via the Aldkheil Esraa5, Berndsen E. Christopher5, Nathan T. Wright5, W-Helix/Transducer Region Leading to Force Production Peter Hackmann3, Bruno Eymard6, Bjarne Udd7, Vincenzo Sorrentino1. Peter Franz1, Wiebke Ewert1, Matthias Preller1,2, Georgios Tsiavaliaris1. 1Molecular and Developmental Medicine, University, Siena, Italy, 1Institute for Biophysical Chemistry, Hannover Medical School, Hannover, 2Neuromuscular Research Center, University and University Hospital, Germany, 2German Electron Synchrotron (DESY), Hamburg, Germany. Tampere, Finland, 3Folkh€alsan Institute of Genetics and Department of According to the model by Lymn and Taylor, the acto-myosin system generates Medical Genetics, University of Helsinki, Helsinki, Finland, 4Molecular mechanical work when myosin executes the power-stroke. However, the Medicine and Genetics, Azienda Ospedaliera Universitaria Senese, Siena, conformational transitions in the myosin motor domain associated with the Italy, 5Chemistry and Biochemistry, James Madison University, force generating step are poorly characterized, e.g. there is lack of information Harrisonburg, VA, USA, 6Neuromuscular Morphology Unit, Groupe regarding the coupling between ATP hydrolysis and weak to strong transitions Hospitalier La Pitie-Salpeˆtrie`re, Paris, France, 7Neuromuscular Research of actin binding preceding the power-stroke. With the aid of mutations intro- Center, Tampere University, Tampere, Finland. duced in the inner core of D. discoideum myosin-2, we were able to decouple

BPJ 8573_8577 Sunday, February 18, 2018 137a the actin-activated product release from force output, thus identifying the W- and power output by a combination of faster cross-bridge cycling kinetics, helix/transducer region as important hub mediating allosteric communication recruitment of cross-bridges, and reduction in drag forces as thin filaments between distant sites, the actin binding region and the converter. Mutant con- slide through the C-zone. structs displayed unchanged basal ATPase activities compared to wild-type, while actin-activated ATP turnover rates were up to 5-fold increased. At the 690-Pos Board B460 same time, the mutations disturbed effective actin translocation by a more The Skeletal Muscle Molecular Clock Regulates Titin Splicing and Protein than 3-fold reduction of sliding velocities in the motility assay. Single and mul- Expression tiple turnover kinetic experiments indicated that increased ATP hydrolysis and Lance A. Riley, Joseph R. Mijares, Xiping Zhang, Karyn A. Esser. accelerated ADP-release rates of the actin-bound states are responsible for the Physiology and Functional Genomics, University of Florida, Gainesville, enhanced steady-state ATPase activity. However, the dissociation kinetics of FL, USA. acto-myosin (A.M) by ATP was highly affected in favor of 3-fold faster forma- The circadian factor BMAL1 is a fundamental transcriptional regulator of cell tion and up to 10-fold longer lived ATP-bound acto-myosin (A.M.T) states. time keeping and expression of cell specific genes important for homeostasis. The achievement of prolonging the duration of the strongly actin-attached Our lab developed the inducible, skeletal muscle specific Bmal1 knockout states relative to a single ATP turnover while decoupling lever-arm movement (iMSBmal1-/-) mouse to determine the role of this protein in adult skeletal mus- provide the experimental basis to assess the biochemical and structural states of cle. Skeletal muscles from these mice exhibit decreased specific tension and myosin that initiate the power stroke. This is currently investigated by crystal- reduced unstimulated tension developed during a fatigability test. Since this lographic approaches and single molecule experiments. change could be caused by changes in the elastic properties of the muscle, we investigated whether titin isoform expression changes in these mice. The tibialis 688-Pos Board B458 anterior muscle of the iMSBmal1-/- mice exhibits a significant shift in expression Sarcomere Breathing: Does Flow within Contracting Myofibrils Influence from a short to long isoform of titin protein (38% long in iMSBmal1-/- vs. 19% in Substrate Delivery? iMSBmal1þ/þ). RNA-Seq from these muscles indicates increased inclusion of Julie A. Cass1, Anette L. Hosoi2, Tom L. Daniel1. 1 2 titin exons 150-155 and 190-225, which are found within titin’s PEVK region. Biology, University of Washington, Seattle, WA, USA, Mechanical Because this region is highly extensible and acts as a spring, we performed Engineering, MIT, Cambridge, MA, USA. immunohistochemistry to test whether changes to this protein could have an ef- Sarcomeric contractions with constant lattice spacing necessitate flow to fect on sarcomere length. An antibody to a-actinin was used to demarcate Z-lines maintain conservation of mass as the sarcomere shortens in its axial direction. in longitudinal sections of the tibialis anterior muscle and sarcomere length was These contractions are driven by (and therefore depend on) ATP hydrolysis. measured as the distance between Z-lines. While average sarcomere length was However, the availability of ATP to the sarcomere is limited by diffusion in not different between iMSBmal1þ/þ and iMSBmal1-/- muscle, variation in sarco- the crowded space of the lattice of thick and thin filaments. We asked whether mere length increased following Bmal1 knockout. To test if increased variability the flow resulting from periodic contractions influences ATP availability in of titin isoform expression similar to the iMSBmal1-/- mouse could lead to var- the sarcomere. Drawing on published estimates of diffusion rates and enzyme iable sarcomere lengths, we are currently transfecting C2C12 myoblasts with U7 kinetics, we modeled the substrate delivery to the interior of myofibrils and snRNPs targeted to titin PEVK regions. Sarcomere length variability will be associated sarcomeric ATPase rates for the diffusion reaction convection measured in these cells following 7 days of differentation. Current studies equation. Despite the linearity and reversibility of the contraction-driven with this model are ongoing and will lead to increased knowledge of titin’s low Reynolds number flow, we found that convection has non-linear effects role in regulating sarcomere length in a skeletal muscle at rest. on the resulting substrate availability. Our models predict that flow improves ATP concentrations in the sarcomere over diffusion alone by 81.9%, 30.8% 691-Pos Board B461 and 19.2% during the first three contraction cycles. We compared the asymp- 3-D Structure of Z-Disks Isolated from the Flight Muscle of Lethocerus totic effects of flow after many cycles, integrated over the cross section at the Indicus m-line, and found that convection consistently enhances ATP consumption Fatemeh A. Yeganeh, Corrine Summerill, Zhongjun Hu, and substrate availability. Convection improves cycle-averaged ATP concen- Hamidreza Rahmani, Dianne W. Taylor, Kenneth A. Taylor. trations and cycle-integrated ATP consumption by 12.2% and 14.5% respec- Molecular Biophyscs, Florida State University, Tallahassee, FL, USA. tively over diffusion alone, suggesting that flow plays an important role in The recent publication of a 3-D image of isolated Z-disks from Apis mellifera, sarcomere energetics. honey-bee, flight muscle [Rusu et al., J. Muscle Res Cell Motil (2017)] used the 689-Pos Board B459 relatively harsh treatment of high KCl/KI to remove both thick and thin fila- MYBP-C Phosphorylation Accelerates Sarcomere Shortening as Thin Fil- ments in a single step that nevertheless produced a specimen suitable for cry- aments Slide through the C-Zone oelectron tomography (cryoET). The reconstruction, the first of its kind Joel C. Robinett, Laurin M. Hanft, Kerry S. McDonald. utilizing isolated Z-disks, confirmed many details of the previously 3-D image Medical Pharmacology and Physiology, University of Missouri, Columbia, obtained from plastic sections. Here we use a different approach for Z-disk MO, USA. isolation from, in this case, flight muscle from the large waterbug Lethocerus Myosin Binding Protein-C (MyBP-C) is a 140-150 kDa thick filament protein indicus that combines 1.4M NaCl and 10 mM pyrophosphate to remove the located in the middle third of each sarcomere, i.e., the C-zone, and is likely a thick filaments and gelsolin to remove the thin filaments. Myofibrils are sus- key regulator of contraction. Phosphorylation of MyBP-C occurs at 4 residues pended in the high salt-pyrophosphate buffer and the I-Z-I brushes are centri- within its N-terminus and PKA phosphorylation of MyBP-C in hearts is asso- fuged gently to remove the dissolved thick filaments. The I-Z-I brushes are ciated with faster cardiac muscle twitch dynamics (Tong et. al. Circ. Res. placed on the grid bar side of a QuantifoilÔ grid and treated with a calcium 2008). Here we investigated potential mechanisms by which MyBP-C phos- insensitive gelsolin construct for 4.5 min after which the broken up thin fila- phorylation regulates myofilament function. We utilized slow-twitch skeletal ments are washed from the grid and the 3 mm diameter Z-disk are visualized muscle fibers since PKA phosphorylates slow skeletal (ss)MyBP-C but not over holes. Tilt series are collected on a Titan Krios electron microscope equip- (ss)troponin I. Permeabilized rat slow-twitch fibers were mounted between ped with a DE-20 camera and merged using PROTOMO. Fourier transforms of a force transducer and motor, and calcium activated to elicit 30% maximal the Z-disks show spots from a hexagonal lattice with a spacing of 520A˚ extend- force. We measured rates of force development and transient force overshoots ing to 87A˚ . The tomograms are similar to those from honey bee with some dif- following a slack re-stretch maneuver and monitored sarcomere shortening ferences. The large solvent channel of honey bee is not as prominent in during load-clamps. Following PKA, force development rates increased Lethocerus Z-disks and this is reflected in weak to absent intensity in the first 40% and there was a near-doubling in the magnitude of transient force over- order diffraction from the hexagonal lattice. We conclude that Z-disks from shoot. We also observed faster shortening velocities at all loads following Apis mellifera and Lethocerus indicus are similar but not identical. PKA treatment; this may arise from (i) faster cross-bridge cycling rates, (ii) Supported by NIH and AHA. greater number of cross-bridges during load clamps, and/or (iii) attenuation of internal drag imposed by binding of MyBP-C to actin. To address these 692-Pos Board B462 mechanisms, we quantified sarcomere length shortening as thin filaments tra- Role of Thick Filament Inactivation during Isotonic Shortening in Striated versed into the C-zone. Before PKA, there tended to be a slowdown as Muscle sarcomeres shortened from 3.05 to 2.90 mm. After PKA, sarcomeres short- Kerry S. McDonald1, Kenneth S. Campbell2. ened a considerably greater distance during similar load clamps and, at times, 1Medical Pharmacology and Physiology, University of Missouri, Columbia, shortening even accelerated as thin filaments slid through the C-zone. Overall MO, USA, 2Department of Physiology, University of Kentucky, Lexington, these results suggest MyBP-C phosphorylation speeds sarcomere shortening KY, USA.

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Loaded shortening and power output are highly dependent on the level of complex in response to Ca2þ The current model of muscle regulation holds Ca2þ activation in permeabilized cardiac myocytes, fast-twitch and slow- that at relaxing (low-Ca2þ) conditions tropomyosin blocks myosin binding twitch skeletal muscle fibers (McDonald, J. Physiol., 2000). In addition, sites on F-actin, whereas at activating (high-Ca2þ) conditions tropomyosin during submaximal Ca2þ activation, isotonic shortening becomes markedly translocation only partially exposes myosin binding sites on F-actin so that curvilinear in all three muscle types, which has been hypothesized to arise binding of rigor myosin is required to fully activate the thin filament (TF). from shortening-induced cooperative inactivation of thin filaments. Consis- Here we used a single-particle approach to helical reconstruction of frozen hy- tent with this hypothesis, maintaining thin filament activation by non- drated native cardiac TFs under relaxing and activating conditions to reveal cycling, strongly binding cross-bridges eliminated both the Ca2þ depen- the azimuthal movement of the tropomyosin on the surface of the native car- dence of loaded shortening velocities and curvilinear shortening (McDonald diac TF upon Ca2þ activation. We demonstrate that at either relaxing or acti- & Moss, Circ. Res., 2000). However, recent studies have supported an addi- vating conditions tropomyosin is not constrained in one structural state, but tional sarcomere control mechanism that relies on mechano-sensitive thick rather is distributed between three structural positions on the surface of the filament regulation whereby cross-bridges transition between ‘‘Off’’ and TF. We show that two of these tropomyosin positions restrain actomyosin in- ‘‘On’’ states (Linari et. al., Nature, 2015). We tested whether thick filament teractions, whereas in the third position, which is significantly enhanced at regulation can explain isotonic shortening patterns from striated muscle high Ca2þ, tropomyosin does not block myosin binding sites on F-actin. preparations using a computational model that incorporates both thin fila- Our data provide a structural framework for the enhanced activation of the ment and thick filament regulatory mechanisms, which may account for cardiac TF over the skeletal TF by Ca2þ and lead to a mechanistic model the progressive slowing of shortening velocity during low loads at submax- for the regulation of the cardiac TF. imal Ca2þ activations. Computer simulations yielded thin and thick filament inactivation, where the extent of both were inversely proportional to the 695-Pos Board B465 isotonic force values. In addition, thin filament inactivation occurred Super-Stable Phosphorylation Dependent Intramolecular Interactions throughout the duration of load clamps, while the re-distribution of cross- Regulate the Structure and Function of MyBP-C bridges to a lower On:Off ratio occurred relatively early in the load clamp. Sheema Rahmanseresht1, Arthur J. Michalek2, James Gulick3, Overall, these results indicate that shortening-induced cooperative inactiva- Jeffrey Robbins3, David M. Warshaw1, Michael J. Previs1. tion may arise from dynamic changes in thin and thick filament processes, 1Molecular Physiology and Biophysics, Cardiovascular Research Institute, both of which are likely important to tune muscle performance. For University of Vermont, Burlington, VT, USA, 2Mechanical and Aeronautical instance, in the heart, shortening-induced reduction of force-generating Engineering, Clarkson University, Potsdam, NY, USA, 3Pediatrics, Heart cross-bridges may help match ventricular wall stress with ventricular pres- Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, sure as chamber size diminishes during systole, and a similar process could OH, USA. assist timely contraction/relaxation cycles of antagonistic skeletal muscles Cardiac myosin binding protein-C (MyBP-C) is a sarcomeric protein that during repetitive motion. regulates cardiac contractility. Phosphorylation of four serines within a hinge region near MyBP-C’s N-terminus (i.e. M-domain) regulates its bind- 693-Pos Board B463 ing partner interactions, i.e with actin and myosin. To test the hypothesis Sex-Related Differences in Sarcomeric Protein Expression in Guinea Pig that site-specific phosphorylation induces conformational changes within Masticatory Muscles the M-domain, we pulled on N-terminal fragments of MyBP-C using atomic Peter J. Reiser, Natalya Belevych, Kelly Doan, Jarid R. Jones, force microscopy (AFM). M-domain extensibility, as a measure of struc- Suarav Kadatane. tural conformation, was probed as a function of phosphomimetic replace- Biosciences, Ohio State University, Columbus, OH, USA. ment of the four serines with combinations of aspartic acid residues. Differences in jaw adductors (closing muscles) between male and female Phosphomimetic substitution of one or more serines resulted in the emer- guinea pigs have been recognized for several decades, but observations gence of a stable M-domain conformation that took 80 pN to unfold, were based primarily on differences in muscle mass and in fiber type compo- and large reductions (up to 12 nm) in the length of the fully extended M- sition, the latter based upon classical histochemical staining patterns. The ob- domain when pulling with 200 pN of force. These large reductions in jectives of this study were to initially construct a detailed map of myosin length that resisted unfolding at high pulling forces suggested the phosphor- heavy chain (MHC) isoform expression within adult male and female ylation creates super-stable intramolecular interactions. Through sequence masseter and temporalis muscles and to subsequently determine whether gazing we identified potential interactions between the negatively charged the isoform expression patterns of other sarcomeric proteins in the same mus- phosphate groups and neighboring clusters of positively charged amino cle compartments correlated with differences in MHC expression. Samples acids which could account for such large length changes. To determine if were obtained from a total of twenty-four sites within the masseter and tem- these interactions were feasible, short (11-21 amino acid) peptides were poralis muscles of three male and three female guinea pigs and were exam- synthesized which contained these moieties and the formation of super- ined with SDS-PAGE and troponin-T (TnT) blots. Major differences in MHC stable peptide dimers was observed by electrospray ionization mass spec- isoform expression between the masseter and temporalis muscles and be- trometry. These data suggest a model in which local, intramolecular inter- tween compartments within the temporalis muscle of both sexes were actions controls the global structure of MyBP-C’s N-terminus to modulate observed. Sex-related differences were also observed, especially in the tem- its intermolecular binding partner interactions. poralis muscle. Differences in fast-type TnT isoform expression were also observed and it appears that MHC and TnT isoform expression is highly co- 696-Pos Board B466 ordinated among jaw adductor muscle compartments. These results suggest Effect of a Unique Polymophism in Tropomyosin-Binding Site 1 of Toad that variations in MHC and TnT isoform expression underlie differences in Slow Skeletal Muscle Troponin T on Cardiac Function motor functions among guinea pig jaw adductors and advance the under- Hanzhong Feng, Shirin Akhter, Hui Wang, Jian-Ping Jin. standing of the marked compartmentalization among mammalian craniofa- Physiology Department, Wayne State University, Detroit, MI, USA. cial muscles. Toad heart exclusively utilizes slow skeletal muscle troponin T (ssTnT) instead of cardiac TnT (cTnT) with increased resistance against afterload Posters: Cardiac Muscle Regulation I to compensate for decreases in blood volume during dry seasons. Sequence alignment revealed a unique Gly in the tropomyosin-binding site 1 of toad 694-Pos Board B464 ssTnT instead of Ala in fast and slow skeletal muscle TnT and Ser in CA2D-Induced Movement of Tropomyosin on Native Cardiac Thin cTnT (Ser125 in mouse cTnT) of all other vertebrate species. We found Filaments Revealed by Cryoelectron Microscopy that S125G substitution in mouse cTnT increases the binding affinity for Cristina Risi1, Betty Belknap1, David H. Heeley2, Howard D. White1, tropomyosin and studied the functional effect of this polymorphism in trans- Gunnar Schro¨der3, Vitold E. Galkin1. genic mouse hearts over-expressing cTnT-125G. Two original transgenic 1Department of Physiological Sciences, Eastern Virginia Medical School, mouse lines were established. Quantitative RT-PCR showed comparable Norfolk, VA, USA, 2Department of Biochemistry, Memorial University levels of cTnT-125G and wild-type (WT) cTnT mRNAs, plausibly represent- of Newfoundland, Newfoundland, NL, Canada, 3Structural Biochemistry ing the heterozygote state during evolutionary selection. cTnT-125G protein (ICS-6), Institute of Complex Systems, Julich,€ Germany. was incorporated into cardiac myofibrils as determined by Western blotting Muscle contraction relies on the interaction of myosin motors with F-actin, and mass spectrometry. Functional studies using ex vivo working heart prep- which is regulated through a translocation of tropomyosin by the troponin arations found increased left ventricular stroke volume as compared with that

BPJ 8573_8577 Sunday, February 18, 2018 139a of WT hearts. An interesting finding was that cTnT-125G mouse hearts pro- dimers, and is independent of the rest of the thick filament. This work duced a significant amount of N-terminal truncated cTnT (cTnT-ND) (14% was supported by NIH AR032961 & AR057220 (DDT), American Heart of total TnT) that was nearly non-detectable in WT hearts. Previous studies Association Scientist Development Grant (JMM), and Graduate Excellence have showed that cTnT-ND is generated by restrictive proteolysis in Fellowship-University of MN (JAR). response to ischemia or high afterload and remains in the cardiac myofila- ments to tune down systolic velocity and elongate ejection time to sustain 699-Pos Board B469 stroke volume against afterload . Therefore, the S125G single amino acid Signaling Pathways Affected in Cardiac Cells by Ibuprofen substitution in cTnT to mimic a feature of toad ssTnT may modulate cardiac Shuchita Tiwari, Aldrin Gomes. muscle contractility via directly altering the interaction with tropomyosin University of California, Davis, Davis, CA, USA. and the production of cTnT-ND. Characterization of this polymorphic site Ibuprofen is a commonly used nonsteroidal anti-inflammatory (NSAID) drug. identifies a potential target for designing approaches to improve cardiac func- The anti-inflammatory and analgesic benefits of ibuprofen are mediated tion for the treatment of heart failure. through the inhibition of prostaglandins production by blocking cyclo- oxygenase (COX) activity. Despite its analgesic and anti-inflammatory ef- 697-Pos Board B467 fects, ibuprofen is associated with increased risk of cardiovascular side Hypertrophic Cardiomyopathy Mutations Disrupt Human Beta Cardiac effects. The mechanism that governs ibuprofen-mediated cardiovascular de- Myosin Intramolecular Interactions Leading to Increased Myosin Activity fects is still not clear. The role of ibuprofen on heart function is not well un- Arjun S. Adhikari, Darshan V. Trivedi, Saswata S. Sarkar, derstood. In this study we used a label-free quantitative proteomic approach as Kathleen M. Ruppel, Spudich A. James. well as several biological assays to compare protein expression and mitochon- Biochemistry, Stanford University, Stanford, CA, USA. drial function between different ibuprofen treated (200mM) and vehicle treated Hypertrophic cardiomyopathy is caused by mutations in beta-cardiac myosin rat cardiac H9c2 cells. H9c2 cells were treated with ibuprofen for 12 days. and is characterized by increased cardiac contractility. Myosin ensemble force Mass spectrometry data (n=4 for each group) was analyzed by MaxQuant depends upon its duty ratio, intrinsic force and the number of available heads and PEAKs. Several processes were found to be statistically significantly (NA). We have hypothesized that NA may be regulated by altering the propor- altered in the ibuprofen treated cells inducing metabolic and developmental tion of myosin heads in an ‘‘off-state’’. Recent work from our lab suggests processes as well as processes involved in intracellular protein localization. that HCM mutations located at the interface between the myosin head (S1) Serine/threonine-protein phosphatase PP1, known to be important in the con- and tail (S2) disrupt the myosin folded back state, potentially switching the trol of apoptosis, cell division, glycogen metabolism, mitosis, muscle contrac- myosin heads from an ‘‘off-state’’ to an ‘‘on-state’’. This may increase NA tion, protein synthesis, and splicing of RNA, was upregulated in ibuprofen and thereby increase myosin contractility at the molecular level. To investi- treated cells. Independent studies measuring mitochondrial complex I, II, gate how HCM mutations at the S1-S2 interface (R249Q, H251N) and the and III activities showed that ibuprofen treated cells affected mitochondrial S1-S1 interface (R719W, D382Y) alter myosin activity, we introduced each function. Immunocytochemistry studies showed ibuprofen increased reactive mutant into two different two-headed myosin constructs containing either a oxygen species (ROS) produced in these cells. These results show that 25-heptad myosin S2 tail (25-hep) or a minimal 2-heptad tail (2-hep). We physiologically relevant amounts of ibuprofen affect mitochondrial function, hypothesized that 25-hep myosin will be able to fold back and form these in- increase ROS production may alter key cellular proteins involved in various tramolecular interactions, thereby sequestering heads and leading to lower cellular processes. Our results suggest that ibuprofen has several effects on myosin catalytic activity. Conversely, the 2-hep myosin, which lacks the the cell unrelated to ibuprofen’s function as a pain reliever or anti- necessary proximal tail, would be unable to form these interactions and would inflammatory action. have higher catalytic activity. We tested this hypothesis using an actin- activated ATPase assay. The ATPase activity of WT 25-hep was 43% than 700-Pos Board B470 that of WT 2-hep, suggesting that the WT 25-hep myosin is in an off-state. Suppressing Detyrosinated Microtubules Improves My-Cute Function in However, for each mutation tested, the 25-hep activity was only 5-15% lower, Human Heart Failure 1 1 2 indicating that these mutations disrupt the myosin intramolecular interactions, Christina Yingxian Chen , Matthew A. Caporizzo , Kenneth Bedi , Michael P. Morley2, Kenneth B. Margulies2, Benjamin L. Prosser1. thereby increasing NA and leading to increased contractility. Moreover, single 1Department of Physiology, Pennsylvania Muscle Institute, University of ATP turnover experiments also suggest that a higher fraction ( 80%) of the 2 HCM-mutant 25-hep myosins are in an open state as compared to WT 25-hep Pennsylvania, Philadelphia, PA, USA, Department of Medicine, University (40%). of Pennsylvania, Philadelphia, PA, USA. Detyrosinated microtubules (dTyr-MT) can increase myocyte stiffness 698-Pos Board B468 and provide an internal resistance during contraction. Levels of dTyr- Detection of the Super-Relaxed State in Cardiac Heavy Meromyosin tubulin are inversely correlated with left ventricular ejection fraction in John A. Rohde, Lien Phung, David D. Thomas, Joseph M. Muretta. patients with hypertrophic cardiomyopathy, yet the functional contribution Department of Biochemistry, Molecular Biology and Biophysics, University of dTyr-MT is unexplored in failing hearts or in humans. To characterize of Minnesota, Minneapolis, MN, USA. cytoskeletal alterations in human heart failure, we performed mass The actin-independent ATP turnover of single-headed myosin (S1) and two- spectrometry of left ventricle tissue samples from patients with heart dis- headed heavy meromyosin (HMM) was detected with mantATP, revealing ease of varying origin and severity. Proteomic analysis revealed a broadly differences consistent with the hypothesized super-relaxed state (SRX) in conserved upregulation and stabilization of intermediate filaments and HMM. Activated muscle generates force as actomyosin rapidly cycles MTs during the progression of human heart failure, with concomitant ATP. In relaxed muscle the turnover of ATP is reduced by two orders of down regulation of sarcomeric actin and myosin. Super-resolution imag- magnitude by preventing actin activation. In the SRX, myosin enters a ing demonstrated a denser and more detyrosinated MT network in sequestered state, with myosin heads folded back against the thick filament, myocytes isolated from failing human hearts, compared to myocytes and the rate of ATP turnover reduced by an additional order of magnitude. isolated from non-failing hearts. Meanwhile, failing myocytes showed In order to determine whether the SRX state exists in isolated myosin di- slower and smaller contractions than non-failing counterparts. Pharmaco- mers in solution, we have applied a mantATP chase experiment in vitro logical suppression of dTyr-MT reduced the viscoelasticity of failing to compare single-headed and double-headed bovine cardiac myosin over myocytes and restored 50% of the lost contractile function, while there a range of conditions: investigating temperature, ionic strength, phosphor- was less of an effect in non-failing myocytes. Similarly, genetically ylation, small molecule effectors, and interactions with myosin-binding reducing detyrosination softened cardiomyocytes and robustly improved protein C (MyBP-C). We find that HMM exhibits significantly decreased contractile kinetics. Taken together, our data demonstrate that a dense basal ATPase activity at low ionic strength, compared to S1, consistent and modified MT network impairs contractility in human heart failure, with a sequestered state involving head-head, head-RLC, and/or head-S2 in- and that targeting dTyr-MTs represent a novel inotropic strategy to improve teractions within HMM. We also find that the ATPase rate of HMM in- cardiac function. creases with increasing ionic strength, matching the ATP turnover rate of S1, while the rate of S1 turnover of ATP is unaffected over the same range 701-Pos Board B471 of ionic strengths. S1’s ATPase rate increases with increasing temperature, Dissecting the Molecular Mechanism for Familial Cardiomyopathies but HMM’s rate remains constant over the same range of temperatures. Sarah R. Clippinger, Lina Greenberg, Michael J. Greenberg. These results are consistent with a sequestered, super-relaxed state present Biochemistry & Molecular Biophysics, Washington University in St. Louis, in isolated HMM that depends on charge interactions within the myosin St. Louis, MO, USA.

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The heart needs to generate sufficient force and power to pump blood to the relation between amount of voluntary exercise and its effects on cardiac func- body and to adjust its power output in response to stresses. Mutations of con- tion in this model mouse. Homozygous DK210 (DCM) mice showed enlarged tractile proteins, such as cardiac troponin T, can lead to familial cardiomy- heart and frequent SD with t1/2 of 70 days. They were divided into 3 groups opathies, the leading cause of sudden cardiac death in people under the based on the frequency of voluntary wheel running at 1-month of age: 1) no ex- age of 30. Two common forms of cardiomyopathy, hypertrophic (HCM) ercise control, 2) intermittent exercise (every 2 day) and 3) daily exercise. To and dilated (DCM) are characterized by thickening or thinning of the ventric- exercise mice, they were housed with a running wheel. Following each dose of ular wall, respectively. However, how changes in contractile proteins at the exercise for 1 month, cardiac function was assessed by echocardiography. Mice molecular level lead to the disease phenotype is not well understood. To were sacrificed and their heart, lung, lower extremity muscles (soleus, plantaris address this problem, we examined two point mutations in human cardiac and gastrocnemius) and body weights were measured. Gene expressions of HF- troponin T (TnT), R92Q and DK210, which cause hypertrophic and dilated related genes were quantified by qPCR analysis.The weight of soleus muscle cardiomyopathy, respectively, in human patients. We have reconstituted was significantly increased in intermittent and daily exercise groups compared the calcium-based regulation of actomyosin in vitro using tissue purified with control. The mortality rates up to 2 months of age decreased to 12% in the and recombinant cardiac proteins. Similar to previous studies, in vitro exercise groups from 39% in the control group (n=18-25). In the echocardiog- motility assays which reconstitute the calcium-dependent interactions be- raphy, the cardiac contractility was greatly improved in the daily group, tween myosin and regulated thin filaments revealed that R92Q is shifted to- whereas somewhat improved in the intermittent group. Expression of HF- wards submaximal calcium activation while DK210 is shifted towards related genes were significantly reduced in both exercise groups. The daily supermaximal calcium activation. We dissected the molecular mechanism voluntary exercise appears to be most effective in preventing HF and SD in of these differential changes using a combination of fluorescence equilibrium DCM mice. titrations and stopped-flow experiments, and show that these effects are due to alterations in the free energies of the biochemical transitions that regulate 704-Pos Board B474 muscle activation. In particular, we see that these mutations differentially Therapeutic Effects of Ghrelin and Des-Acyl Ghrelin on Doxorubicin- affect calcium-dependent positioning of tropomyosin along the thin filament. Induced Cardiotoxicity We propose that these changes affect the regulation of force production by Miki Nonaka1, Nagomi Kurebayashi2, Takashi Murayama2, the molecular motor myosin, leading to alterations in mechanosensing by Masami Sugihara3, Hiroshi Hosoda4, Shosei Kishida5, Kenji Kangawa6, heart tissue. Takashi Sakurai2, Yasuhito Uezono1,7. 1Division of Cancer Pathophysiology, National Cancer Center Research 702-Pos Board B472 Institute, Tokyo, Japan, 2Department of Cellular and Molecular The EPAC2 Inhibitor ESI-05 Prolongs the Action Potential and Increases Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, Susceptibility to EAD Arrhythmias Japan, 3Department of Clinical Laboratory Medicine, Juntendo University Hannah M. Kirton1, Moza Al-Owais2, Chris Peers2, Derek S. Steele1. School of Medicine, Tokyo, Japan, 4Department of Regenerative Medicine 1 University of Leeds, School of Biomedical Sciences, Leeds, United and Tissue Engineering, National Cerebral and Cardiovascular Center 2 Kingdom, University of Leeds, Division of Cardiovascular and Diabetes Research Institute, Osaka, Japan, 5Department of Biochemistry and Genetics, Research, Leeds, United Kingdom. Kagoshima University Graduate School of Medical and Dental Sciences, 6 In the heart, the inotropic and lusitropic effects of b1-adrenergic stimulation Kagoshima, Japan, National Cerebral and Cardiovascular Center Research are known to involve the second messenger cAMP acting via protein Institute, Osaka, Japan, 7Division of Supportive Care Research, National kinase A (PKA) to increase phosphorylation of intracellular targets Cancer Center, Exploratory Oncology Research & Clinical Trial Center, including the L-type calcium channel, phospholamban and RyR2. In addi- Tokyo, Japan. tion, cAMP activates ‘exchange protein directly activated by cAMP’ Anthracycline doxorubicin (DOX), which has been used for cancer chemo- (Epac): a cAMP-activated guanine nucleotide exchange factor (GEF). How- therapy, often causes cardiotoxicity. Recent reports indicate that orexigenic ever, little is known about Epac signalling via its role as a GEF and effects hormone ghrelin and des-acyl ghrelin, which is a non-octanoylated form of mediated via downstream effectors such as the small GTPase Rap1. In the ghrelin, inhibit DOX-induced cardiotoxicity. However, molecular mecha- present study on adult rat ventricular myocytes (ARVMs), the Epac2 inhib- nisms of ghrelin and des-acyl ghrelin remain unclear. In the present study, itor ESI-05 induced activation of the late sodium current (INalate), prolonga- to provide mechanistic experimental evidence of these peptides against tion of action potential (AP) and early afterdepolarisation arrhythmias DOX-induced cardiotoxicity, we examined the effects of ghrelin and des- (EADs). Ranolazine inhibited both activation of INalate and AP prolongation acyl ghrelin on cell damage, cell death, apoptosis, and cardiac dysfunction by ESI-05. Active Rap1 (Rap1GTP) was detectable under basal conditions using an in vitro and in vivo system. H9C2 cardiomyocyte cells were in ARVMs and was reduced 50% by ESI-05. Moreover, the electrophysi- treated with DOX (0-1 mM) for 72h with and without ghrelin or des-acyl ological effects of ESI-05 were mimicked by downstream inhibition ghrelin (1 mM each). Ghrelin and des-acyl ghrelin significantly reduced of Rap1GTP activation with GGTI-298, suggesting that the electrophysio- the DOX-induced cell damage and cell death, with greater effects at des- logical effects of the drug may reflect impaired Epac2-Rap1 signalling. acyl ghrelin. In addition, ghrelin and des-acyl ghrelin prevented cell dam- ESI-05 induced AP prolongation was also blocked by preincubation with age and cell death through apoptosis, and des-acyl ghrelin but not ghrelin the mitochondria targeted antioxidant mitoTEMPO or inclusion of pyruvate significantly decreased ROS generation. In in vivo experiment, DOX was in the physiological saline. These data suggest that ESI-05 induced administered once at a dose of 15 mg/kg to 8 weeks old male C57BL/6 inhibition of Epac2-Rap1 signalling is associated with increased mitochon- mice intraperitoneally. At 1 day before DOX treatment, we started drial ROS production, activation of INalate, AP prolongation and EAD subcutaneous administration to mice twice daily with des-acyl ghrelin arrhythmias. (100 mg/kg) for 8 consecutive days. The in vivo experiment showed that des-acyl ghrelin prevented the progression in left ventricular ejection frac- 703-Pos Board B473 tion decrease in DOX-treated mice probably because of the decrease Relation between Voluntary Exercise Frequency and Cardiac Function in expression levels of non-phosphorylated Cx43. Our study showed that Dilated Cardiomyopathy Model Mice ghrelin and des-acyl ghrelin may have therapeutic effects in DOX- Masami Sugihara1, Ryo Kakigi2, Takashi Murayama3, Takashi Miida1, induced cardiotoxicity. Further, des-acyl ghrelin may have greater benefi- Takashi Sakurai3, Sachio Morimoto4, Nagomi Kurebayashi3. cial effects for the reduction of cellular ROS generation and expression 1 Department of Clinical Laboratory Medicine, Juntendo University, Tokyo, level of non-phosphorylated Cx43. Further studies are required to clarify 2 Japan, Department of Physiology, Juntendo University, Tokyo, Japan, the mechanisms how des-acyl ghrelin prevents the DOX-induced cardio- 3 Department of Phamacology, Juntendo University, Tokyo, Japan, toxicity and such experiments are underway. 4Department of Physical Therapy, International University of Health and Welfare, Fukuoka, Japan. 705-Pos Board B475 Dilated cardiomyopathy (DCM) is one of major causes of heart failure (HF). Role of Electrostatic Interactions in the Isoform-Specific Rate of ADP Though recent studies have demonstrated that exercise has beneficial effects Release from Human Cardiac Myosin on cardiac function of HF patients, effects of exercise on patients with inherited Akhil Gargey1, Jinghua Ge2, Yaroslav Tkachev3, Yuri Nesmelov2. DCM has not been established because DCM is associated with high risk of 1Biology, University of North Carolina at Charlotte, Charlotte, NC, USA, worsening HF and sudden death (SD). A knock-in mouse model of human in- 2Department of Physics and Optical Science, University of North Carolina at herited DCM, TNNT2 DK210, shows similar characteristics to DCM patients Charlotte, Charlotte, NC, USA, 3Engelhardt Institute of Molecular Biology and is useful model for evaluating therapeutic effects. We aimed to examine RAS, Moscow, Russian Federation.

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Human cardiac myosin has two isoforms, alpha and beta, coexisting in tion affected cation-induced conformational changes within troponin. healthy muscle. These isoforms share significant sequence similarity, but Our data demonstrated that the K15N mutation in Tpm1.1 affected Ca2þ- they are different in kinetics: ADP release from actomyosin is an dependent thin filament regulation while the R21H mutation had almost order of magnitude faster in alpha myosin isoform. Apparently, small dif- no effect. ferences in sequence are responsible for distinct local inter-residue interac- tions within alpha and beta isoforms, leading to particular rate of ADP 707-Pos Board B477 release. Previously we analyzed structural differences of alpha and beta iso- Very-Low-Density Lipoprotein of Metabolic Syndrome Suppresses Store- forms with molecular dynamics simulations, and found several places Operated Calcium Entry through Modulations of STIM1 in HL-1 Atrial within myosin head with distinct dynamics of local structural elements. Myocytes We hypothesize that isoform-specific electrostatic interactions play a role Hsiang-Chun Lee, Yi-Lin Shiou, I-Chieh Huang. in that difference. In this work we examined one of those places within Kaohsiung Medical University, Kaohsiung, Taiwan. myosin head, the force-generation region, and particularly helix SH1. Introduction: Metabolic syndrome (MetS) is a major risk for atrial myopathy According to our simulations, the helix experiences helix-loop transition and atrial fibrillation. Very-low-density lipoprotein (VLDL) of MetS (MetS- in alpha isoform. Our analysis shows that, within the force-generating VLDL) exerts in vivo and in vitro cytotoxicity to atrial myocytes. region, R694 displays one of the most marked differences between iso- Calcineurin-NFAT pathway, which is regulated by STIM1/Orai1-mediated forms. In beta isoform, this residue forms two permanent salt bridges, store-operated calcium entry (SOCE), is pivotal in mediating adaptive cardiac which are not present in alpha isoform. We prepared R694N mutant of hypertrophy. We hypothesized that MetS-VLDL can suppress SOCE and its beta isoform of human cardiac myosin to destabilize two salt bridges downstream calcineurin-NFAT pathway. in the force-generating region. The mutant supports ATPase activity. Methods & Results: The normal-VLDL and MetS-VLDL samples were We report on the kinetics of ADP release from actomyosin with and isolated from peripheral blood of healthy volunteers and individuals without mutation, and conclude on the role of electrostatic interactions with MetS, and used to incubate HL-1 atrial myocytes for 16 hours. Calcium imaging was facilitated with fura-2AM Ca2þ dye. After sarco- within the force-generating region on the kinetics of ADP release from 2þ actomyosin. plasmic reticulum (SR) Ca store was depleted with perfusion of thapsi- garin and caffeine in a Ca2þ -free solution, SOCE was triggered upon reperfusion of a 1.8 mM Ca2þ solution. Protein expression of STIM1, 706-Pos Board B476 Orai1, camodulin, CaMKII, NFAT, and calcineurin were quantified by Cardiomyopathy-Linked Mutation K15N in Tropomyosin Alters Calcium- Western blot. O-GlcNAylation of STIM1 and Orai1 were assessed by Dependent Regulation of Reconstituted Cardiac Thin Filaments þ immunoprecipitation. The SR Ca2 release was not changed in VLDLs- Thu N. Ly1, William Schlecht1,2, Mert Colpan1,3, Wen-Ji Dong1,2, treated groups. SOCE was significantlyattenuatedbyMetS-VLDL(0.59 Alla S. Kostyukova1. 5 0.03 folds to control, p< 0.0001), through reduced membranous expres- 1Voiland School of Chemical Engineering and Bioengineering, Washington sion of STIM1 (0.32 5 0.03 folds to control, p= 0.025), in conjunction with State University, Pullman, WA, USA, 2Department of Integrated increased O-GlcNAylation modification. MetS-VLDL also reduced nuclear Neuroscience and Physiology, Washington State University, Pullman, WA, translocation of NFAT and intra-nuclear calcineurin (all p< 0.05). These USA, 3Department of Cellular and Molecular Medicine, University of changes were associated with derangement of myofilament proteins Arizona, Tucson, AZ, USA. expression. In striated muscle, actin-based thin filaments and myosin-based thick fila- Conclusions: The adaptive hypertrophy response in atrial myocytes was ments are organized into basic contractile units called sarcomeres. Tropo- þ hampered by MetS-VLDL through inhibiting Ca2 -calcineurin-NFAT myosin (Tpm), a coiled-coil dimer, polymerizes head-to-tail along the pathway. The VLDL-induced STIM 1 modulation and SOCE suppression thin filament, stabilizes the filament and, together with the troponin þ may contribute to atrial myopathy in MetS. complex, regulates Ca2 -dependent actomyosin interactions which are mo- lecular prerequisites for muscle contraction. Troponin, consisting of 708-Pos Board B478 troponin I (TnI), troponin C (TnC), and troponin T (TnT), initiates muscle A Molecular Approach to Understand the Super-Relaxed State of Myosin contraction through a series of structural changes within the complex, Observed in Cardiac Muscle which is triggered by the binding of Ca2þ to TnC. The structural changes Saswata S. Sarkar*, Darshan V. Trivedi*, Makenna M. Morck, spread through TnT which in turn move Tpm’s position on the thin fila- Arjun S. Adhikari, Kathleen M. Ruppel, James A. Spudich. ment, enabling actomyosin interactions. In this work, we studied two Biochemistry, Stanford University, Stanford, CA, USA. missense mutations in striated muscle tropomyosin (Tpm1.1), K15N and *Equal Contribution R21H, which are associated with dilated cardiomyopathy and hypertrophic Cardiac myosin can undergo ATP hydrolysis at two different rates in cardiomyopathy, respectively. We investigated the mutations’ effects on relaxed cardiac muscle. The presence of the slower rate assigned to the Tpm1.1’s affinity to F-actin and on the Ca2þ-dependent regulation of recon- Super-relaxed state (SRX) of myosin is believed to be a novel way to mini- stituted cardiac thin filaments using Fluorescence Resonance Energy Trans- mize the energy consumption in the heart. In muscle, the cardiac myosin fer (FRET). We labeled residues 151 and 167 of cardiac TnI with can adopt a folded conformation where the head domains interact with their IAEDANS as FRET donors, and residue 89 of cardiac TnC with DDPM own tail. The presence of the folded state of myosin is hypothesized as FRET acceptor. We found that while both mutations reduced Tpm1.1’s to be the origin of SRX. Recent studies in the lab established that affinity to F-actin, the K15N mutation decreased Ca2þ-sensitivity our 2-headed cardiac myosin construct that contains the myosin tail domain and Ca2þ-dissociation-induced kinetics of reconstituted cardiac thin fila- can form the folded state. Here, we tested the formation of SRX using ments. In the presence of the K15N mutant, the interprobe distances were two 2-headed constructs differing in their ability to form the folded state greater than in the presence of wild type Tpm1.1, indicating that the muta- of myosin.

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Posters: Actin Structure, Dynamics, and difference in Myosin distribution between AWs and GC itself. Depletion of membrane Cholesterol and inhibition of RhoGTPases decreases AWs’ fre- Associated Proteins quency and velocity. Our results point out that AWs could have a main role in maintaining the GC active during the environment exploration. We also pro- 709-Pos Board B479 vide evidence that Myosin II, the membrane tension and small RhoGTPases act Insights into the Cooperative Nature of ATP Hydrolysis in Actin Filaments as important players in the regulation of the AW dynamics. Harshwardhan H. Katkar1, Aram Davtyan2, Aleksander E.P. Durumeric1, Glen M. Hocky1, Anthony Schramm3, Enrique M. De La Cruz3, 712-Pos Board B482 Gregory A. Voth1. The Mechanosensitivity of Actin Bundles 1University of Chicago, Chicago, IL, USA, 2Rice University, Houston, TX, Emiko Suzuki, Antoine Jegou, Guillaume Romet-Lemonne. USA, 3Yale University, New Haven, CT, USA. Institut Jacques Monod, Paris, France. Actin filaments continually assemble and disassemble in the cell cytoplasm, dur- Cells’ ability to sense their environment is essential for many cellular processes ing which they age as the nucleotides bound to newly added actin sub-units hy- including cell division, migration and morphogenesis. The actin cytoskeleton, drolyze from ATP to an intermediate ADP-Pi, followed by a slower release of Pi which has been shown to be mechanosensitive, is organized into different archi- to form ADP. This results into a spatial distribution of all three states of the bound tectures that carry out various functions within the cell. Filopodia, which are nucleotide along the growing filament. It is not fully understood whether ATP finger-like structures consisting of actin filaments bundled in parallel, emerge hydrolysis and Pi release in a growing actin filament are spatially cooperative at the cell front and orient the cell in response to its mechanical environment. or purely random. Using a bottom-up multi-scale coarse-grained modeling strat- These actin filaments are elongated at their barbed ends by formins and Ena/ egy, we design simulations to study cooperativity in these reactions and find that VASP and cross-linked by the bundling protein fascin. These two machineries the reactions are indeed cooperative to a certain extent. are thought to collaborate to design a unique type of actin network that governs At first level of coarse-graining, we construct an ultra-coarse-grained (UCG) filopodium dynamics, yet the exact mechanism by which these two key proteins model based on atomistic simulations of short actin filaments. The UCG model synergize, and how mechanosensing is achieved in actin bundles, are not well agrees with experiments in terms of mechanical properties such as persistence understood. Core questions such as: how actin filaments self-assemble in a length, which is governed by the bound nucleotide’s state. The model also predicts bundle; how forces are transmitted along filopodia; how fascin and formin syn- that states of the bound nucleotides of neighboring sub-units significantly modu- ergize to control the growth of actin filament in filopodia remain to be addressed. late the reaction kinetics, implying cooperativity especially in the slower Pi release To tackle these questions, we use a microfluidics-based approach to reconstitute, reaction. We further coarse-grain the system into a Markov state model that incor- in vitro, a minimal system to recapitulate the mechanosensitivy of actin bundles. porates assembly and disassembly, and the cooperativity predicted by the UCG We have probed the activity of the actin binding proteins formins and fascins in a model. The model reveals that cooperativity in ATP hydrolysis and Pi release large range of biochemical conditions : first on single filaments, then scaling up has significant effects on the filament growth dynamics, but only near the critical to bundles of several filaments. We have incorporated fluorescence polarization g-actin monomer concentration. Filament dynamics are robust to the mechanism into the microfluidics setup to characterize the rotational behavior of actin fila- of hydrolysis far from the critical concentration and both cooperative and random ments elongated by formin and bundled by fascin. This bottom-up approach al- mechanisms show similar growth dynamics. On the contrary, the filament compo- lows us to understand actin bundles mechanosensitivity and bring us closer to sition interms of the bound nucleotide distribution varies significantly at all mono- obtaining a comprehensive description of force generation and transmission in mer concentrations studied. These results provide new insights into the formin and fascin generated bundles. mechanism of ATP hydrolysis and its implications on actin filament properties. 713-Pos Board B483 710-Pos Board B480 Electrophoretic Cytometry Elucidates Stress-Induced Actin Cytoskeletal Nucleotide and Polymerization Effects on the Structure and Dynamics of Reorganization Actin Julea Vlassakis, Ryo Higuchi-Sanabria, Andrew Dillin, Amy E. Herr. Lauren Jepsen1, David Sept2. UC Berkeley, Berkeley, CA, USA. 1Department of Computational Medicine and Bioinformatics, University of The actin cytoskeleton dramatically reorganizes during many cellular pro- Michigan, Ann Arbor, MI, USA, 2University of Michigan, Ann Arbor, MI, cesses, such as endocytosis, cell division, and movement, and in response to USA. stress, such as heat stress, mechanical stress, and aging. Existing tools for Actin is one of the most highly conserved and abundant proteins found in eukary- measuring the distribution of filamentous (F) versus monomeric (G) actin otic cells. It participates in a vast number of protein-protein interactions, many of cannot accurately quantify F- and G-actin levels in individual cells. Fluorescent which are involved in the strict regulation of the polymerization and disassembly small molecule dyes, such as labeled phalloidin, are qualitative and subject to of actin filaments. Actin’s affinity for these interactions, as well as for polymer- competitive binding artifacts. Quantitative measurements rely on bulk ultracen- ization, is strongly dependent upon nucleotide state. To examine the effects of trifugation assays, which pellet (F-actin). However, bulk ultracentrifugation re- both nucleotide and polymerization on actin structure and dynamics, we per- quires millions of cells, averaging out cell-to-cell variation in actin distribution. formed multi-microsecond molecular dynamic simulations on both ADP and We present a single-cell electrophoretic cytometry assay that separates F- and ATP muscle G-actin, as well as two of the more recent F-actin models (2ZWH G-actin from individual cells. In a unique multi-layered microfluidic device, we and 3MFP). We find clear nucleotide state dependence on the structure and dy- isolate single cells in microwells stippled in a 40 micron-thick polyacryl- namics of actin’s target-binding cleft, where the majority of actin binding pro- amide gel. We sequentially apply F-actin stabilizing lysis buffer to the teins dock. In this cleft, both the C-terminal hinge loop (A331-Y337) and the microwell array, electrophorese, and immobilize G-actin in the gel. F-actin is FQQ-loop (S348-W356) have altered structure and dynamics, with the FQQ- size-excluded from entering the gel until a depolymerization buffer is applied, loop moving nearly 6 A˚ in the ATP state to partially close the cleft. The F-actin and the depolymerized actin is electrophoresed in the opposite direction of the simulations also reveal both structural and dynamic differences between the G-actin prior to immobilization. Both F- and G-actin species from hundreds of barbed and pointed ends that provide insight into differences in phosphate single cells are detected in-gel by immunofluorescence with minimized post- release and polymerization rates at the two ends of the filament. lysis handling. Validation of the cytometry assay is performed with cells treated with Latrunculin, a drug known to reduce the F-actin fraction in cells. 711-Pos Board B481 This quantitative assay will be applied to the measurement of actin reorganiza- Functions and Dynamics of Actin Waves tion upon heat shock. Moreover, we will determine the role of the master tran- Simone Mortal1, Federico Iseppon1, Andrea Perissinotto1, Elisa D’Este2, scriptional regulator, heat-shock factor 1 (HSF-1), in regulation of actin Dan Cojoc3, Luisa M.R. Napolitano1, Vincent Torre1. 1 2 integrity under conditions of heat stress by quantitatively measuring F- and International School for Advanced Studies, Trieste, Italy, Department of G-actin in cells harboring knockdown or overexpression of HSF-1. We antici- NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, 3 pate electrophoretic cytometry will become a critical tool in probing the distri- Go¨ttingen, Germany, CNR-IOM, Trieste, Italy. bution of the actin cytoskeleton in poorly understood cellular stress responses. During neurite development, Actin Waves (AWs), growth cone-like structures, emerge at the neurite base and move up to its tip, causing a transient retraction 714-Pos Board B484 of the Growth Cone (GC). Using long term live cell-imaging, we show that Cytoskeletal Remodeling during Oxidative and Thermal Stress AWs do not boost neurite outgrowth: even neurites without AWs can elongate Federico Sesti, Rahul Patel. for several hundred microns. Inhibition of Myosin II abolished GC retraction Neuroscience and Cell Biology, Rutgers, Piscataway, NJ, USA. and strongly modify the AWs morphology. Consistently, STED nanoscopy We used the nematode C. elegans to gain insight into the role of Rho signaling highlighted that Myosin II contribute to AWs ultrastructure and there is a clear during two common environmental challenges, oxidative and thermal stress. In

BPJ 8578_8581 Sunday, February 18, 2018 143a response to heat shock (HS), wild type (N2) worms demonstrated reduced 4Advanced Clinical Biosystems Institute, Cedar-Sinai Medical Center, Los viability which was rescued by genetic suppression of CDC42 and RHO-1. Visu- Angeles, CA, USA. alization of F-actin by phalloidin-rhodamine underscored a strict correlation be- Every biological process, ranging from cell migration to embryogenesis, relies tween the levels of F-actin following GTPase suppression and survival. on the cell’s ability to adapt to changing mechanical environments. By studying Additionally, genetic ablation of OSG-1, a Guanine Nucleotide Exchange Factor the model shape change process cytokinesis in Dictyostelium, we find that the (GEF) previously implicated in oxidative stress, was associated with constitu- cell is a finely tuned control system, with proteins that modulate their behavior tively lower levels of F-actin and increased mortality. However, upon an oxida- in response to mechanical and biochemical signals. Although we know many of tive insult F-actin stability decreased in N2 worms, a rescue of this affect was the players involved in this control system, their biochemical interactions that observed in OSG-1 null worms, consistent with the resistance exhibited by these allow force propagation through the cortical network are still poorly defined. worms to oxidative stress (OS). Together these data suggest that during condi- To identify the direct interactions that govern a cell’s mechanical response, tions of thermal or oxidative stress Rho signaling promotes vulnerability by we performed immunoprecipitation followed by mass spectrometry on two altering actin dynamics. Thus, the stability of the actin cytoskeleton, in part key nodes of the control system, the scaffolding protein IQGAP2 and the actin through a conserved mechanism mediated by Rho signaling, is a crucial factor crosslinker cortexillin I. This analysis identified myosin II as a biochemical in- for the cell’s survival to environmental challenges. teractor of both cortexillin I and IQGAP2. Using fluorescence cross-correlation spectroscopy (FCCS) to measure concentrations and apparent binding coeffi- 715-Pos Board B485 cients, we have confirmed these interactions in live cells. Interestingly, the Effects of Mechanical Stress on Periodontal Ligament myosin II-IQGAP2 and cortexillin I-IQGAP2 interaction in vivo KD’s decrease Ayano Fujita1, Masatoshi Morimatsu1, Masayoshi Nishiyama2, by approximately 5-fold in an iqgap1 null. This, in combination with stoichi- Shogo Takashiba1, Keiji Naruse1. ometry information from Single Molecule Pulldown, suggests a potential 1 Okayama University Graduate School of Medicine, Dentistry and mechanism for the role of IQGAP1 as a negative regulator on the myosin II- 2 Pharmaceutical Sciences, Okayama City, Japan, Human Health Sciences, cortexillin I mechanoresponsiveness. In addition, we found a few unusual inter- Graduate School of Medicine, Kyoto University, Kyoto City, Japan. actors by mass spectrometry analysis, such as RNP1, discoidins, and methylma- Periodontal ligament (PDL), which connects the teeth to the alveolar bone, is always lonyl semialdehyde dehydrogenase (mmsdh), which we previously identified in exposed to mechanical stress such as occlusal force. The occlusal pressure due to genetic suppressor screens, providing greater evidence for their function in the chewing is 10 MPa. Mechanical stress modifies periodontitis caused by bacterial network. Using the cytokinesis control system as a model, we are identifying a infections. The response of PDL cells to mechanical stress has been studied by many quantitative interaction map, complete with new interactions, that is uncover- dentists and researchers. However, it was difficult to obtain the quantitative data ing new biochemistry associated with the mechanobiome. related to mechanical stress effect on PDL in vivo. Here we studied the effects of stretch and pressure on PDL cells. First, we cultured PDL cells under uniaxial cyclic 718-Pos Board B488 stretch for 16 hours. After 4 hours, PDL cells and actin fibers were aligned in the PKC Phosphorylation and mu-calpain Truncation of the C-Terminal End vertical direction to the stretch axis. These results suggested that mechanical stress Segment of SM22alpha Regulates its F-Actin Binding and Mechanical regulate the orientation of PDL cells to support the teeth. Second, we used high hy- Tension Modulated Degradation drostatic pressure microscope to observe PDL cells under high pressure in real time. Hui Wang, M. Moazzem Hossain, Jian-Ping Jin. As a result, high hydrostatic pressure (> 20 MPa ) contracted PDL cells, but did not Physiology, Wayne State University, Detroit, MI, USA. change the actin bundle structure. The length of minor axis decreased to 60 %. SM22alpha, also named transgelin, is an actin filament-associated protein spe- After the release of pressure, contracted cells restarted spreading on the surface. cifically expressed in mature smooth muscle cells and myofibroblasts. Three Our data suggested that excessive occlusal force induces the collapse of PDL and decades after its discovery, the biological function of SM22alpha remains un- occlusal force at biological range affects homeostasis of PDL. der investigation. We recently reported a novel finding that the gene expression and proteolytic degradation of SM22alpha are both regulated by mechanical 716-Pos Board B486 tension in the cytoskeleton (Liu et al., Biochemistry, in press, 2017). Following Modeling of Actomyosin Networks with a Molecular Underpinning of mass spectrometry characterization of an intermediate product of SM22alpha Cross-Linker Proteins degradation in tension-unloaded mouse aorta revealing a mu-calpain catalyzed James Liman1,2, Yossi Eliaz2,3, Herbert Levine1,2, Margaret S. Cheung2,3. truncation of the C-terminal 22 amino acids, here we constructed a mutant 1Department of Bioengineering, Rice University, Houston, TX, USA, 2Center cDNA to express the C-terminal truncated SM22alpha in E. coli culture and for Theoretical Biological Physics, Rice University, Houston, TX, USA, studied the purified protein for PKC-catalyzed phosphorylation and F-actin 3Department of Physics, University of Houston, Houston, TX, USA. binding activities. The results have shown that the C-terminal truncation deletes Dendritic spines are small membranous protrusions of actomyosin networks a PKC phosphorylation site. With an actin-binding site in the C-terminal attached to neuronal dendrites whose morphological plasticity is hypothesized segment of SM22alpha demonstrated in amino acid sequence alignment with to underlie learning and memory formation. In this work, we present a novel calponin, anther mechanoregulatory protein in the cytoskeleton, the C-terminal computational and theoretical approaches to model contractility and growth in truncated and PKC phosphorylated SM22alpha are examined for their binding actin networks to evaluate the spatiotemporal patterns of the system due to actin to F-actin. These experimental studies aim at determining the role of PKC phos- remodeling. We consider cases where both motor and cross-linker proteins are pre- phorylation and mu-calpain truncation of the 22 amino acids C-terminal end sent inthe systems and their contractility are observed. Then we demonstrate a fila- segment in regulating the association of SM22alpha to the actin cytoskeleton mentous actin network projection to outline a simple physical model for under mechanoregulation. The results will serve as groundwork for understand- contractility. On top of that we introduce a computational and theoretical assess- ing the functions of SM22alpha/transgelin in the contractility of smooth muscle ment of the network entropy, its information capacity and evolution. Lastly, we cells and the differentiation and function of myofibroblasts in various patholog- propose a landscape model for the entropy and a stochastic time dependent evolu- ical conditions such as tumorigenesis, wound healing, myocardial remodeling, tion model from which emerge tractable reaction coordinates. Furthermore, we lay fibrosis and calcified aortic valve disease. out the foundations for 2D and 3D contraction, also known as surficial and volu- metric contraction respectively for better understanding the network-boundary 719-Pos Board B489 interaction in the limit of a flexible boundary. This theory introduces a comprehen- Modeling Pulling Force Generation by Ensembles of Polymerizing Actin sive model toanalyze the time evolution and statistics of actomyosinnetworks. We Filaments expect these new approaches to give an insight into the important first steps of den- Fowad Motahari, A.E. Carlsson. dritic spines morphological formation enabled by contractility and growth of the Physics, Washington University in St. Louis, St. Louis, MO, USA. actin cytoskeleton in response to incoming synaptic signals due to synaptic activ- Actin polymerization in yeast is the primary mechanism for overcoming the ity. This work is supported by the National Science Foundation CHE 1743392 and large turgor pressure that opposes clathrin-mediated endocytosis. While gen- the Center for Theoretical Biological Physics PHY 1427654. eration of pushing forces by actin polymerization is fairly well understood, it is not clear how actin polymerization produces pulling forces. Previous work 717-Pos Board B487 using a finite element approach has suggested that continuous spatial variation Visualizing Direct Interactions in the Mechanobiome of the polymerization rate can generate pulling forces1. We extend this work Priyanka Kothari1, Vasudha Srivastava2, Vasudha Aggarwal3, by performing calculations on a discrete system of actin filaments with all Irina Tchernyshyov4, Jennifer Van Eyk4, Taekjip Ha3, Douglas N. Robinson1. subunits explicitly treated. We stochastically simulate growing arrays of up 1Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD, USA, to 200 semiflexible actin filaments in a square network, where each filament 2University of California San Francisco, San Francisco, CA, USA, interacts with the membrane via an interaction potential that has both attrac- 3Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA, tive and repulsive components. The crucial protein Sla2, which binds actin

BPJ 8578_8581 144a Sunday, February 18, 2018

filaments to the membrane, is assumed to contribute a strongly attractive attached to the end. FH2-barbed end complexes exist in a rapid equilibrium be- component to the potential. The polymerization and depolymerization rates tween polymerization-competent and -incompetent states, so polymerization depend on the filament-membrane gap. We model the elasticity of the actin mediated by FH2 domains is relatively slow. This effect can be overcome by bind- network by linear springs connecting adjacent filaments to each other. The in- ing of actin monomers to profilin, which also binds polyproline tracts in formin’s ner filaments of the array are taken to correspond to a Sla2 patch. They are flexible FH1 domain, located directly N-terminal to the FH2 domain. Diffusion of thus bound more strongly to the membrane, and grow more slowly. The simu- FH1 domains brings associated profilin-actin complexes into contact with the lation results show that the outer filaments push on the membrane, while the FH2-bound barbed end, promoting direct transfer of actin and speeding elonga- inner filaments pull on it. We calculate the total pulling force as a function of tion. As such, formins present multiple pathways for the addition of incoming several model parameters, including the potential depths, the free filament on- monomers to an elongating barbed end. We investigated the relative contributions and off-rates, the numbers of fast- and slow-growing filaments, the filament of FH1- and FH2-mediated pathways for elongation to the overall rate of polymer- tip stiffness, and the network rigidity. ization promoted by Bni1p, a formin from S. cerevisiae. We found that the elon- [1] A. E. Carlsson and P. V. Bayly, Biophys. J. 106:1596-1606(2014). gation rates mediated by the FH1 and FH2 domains are additive, suggesting that these domains contribute to elongation in an independent manner. In contrast, the 720-Pos Board B490 fourpolyproline tracts in the FH1 domain ofBni1promote elongation in a compet- Formin’s Processivity under Applied Force itive manner. This competition is not position-specific, and thus does not result LuYan Cao, Mikae¨l Kerleau, Antoine Jegou, Guillaume Romet-Lemonne. from steric clashes arising from binding of profilin-actin to neighboring binding Institut Jaques Monod, Paris, France. sites. Rather, our results suggest that multiple FH1-bound profilin-actin com- Formin, a key actin regulator, is involved in a number of pathologies. It is able plexes compete for delivery to the same binding site at the barbed end. This to keep tracking the barbed end of actin filaments for a finite time, while accel- competition among polyproline tracts may limit the number of profilin binding erating actin polymerisation in the presence of profilin. In cells, formin’s sites in formin FH1 domainsand may tie this number to FH2 domain gating, which behaviour is precisely regulated by different factors, including local actin/pro- controls the availability of the barbed end for binding. filin concentration, mechanical forces and other actin associated proteins. Clar- ifying the mechanism of formin’s processive movement is essential in order to 723-Pos Board B493 understand formin’s behaviour. However, so far, how formin’s processivity re- Multiscale Model of the Formin Homology 1 Domain Illustrates its Role in sponds to various chemical or mechanical conditions is still unclear. Regulation of Actin Polymerization Here, we investigated how formin dissociates from the barbed end for different Brandon G. Horan1,Gul€ Zerze2, Gregory L. Dignon2, Young C. Kim3, actin/profilin concentration and/or for various applied pulling forces, in order to Dimitrios Vavylonis1, Jeetain Mittal2. answer 1) whether profilin has an impact on formin’s processivity; 2) how for- 1Physics, Lehigh University, Bethlehem, PA, USA, 2Chemical and min’s processivity responds to applied forces. Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA, 3Center We found that formin’s dissociation rate (koff) from actin barbed end has a pos- for Materials Physics and Technology, Naval Research Laboratory, itive correlation with its elongation rate (Velong) at fixed profilin concentration. Washington, DC, USA. However, when the concentration of profilin is increased, koff/Velong is Formins are important actin regulators. Formins bind profilin to polyproline tracks decreased. These results indicate that formin’s processive movement is of its believed flexible Formin Homology (FH) 1 domain. The FH2 domains wrap improved by the presence of profilin. around the barbed end of the actin filament and elongate the filament processively. Also, we found that formin’s processivity is very sensitive to applied pulling Profilin-actin complexes on the FH1 domain are modeled to transfer to the barbed forces. Formin’s dissociation rate increases exponentially with force, even at end; however, the mechanism is not known. Previous models of the FH1 domain < relatively small force ( 10 pN) is applied. Moreover, we found that force have not captured sequence-specific effects such as the length and distribution of has a dominant impact on formin’s processivity, for all actin/profilin concentra- the polyproline tracks and possible variety in mechanosensitivity and response to tion that we tested, irrespective of the elongation rate. bound profilin/profilin-actin. To remedy this, we perform simulations of the FH1 Finally, our study of formin’s processivity will contribute to develop a more domain of well-studied formins: the mouse formins mDia1 and mDia2, the comprehensive model to describe formin’s behaviour, especially how formin budding yeast formins—Bni1 and Bnr1, and the fission yeast formins—Cdc12, responds to mechanical forces in cells. Fus1, and For3. We perform all-atom molecular dynamics simulations of each 721-Pos Board B491 of these FH1 domains and show that FH1 is a typical intrinsically disordered pro- MDIA1 Senses Both Force and Torque during F-actin Filament tein (IDP), with the polyproline tracks forming high propensity poly-L-proline he- Polymerisation lices. We develop an alpha-carbon coarse-grained model that retains the Miao Yu1, Xin Yuan1, Michael Sheetz1,2, Alexander Bershadsky1,3, sequence-specificity of the FH1 domain which is consistent with the IDP notion Jie Yan1,4. of FH1, and use this to study the FH1 domain in the context of its biological 1Mechanobiology Institute, Singapore, Singapore, 2Columbia University, role. We use the coarse-grained model to investigate the response of FH1 to force New York, NY, USA, 3Weizmann Institute of Science, Rehovot, Israel, and bound profilin. We show how bound profilin/profilin-actin may extend the 4Department of Physics, National University of Singapore, Singapore, FH1. We show definitively that multiple profilin-actin complexes can simulta- Singapore. neously bind to the FH1, which may be biologically important given the relatively Formins play a crucial role in regulating actin polymerization. A formin dimer high concentration of profilin-actin in vivo. We examine the transfer mechanism binds to the barbed end of F-actin through its ring-like dimerized FH2 domains in further detail. We show how the FH1 may be affected by being on the FH2- and recruits G-actin monomers through its proline-rich FH1 domains in a pro- bound actin filament barbed end rather than in isolation and examine the geometry filin dependent manner. Formins are often subject to mechanical stretch due to of this system with bound profilin-actin. actomyosin contraction; therefore its function has been believed to be depen- dent on mechanical force. We have recently investigated the effects of force 724-Pos Board B494 on formin-dependent actin polymerization. In both the presence and the The Drosophila Formin FHOD Nucleates Actin Filaments 1 2,3 1 absence of profilin, we observed that force could strongly promote actin poly- Aanand A. Patel , Zeynep A. Oztug Durer , Aaron P. van Loon , 2 2,4 merization, which strictly required that the actin filament could freely rotate Kathryn V. Bremer , Margot E. Quinlan . 1Molecular Biology Interdepartmental Doctoral Program, University of around the formin. These results revealed that formins senses not only tensile 2 force but also torque in the filament. Together, these results provide important California, Los Angeles, Los Angeles, CA, USA, Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA, new insights into the mechanosensing functions of formin-dependent actin or- 3 ganization in living cells. USA, Department of Biophysics, Acibadem University School of Medicine, Istanbul, Turkey, 4Molecular Biology Institute, University of California, Los 722-Pos Board B492 Angeles, Los Angeles, CA, USA. Competition Among Multiple Pathways for Subunit Addition in Formin- Formins are a conserved group of proteins that nucleate and processively elon- Mediated Actin Filament Elongation gate actin filaments. Among them, the formin homology domain-containing Mark E. Zweifel, Naomi Courtemanche. protein (FHOD) family of formins contributes to contractility of striated muscle Genetics, Cell Biology and Development, University of Minnesota, and cell motility in several contexts. However, the mechanisms by which they Minneapolis, MN, USA. carry out these functions remain poorly understood. Mammalian FHOD pro- The formin family of proteins nucleates and directs the growth of unbranched teins were reported not to accelerate actin assembly in vitro, and they have actin filaments. Formin-mediated elongation of actin filaments is accomplished instead been suggested to act as barbed end cappers or bundlers. Here, we via association of dimeric Formin Homology 2 (FH2) domains with filament show that purified Drosophila Fhod and human FHOD1 both accelerate actin barbed ends, where they enable subunit addition while remaining processively assembly by nucleation. FHOD1’s nucleation activity is restricted to

BPJ 8578_8581 Sunday, February 18, 2018 145a cytoplasmic actin, whereas Drosophila Fhod potently nucleates both cyto- demonstrated that FAB domain favors to interact with the cleft between subdo- plasmic and sarcomeric actin isoforms. We found that Drosophila Fhod binds main (SD) 1 and SD 3 of the actin subunits, which is in a good agreement with a tightly to barbed ends, where it slows elongation in the absence of profilin and previous hypothesis. To understand the factors affecting the processivity of allows elongation in the presence of profilin. Fhod protects barbed ends from Ena/VASP at the barbed end of crosslinked filaments, we generated coarse- capping protein, but dissociates from barbed ends relatively quickly. Finally, grained (CG) model of this structure by using a bottom-up approach, and we used cosedimentation assays to determine that Fhod binds the sides of actin then elaborated this CG structure to include four arms and two actin filaments filaments and bundles filaments. This work establishes that Fhod shares the ca- connected with a cross-linker. We varied the number of arms in the CG model pacity of other formins to nucleate and bundle actin filaments, but is notably and investigated differences between their behaviors to understand the under- less effective at processively elongating barbed ends. lying mechanism of decreasing processivity with reduced number of arms as observed in the experiments. Our results showed that Ena/VASP becomes 725-Pos Board B495 less stable on the filament when the number of arms decrease, which might Cofilin Induces a Local Change in the Twist of Actin Filaments be one of the factors that causes reduced processivity of Ena/VASP. The find- Andrew R. Huehn, Wenxiang Cao, W. Austin Elam, Enrique De La Cruz, ings of this work can be used to resolve the underlying molecular mechanisms Charles V. Sindelar. of actin network assembly/disassembly interacting with Ena/VASP. Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. 728-Pos Board B498 Cofilin is a small protein that binds and severs actin filaments near the bound- Binding of the N2A Region of Titin to Actin Filaments aries between cofilin-decorated and bare actin segments. Cofilin binds cooper- Christopher M. Tsiros, Humra Athar, Matthew Gage. atively to actin by inducing conformational changes that propagate along the UMass Lowell, Lowell, MA, USA. helical lattice to neighboring bare actin segments, favoring cofilin binding. The sliding filament model has been the foundation of our understanding of However, the extent to which these cooperative conformational changes prop- muscle contraction, explaining contraction as the calcium-dependent formation agate remains unclear and prior estimates for the propagation length vary of cross-bridges between actin and myosin filaments. While this has been the widely; it has been suggested that anywhere from 2 to 24 actin subunits extend- prevailing model for muscle contraction for over 50 years, a weakness of model ing into the bare region could be affected. We address this unresolved question is its inability to account for all the measurable forces observed in muscle by imaging partially cofilin-decorated actin filaments using cryo-electron mi- contraction. Recently, it has been suggested that interactions between the titin croscopy and determining the location of boundaries between decorated and filament and actin could be the missing component in existing models. The undecorated regions at the subunit level using a novel particle subtraction N2A region of titin sits in a unique region of titin, between the Ig-domain re- and 3D classification strategy. We used characteristic changes in filament twist gion that is elongated under low forces and the PEVK region, which is extended induced by cofilin as a marker for cooperative changes in actin, and tracked under higher forces. In addition, the muscular dystrophy with myositis (mdm) these changes with respect to the boundary. Our results indicate that cofilin in- mice contain a deletion at the junction between the N2A and PEVK regions and duces a change in twist that propagates at most 2 subunits away from the muscles from these mice exhibit altered contractile properties. Based on these boundary, irrespective of the polarity of the boundary (i.e. whether the bare observations, we hypothesized that the N2A region might be a site of interac- side of the boundary extends toward the pointed or barbed end of the actin fila- tion between titin and actin and have used actin co-sedimentation and actin ment). These observations provide direct experimental support for nearest- motility assays to investigate this possibility. We have demonstrated that neighbor models describing cofilin-binding cooperativity, and inform mecha- recombinantly-expressed N2A constructs co-sediment with filamentous actin, nistic models for cofilin-mediated actin severing. suggesting an association between N2A and actin. In addition, in vitro motility 726-Pos Board B496 data demonstrates reduced actin filament velocity in the presence of N2A, Mechanotransmission and Mechanosensing of Human Alpha-Actinin 1 further supporting a binding interaction between N2A and actin. This work Shimin Le1, Xian Hu2, Mingxi Yao2, Hu Chen3, Michael P. Sheetz2, demonstrates that binding between actin and titin occurs in vitro and future Jie Yan1. work will be focused on demonstrating that this interaction also occurs in 1Department of Physics, National University of Singapore, Singapore, vivo to elucidate the role of this interaction in muscle contraction. Singapore, 2Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 3Department of Physics, Xiamen University, Xiamen, 729-Pos Board B499 China. Stochastic Simulations of Tropomyosin Binding and Diffusion on Alpha-actinins play crucial roles in organizing the framework of the cytoskel- Filamentous Actin Mikkel H. Jensen1, Ashley Luiz1, Hai Tran2. eton through crosslinking the actin filaments. However, the molecular mecha- 1 2 nisms underlying its functions are still incompletely understood. In this work, Physics, California State University, Sacramento, CA, USA, Chemistry, by mechanical manipulation of single human alpha-actinin 1 using magnetic California State University, Sacramento, CA, USA. tweezers, we determined the mechanical stability and kinetics of the functional The actin-binding protein tropomyosin plays a key role in regulating both the domains in alpha-actinin 1 as well as the mechanical strength of the alpha- interaction of filamentous actin (F-actin) with other binding proteins, as well actinin 1 dimerization interaction. Moreover, we identified the force- as the bending rigidity and biochemistry of F-actin itself. Tropomyosin binds dependence of vinculin binding to alpha-actinin 1, with the demonstration to F-actin in a 1:7 ratio, with one tropomyosin unit covering 7 actin monomers. that force is required to expose the high-affinity binding site for vinculin bind- Tropomyosin units can form end-to-end bonds with their neighbors, and have ing. Based on the mechanical stability and kinetics of alpha-actinin 1, a novel been experimentally observed to nucleate and form tropomyosin chains on the role of the alpha-actinin 1 as molecular shock absorber for the cytoskeleton actin filament, which can elongate and decorate the actin filament. However, network is revealed. Our results provide the first comprehensive analysis of since tropomyosin spans 7 actin monomers, two such tropomyosin chains on the force dependent stability and interactions of alpha-actinin 1, which sheds the same actin filament have a 6-in-7 chance of being out of register, leaving new light on the molecular mechanisms underlying its mechanotransmission a gap in decoration of one or more actin monomers where they meet. and mechanosensing functions. Lateral diffusion of tropomyosin chains on F-actin was recently proposed as a potential mechanism by which gaps in decoration could be resolved. Here, we 727-Pos Board B497 present stochastic computer simulations of tropomyosin binding to F-actin. We Computational Modeling of Ena/VASP Interacting with Actin Filament to develop a theoretical framework based on the hydrodynamic drag experienced Understand its Processivity by tropomyosin during diffusion, and simulate tropomyosin’s decoration to F- Fikret Aydin, Aleksander Durumeric, Harshwardhan Katkar, actin and the development and resolution of gaps in decoration over time. Our Gregory A. Voth. results support lateral tropomyosin diffusion on F-actin as a potentially essen- University of Chicago, Chicago, IL, USA. tially mechanism by which tropomyosin molecules can decorate filamentous Ena/VASP proteins enhance formation of filopodia in the cell membrane, actin without leaving gaps in decoration. which is composed of long and straight actin filaments. These proteins proces- sively bind to the barbed end of actin filaments and increase actin elongation 730-Pos Board B500 rate by two to three times. In this work, we first built an all-atom model of C1 IG-Domain of Myosin Binding Protein-C Activates Cardiac Thin Fila- Ena/VASP interacting with actin filament (F-actin) by using computational ment by Means of Thethering Tropomyosin to the Subdomain-1 of Actin protein design techniques since a complete structure of Ena/VASP interacting Cristina Risi1, Betty Belknap1, Tyler Glendrange1, Samantha Harris2, with F-actin is currently not available. We assessed the correct binding site of Howard D. White1, Gunnar Schro¨der3, Vitold E. Galkin1. F-actin binding domain (FAB) of Ena/VASP on the F-actin by using well- 1Eastern Virginia Medical School, Norfolk, VA, USA, 2University of tempered metadynamics (WTMetaD) simulations. The WTMetaD simulations Arizona, Tucson, AZ, USA, 3Institute of Complex Systems, Julich,€ Germany.

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Cardiac muscle contraction is based on the interaction between two filamentous rangements of the protein that initiates a vectorial translocation of a proton out of systems: the thick filament, which is comprised of myosin and its accessory the cell. aR4 has not only a retinal as the premier chromophore, but also a bac- protein, cardiac myosin binding protein C (cMyBP-C) and the thin filament terioruberin as the second chromophore. Furthermore, aR4 has an opposite tem- (TF), which is comprised of troponin (Tn), tropomyosin (Tpm), and filamen- poral order of proton uptake and release at neutral pH as compared with bR. In tous actin (F-actin). Mutations in the gene encoding cMyBP-C are one of the order to elucidate how the retinal cis-trans thermal equilibrium is maintained most common causes of hypertrophic cardiomyopathy (HCM), a disease that and modulated by the second chromophore, and further affect the proton uptake affects approximately 1 in 500 people. The interaction between thick and and release order, the photocycle kinetics, and the energy conversion efficiency thin filaments is regulated through translocation of Tpm cable by the Tn- in the claret membrane, in situ 2D solid-state NMR of the specifically labelled complex in response to Ca2þ followed by additional Tpm azimuthal movement receptors in the claret membranes, reinforced with many single point mutation upon binding of rigor myosin cross-bridges to F-actin. Therefore, activation of analyses and function assays were carried out. Hydrogen bonding and hydropho- the TF is a two-step process which depends on both Ca2þ-induced translocation bic interactions were identified as the mechanistic origin of multiple outcomes, of the Tpm and subsequent binding of rigor myosin heads. The N-terminal including direct electromechanical coupling to the dynamics of conformational domain of cMyBP-C is comprised of three Ig-domains (C0, C1 and C2) and changes within the receptor, direct monomer interactions, and energy conversion a regulatory linker (M-domain). We show that C1 Ig-domain is the only Ig- efficiency in native membranes. These new insights may be generalized to other domain that can activate the TFs at low Ca2þ. 3D-reconstruction of frozen hy- receptors and proteins in which metastable thermal equilibria have been identi- drated cardiac TFs decorated with C1 Ig-domain shows that the C1 Ig-domain fied and perturbed by ligand binding and downstream signaling. on its own can translocate the Tpm cable on the surface of F-actin to the same þ extent as the combination of Ca2 and rigor myosin by tethering the Tpm cable 733-Pos Board B503 to the subdomain-1 (SD1) of actin. Disruption of the interaction of the C1 Measuring Transport Kinetics of Light Driven Membrane Protein, Ig-domain interaction with either the SD1 of actin or Tpm cable by point mu- Halorhodopsin tations in C1 Ig-domain results in a complete loss of the C1-induced TF activa- Hasin M. Feroz1, Bryan Ferlez2, Cecile Lefoulon3, þ tion at low Ca2 . These data suggest how cMyBP-C can modulate the Hossein Mohammadiarani4, Tingwei Rei1, Carol S. Baker2, Peter J. Butler5, activation of the TF in heart muscle. Jonas Huhn€ 6, Cheryl A. Kerfeld7, Nigel J. Burroughs8, Harish Vashisth4, Wolfgang Parak6, Mike Blatt9, John Golbeck2, Manish Kumar1. 731-Pos Board B501 1Chemical Engineering, Penn State University, University Park, PA, USA, Structural Basis for High-Affinity Actin Binding Revealed by a B-III-Spec- 2Biochemistry and Molecular Biology, Penn State University, University trin SCA5 Missense Mutation Park, PA, USA, 3Plant Biology and Biophysics, University of Glasgow, 1 1 2 2 Michael E. Fealey , Adam W. Avery , Fengbin Wang , Albina Orlova , Glasgow, United Kingdom, 4Chemical Engineering, University of New 1 2 1 Andrew R. Thompson , Edward H. Egelman , Thomas S. Hays , Hampshire, Durham, NH, USA, 5Biomedical Engineering, Penn State David D. Thomas1. 6 1 2 University, University Park, PA, USA, Physics and Chemistry, Philipps University of Minnesota, Minneapolis, MN, USA, University of Virginia, University of Marburg, Marburg, Germany, 7Biochemistry and Molecular Charlottesville, VA, USA. Biology, Michigan State University, East Lansing, MI, USA, 8University of Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disorder stem- Warwick, Warwick, United Kingdom, 9Molecular Cell and Systems Biology, ming from several distinct mutations in the protein b-III-spectrin, including a University of Glasgow, Glasgow, United Kingdom. leucine-to-proline missense mutation (L253P) in the actin-binding domain Despite growing interest in light-driven ion pumps for use in optogenetics, (ABD). This mutation causes a 1000-fold increase in affinity for actin and current estimates of their transport rates span three orders of magnitude likely contributes to pathogenesis. However, the structural basis for the increase ˚ due to challenges in measuring slow transport processes, determining protein in affinity is unknown. Here, we report a 6.9 A cryo-EM structure of F-actin concentration and/or orientation in membranes in vitro.Weprovidethree complexed with the b-III-spectrin ABD, harboring the L253P SCA5 mutation. orthogonal measurements to determine the light-driven Cl– transport of This structure, along with co-sedimentation and pulsed-EPR measurements, the anion pump halorohodopsin from Natronomonaspharaonis (pHR). First, demonstrates that high-affinity binding caused by the CH2-localized mutation using a fluorimetric liposome-based assay we measured a transport rate of is due to an opening of the two CH domains (Avery et al., Nature Commun, 442 (5 98) Cl–/protein/s for a photon flux of 1450 photons/protein/s. 2017). This opening allows CH1 to bind actin aided by an unstructured N-ter- Second, using light-interfaced voltage clamp measurements on pHR- minal region that becomes a-helical upon binding. This N-terminal helix is expressing oocytes we obtained a transport rate of 219 (5 98) Cl–/pro- crucial for association with actin, as its removal eliminates binding. In tein/s for a photon flux of 650 photons/protein/s. Both measurements are drosophila, the SCA-5 missense mutation results in aberrant localization of consistent with the literature reported quantum efficiency of 30% for pHR, b-III-spectrin. Rather than being found throughout dendritic spines, b-III-spec- 0.3 isomerizations per photon absorbed [36-38]. Finally, using novel consec- trin is limited to the base of spine structures. This phenotype is consistent with utive single-turnover flash experiments we proved that continuous illumina- the L253P mutation causing increased affinity for actin and, consequently, tion helps pHR bypass the earlier reported longest step in the pHR limiting b-III-spectrin’s localization in neurons (Avery et al., Proc Natl Acad photocycle. Assuming that the next longest step is rate-limiting results in Sci, 2017). Collectively, these results provide novel structural insights into a pHR turnover rate of 400/s which is in excellent agreement with our the disease-causing SCA5 mutation. This work was supported in part by grants measured transport rates of pHR. from NIH to DDT (AR032961, AR007612), TSH (GM44757), and EHE (GM81303). 734-Pos Board B504 The Elusive Proton in the Gastric Proton Potassium ATPase Posters: Membrane Pumps, Transporters, and Vikas Dubey1, Kazuhiro Abe2, Ilia Solov’yov3, Himanshu Khandelia1. Exchangers I 1Memphys: Center for Biomembrane Physics, University of Southern Denmark, Odense M, Denmark, 2Cellular and Structural Physiology Institute, 732-Pos Board B502 Nagoya University, Nagoya, Japan, 3Physics Chemistry and Pharmacy, Thermal Equilibrium and Energy Conversion of a New Photoreceptor University of Southern Denmark, Odense M, Denmark. with Two Chromophores as Studied by In Situ Spectroscopy The proton potassium pump (HKA) acidifies the stomach by pumping protons Xiaoyan Ding1,2, Chao Sun1, Haolin Cui1, Yujiao Gao1, Juan Wang1, against a million-fold proton concentration gradient while counter-transporting Yanan Yang1, Xin Zhao1. Kþ ions into the cytoplasm. The pump alternates between and cytoplasm-open 1Department of Physics, East China Normal University, Shanghai, China, E1 states and gastric-lumen open E2 states. We use molecular dynamics (MD) 2Department of Biochemistry and Molecular Biology, Penn State College of simulations at the microseconds time scales, and free energy calculations of the Medicine, Hershey, PA, USA. E2 states to investigate the energetics of ion binding and suggest protonation Archaerhodopsin-4 (aR4), an unknown structured membrane protein from Hal- states of the ion-occluded and the E2P outside-open states based on our simu- obacterium species XZ515 found in a salt lake in China, functions as a proton lations. Based on dynamic pKa correlations, we infer possible proton transfers pump similar to that of bacteriorhodopsin (bR). It has a seven-transmembrane and rearrangements inside the binding site. Our results are in agreement with topology and a protonated Schiff base formed by the retinal chromophore published biochemical studies where Kþ affinity and ATPase activity were with the lysine 217 on the helix G through a covalent bond linkage. Absorption measured for several acidic amino acids in the ion-binding sites. We make of a photon causes photoisomerization of the retinal chromophore from the all- experimentally testable predictions about the possible paths a proton can take trans to the 13-cis, 15-anti configuration and triggers a series of structural rear- to enter and leave the ion-binding sites.

BPJ 8578_8581 Sunday, February 18, 2018 147a

735-Pos Board B505 levels. We study all possible a-b combinations with the neuronal exclusive Properties of Oscillating Electrical Pulse Induced Na/K Pump Current on a3 isoform. We found that the different b-subunits have a modest effect on Single Frog Skeletal Muscle the total amount of charge moved by the slow component, with b1 and b3 car- Pengfei Liang, Wei Chen. rying the largest and smallest current respectively. In the presence of b-3 the Physics, University of South Florida, Tampa, FL, USA. voltage distribution of the transient pump currents was shifted towards more By applying well designed oscillating electrical pulse to a single frog skeletal depolarize potentials. b-2 increased the deocclusion rate of the last Naþ to muscle with voltage-clamp and double vaseline gap techniques, we obtained the extracellular solution compared to b1 and b3. We are now determining pump current on both positive and negative half cycle. The frequency of the the effect of the different a3-b combinations on the medium and fast kinetic oscillating pulses remains at 50 Hz which is comparable with the physiological components reflecting the binding/release of the first and second Naþ. turnover rate of the Na/K pump. Different magnitudes of voltage (from 30mV to 65mV with a 5mV step) were applied to the muscle. The results showed that 738-Pos Board B508 the pump current as well as the charge mediated by pump moved across the Crystal Structures of SERCA2a and SERCA2b plasma cell membrane were voltage dependent and reached a saturation status Haruo Ogawa, Yoshiki Kabashima, Rie Nakajima, Chikashi Toyoshima. when the pulse was greater than 55 mV. In this situation, the total amount of Center for Structural Biology of Challenging Proteins, IMCB, The University pump-mediated charge remained roughly the same even with the changing of Tokyo, Tokyo, Japan. Sarco(endo)plasmic reticulum Ca2þ-ATPase (SERCA) plays a crucial role in magnitude of the activation pulses. The charge on the positive half cycle and 2þ negative half cycle which represent the Na extrusion and K intrusion respec- maintaining Ca concentration that controls many vital cell functions. SERCA tively, has a ratio less than but close to 3:2. After adding ouabain, the currents family consists of highly conserved three subgroups (SERCA1, 2, and 3), were recorded each 5 minutes. The results showed that the total pump-mediated which are alternatively spliced, in both tissue-specific and developmentally charge decreased monotonically and reached minimum which was around 0 af- regulated manner. Two subtypes exist in SERCA2. While SERCA2a mainly ter 20 minutes. This indicated that this oscillating electrical pulse induced cur- populate cardiac SR, SERCA2b, the housekeeping variant, is present in the ER of all cell types. Compared to SERCA2a, SERCA2b exhibits a 2-fold appar- rent was highly sensitive to and can be totally eliminated by ouabain, a specific 2þ Na/K pump blocker. ently higher apparent affinity for Ca and lower catalytic turnover rate. These unique functional properties must be related to an additional 11th transmem- 736-Pos Board B506 brane helix (TM11) and a short luminal extension (LE) at its C-terminus that Contribution of the Hydroxyl Group and Phenol Ring of Tyrosine 780 of do not exist in SERCA2a. However, how these additional components cause the Alpha-Subunit to NAD Binding by the Na/K Pump the functional differences are unknown due to lack of the crystal structures Kerri Spontarelli1, Daniel Infield2, Chris A. Ahern2, Pablo Artigas1. of SERCA2a and b. Here we present crystal structures of SERCA2a and b. 1Cell Physiology and Molecular Biophysics, Texas Tech University Health The TM11 and the LE are clearly resolved in the electron density maps. Their Sciences Center, Lubbock, TX, USA, 2University of Iowa, Iowa City, IA, positions are different from those proposed previously. USA. 739-Pos Board B509 The Na/K pump is a heterodimeric(ab) P-type ATPase that exports three Naþ þ Discovery of SERCA2a/PLB Activators and Inhibitors by Structure-Based and imports two K against their electrochemical gradients using the energy of High-throughput Screening using Live Cell FRET Biosensors ATP hydrolysis. Structural studies confirm three ion-binding sites in the cata- þ þ Daniel R. Stroik, Samantha L. Yuen, Kevyn A. Janicek, Tory M. Schaaf, lytic a-subunit. Site-I and Site-II bind either Na or K , while Site-III, the Razvan L. Cornea, David D. Thomas. focus here, exclusively binds Naþ. To determine the relative energetic contri- þ Biochemistry, Molecular Biology and Biophysics, University of Minnesota, bution to Na binding of the hydroxyl group or the p-electrons of Tyr780 in Minneapolis, MN, USA. site-III, we introduced the mutation Y780tag into the a-subunit cDNA for We have developed a cell-based high-throughput screening (HTS) method us- the purpose of using nonsense suppression. Y780tag-a- and b3-cRNA were ing time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding co-injected in Xenopus oocytes with synthetic tRNA (ligated to Tyr, Phe or and structural dynamics of the SERCA2a/PLB complex. Overwhelming evi- Phe derivatives) to compare the functional consequences of the chemical mod- dence establishes the membrane protein complex between the sarcoplasmic re- ifications at this position using two-electrode voltage clamp. Square voltage þ þ þ ticulum Ca-ATPase 2a (SERCA2a) and its regulator phospholamban (PLB) as pulses in the absence of external K (K o) and presence of external Na þ a therapeutic target for reversing cardiac contractile dysfunction caused by (Na o) produce transient currents. The voltage dependence of steady-state aberrant calcium handling. Unfortunately, efforts to discover compounds charge moved by these transients is described by a Boltzmann distribution þ with specificity for this complex have yet to yield an efficacious drug. GFP- with a center (V1/2) related to the relative apparent affinity for Na o (DV1/2= SERCA2a (donor) was co-expressed in the endoplasmic reticulum of -25 mV per 2-fold reduction in affinity). The centers of the distribution where HEK293 cells with and without RFP-PLB (acceptor), and FRET was measured 5 5 V1/2= -46.9 2.5 mV (n=10) for Tyr, V1/2= -123 9 mV for Phe, and V1/2= in a fluorescence lifetime microplate reader. We carried out a triplicate screen 5 54.6 2.1 mV (n=5) for mono-fluorinated at the 3’ position (mono-F)-Tyr. against a small chemical library (LOPAC) and identified 21 compounds that þ These results indicate 8-fold reduction in apparent affinity for Na o by reproducibly changed FRET by >3 SD. Dose-response of FRET and ATPase removal of the hydroxyl group and a smaller reduction ( 2-fold) by partial activity readouts reveal that ten hits reproducibly alter SERCA2a/PLB structure disruption of the cation-p interaction capability by monofluorination. Surpris- þ þ and function. One compound increases SERCA2a Ca affinity and activity ingly, the apparent affinity for K o estimated from the [K ]o dependence of þ (EC50 =23mM) at physiological calcium levels in cardiac SR, but not in skel- pump current activation in the absence of Na o was reduced by both Phe etal SR, suggesting that the compound is acting specifically on the SERCA2a/ ( 4-fold) and mono-F-Tyr ( 1.5-fold). Measurements with other derivatives PLB complex. These properties characterize a drug that could reverse calcium are underway. NSF-MCB 1515434. mishandling. The excellent Z’ factor and correlation between structural and functional assays validate this method to discover SERCA2a-PLB complex ef- 737-Pos Board B507 D fectors for large-scale HTS campaigns. This work was supported by NIH grants Beta-Dependent Modulation of Na Binding and Release of the Alpha-3 (GM27906, HL129814, AR07612, and DA037622). NaDKDPump Cristina Moreno Vadillo, Miguel Holmgren. 740-Pos Board B510 NINDS, Bethesda, MD, USA. Binding Affinity of Serca Regulatory Complexes Quantified by Steered The NaþKþ pump is a P-type ATPase protein complex that uses the energy of Molecular Dynamics Simulations ATP hydrolysis to export 3Naþ from the cell and import 2Kþ in each cycle. The Nikolai Smolin, Seth L. Robia. transport cycle creates an electrochemical gradient across the membrane that is Department of Cell and Molecular Physiology, Loyola University Chicago, used for a variety of cellular processes including neuronal signaling, transport Maywood, IL, USA. and volume regulation. The NaþKþ pump is composed of two subunits: the cat- The regulatory peptide phospholamban (PLB) binds its target, the SERCA cal- alytic/transporter a- and the modulatory b-subunit. Four alpha (a1-4) and three cium pump, with high affinity when the pump is in a calcium-free conformation beta (b1-3) isoforms have been identified in humans. NaþKþ pump subunits are and with lower affinity when the Ca binding sites are occupied. To probe the differentially expressed across the body suggesting tissue-specific functions. energetics of PLB binding to the canonical (M6) binding site and several hypo- However, the details of the b-modulation remain to be elucidated. thetical alternative binding sites on SERCA, we performed steered molecular Here, we take advantage of the cut-open voltage-clamp technique that can dynamics simulations. We measured the force required to rupture the regulato- clamp the oocyte membrane in 20-40ms to measure in isolation the transitions ry complex as an index of PLB-SERCA binding affinity. Rupture force varied (slow, medium and fast) associated with the sequential binding/release of the according to the binding site, the conformation of SERCA, and the axial rota- three Naþ by removing internal and external Kþ and maintaining high ATP tion of the PLB transmembrane helix. We have previously hypothesized that

BPJ 8578_8581 148a Sunday, February 18, 2018

PLB may bind to an alternative site on SERCA helix M9 in a configuration cation of substrates across biological membranes. We use all-atom molecular analogous to the complex of the Naþ/Kþ-ATPase (NKA) with its regulatory dynamics (MD) simulations to investigate the mechanisms underlying the func- partner FXYD2. Steered molecular dynamics results were not consistent with tional working cycle of the ABC transporter TM287/288, a prototypical heter- that hypothesis, as PLB interacted significantly more strongly with M6 than odimeric ABC exporter. Unbiased multi-microsecond simulations in an explicit M9 for all SERCA conformations tested. We also evaluated a structural model membrane/water environment reveal that in response to ATP binding, TM287/ of dwarf open reading frame (DWORF) bound to SERCA and observed that 288 undergoes a spontaneous alternating-access type conformational transition this non-inhibitory micropeptide binds with an apparent affinity that is compa- from the inward-facing state seen in the x-ray crystal structure via a metastable rable to PLB. The results provide insight into competitive binding of peptide occluded intermediate to an outward-facing state. For the latter two states, regulators of SERCA, and reveals new information about dynamic changes which could thus far not be structurally characterized for heterodimeric ex- in regulatory complex stability during the Ca transport cycle. porters, the structures obtained from our simulations are reminiscent to x-ray structures of the homologous transporters PCAT1 and Sav1866, respectively. 741-Pos Board B511 These results allow us to describe the large-scale conformational transition un- The Allosteric Coupling of the Cytoplasmic Phosphorylation and Trans- derlying the working cycle of the ABC exporter in atomic detail. membrane Domain Deocclusion in the Ca2D SERCA Pump Elucidated by Free Energy Simulations 744-Pos Board B514 Huan Rui, Avisek Das, Benoit Roux. Lipid-Dependent Alternating Access Mechanism in ABC Exporters Department of Biochemistry and Molecular Biology, University of Chicago, Revealed using Microsecond-Level Molecular Dynamics Simulations Chicago, IL, USA. 2þ Mahmoud Moradi, Jeevapani Hettige. SERCA Ca pump belongs to the P-type ATPase family, which mediates active Department of Chemistry and Biochemistry, University of Arkansas, ion transport using the energy from ATP hydrolysis. It is a single chain protein Fayetteville, AR, USA. with 994 amino acids. It has a transmembrane (TM) region of 10 helices as ATP-binding cassette (ABC) transporters have been extensively studied struc- well as three cytoplasmic domains (N, P, A), named after their principal functions. turally over the last decade due to their importance in several biological pro- The SERCA pump works by alternating between luminal-facing E2 and cytosol- 2þ cesses and biomedical applications. We have performed the most extensive facing E1 conformations to transport Ca and proton across the sarcoplasmic/ set of molecular dynamics (MD) simulations on an ABC exporter, namely endoplasmic reticulum (SER) membrane. ATP hydrolysis occurs during the E1 Sav1866 in various membrane environments to characterize the lipid- to E2 transition. During this transition, the E1 conformation binds and occludes 2þ dependent behavior of protein structural dynamics. Despite the availability of with two Ca ions. After the occlusion, ATP hydrolyzes and its g-phosphate is various crystal structures, the degree of opening of the cyto-/periplasmic gate transferred to Asp351 in the P domain, resulting in its phosphorylation. The infor- in the inward-/outward-faciung (IF/OF) state has been a controversial issue. mation of this chemical modification is communicated to the distant TM region, 2þ For instance, this degree of opening varies considerably in different crystal which then opens to the luminal side to release the Ca . It is evident that there structures, even for those associated with the same protein (e.g., MsbA trans- exists a bi-directional information flow between the cytoplasmic and the TM re- porter, which is crystallized in two substantially different IF conformations). gions of SERCA. How this occurs within the three-dimensional structure, howev- While many biochemical studies suggest a relatively narrow opening, the er, has yet to be demonstrated. To elucidate the allosteric coupling mechanism resolved crystal structures and other biophysical data are often in favor of a between the cytoplasmic and the TM regions, we compute the free energy land- more open conformation for the IF state. Different experiments are typically scape of the cytoplasmic domain organization upon phosphorylation during the done in different lipidic or non-lipidic environments. We have now performed E1 P to E2-P transition using free energy simulations. The results show that an extensive set of microsecond-level all-atom MD simulations of Sav1866 with Asp351 phosphorylated, the energy cost going from E1 PtoE2-Pisdramat- with an aggregate simulation time of about 100 microseconds. These simula- ically reduced, therefore favoring such a transition. tions suggest that the discrepancies observed experimentally are at least partly 742-Pos Board B512 due to the difference in the protein environment in different experimental con- Is ATP Hydrolysis the Power Stroke in ABC Transporters? ditions. Our simulations provide strong evidence that the alternating access Hendrik Go¨ddeke1, Marten Prieß1, Gerrit Groenhof2, Lars Sch€afer1. mechanism in ABC exporters is indeed lipid-dependent. The molecular basis 1Center for Theoretical Chemistry, Ruhr-University Bochum, Bochum, of this lipid-dependent behavior is also explained. Our study sheds light on Germany, 2Nanoscience Center and Department of Chemistry, University of the mechanistic details of alternating access mechanism and provides molecu- Jyv€askyl€a, Jyv€askyl€a, Finland. lar details regarding the conformational transition of ABC exporters during Hydrolysis of nucleotide triphosphate (NTP) plays a key role in the function of their OF to IF structural transition. many proteins. However, the chemistry of the catalytic reaction in terms of an atomic-level understanding often remains elusive. We studied the molecular 745-Pos Board B515 mechanism of adenosine triphosphate (ATP) hydrolysis in the ATP-binding Study of Conformational Transition of a Protein Secretion ABC Trans- cassette (ABC) transporter BtuCD-F. Free energy profiles obtained from hybrid porter using Molecular Dynamic 1 2 2 quantum mechanical/molecular mechanical (QM/MM) molecular dynamics Ahmad Raeisi Najafi , Reza Dastvan , Hassane S. Mchaourab , 3 (MD) simulations reveal that the hydrolysis reaction proceeds in three steps. Emad Tajkhorshid . 1Department of Mechanical Engineering & Mechanics, Drexel University, The first step is the nucleophilic attack of an activated lytic water molecule 2 þ 2- Philadelphia, PA, USA, Department of Molecular Physiology and at the ATP-phosphate to yield ADP HPO4 as intermediate product. A gluta- 3 mate residue that is located very close to the phosphate transiently accepts a Biophysics, Vanderbilt University, Nashville, TN, USA, Department of proton and thus acts as catalytic base. In the second and third step, this proton Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA. 2- - Among the various ABC transporters, those involved in protein secretion have is transferred back to HPO4 , yielding ADP þ H2PO4 as final products. The rate estimated from the computed free energy barriers is in very good agree- a unique structure in addition to the transmembrane (TMDs) and nucleotide- ment with experiments. The overall free energy change associated with the re- binding (NBDs) domains which are common in all ABC transporters. This fam- action is positive, suggesting that ATP hydrolysis itself does not provide the ily of transporters, known as the peptidase-containing ABC transporters power stroke for substrate transport in BtuCD-F. The proposed mechanism is (PCATs), contain additional peptidase domains (PEPs), the main function of likely relevant for all ABC transporters and might have implications also for which is processing of polypeptide substrates. In this study, we use molecular other NTPases, thus representing a key step towards understanding chemo- dynamics (MD) simulations to characterize and compare the dynamics of mechanical energy conversion in NTP-driven molecular machines. PCAT1 in the absence and presence of ATP. Using the simulations, we monitor the separation of the NBDs, TMDs, and PEPs to capture the conformational 743-Pos Board B513 response of PCAT1 to ATP binding. We also aim to characterize the alternate Mechanism of Large-Scale Alternating Access Conformational Transition conformation of PCAT1 formed during the transport cycle using non- in the ABC Exporter TM287/288 equilibrium MD simulations. To investigate the conformational changes, we Hendrik Go¨ddeke1, Mikko Karttunen2, Lars V. Schafer€ 1. consider three states: an IF model (unknown), an occluded state, and an 1Theoretical Chemistry, Ruhr-University Bochum, Bochum, Germany, outward-facing (OF) model (unknown). The IF state is constructed based on 2Department of Chemistry and Department of Applied Mathematics, Western the semi-open state of PCAT1 (PDB code 4RY2) while the OF state is rebuilt University, London, ON, Canada. using a homology model based on a structural template from the OF state of the þ ATP-binding cassette (ABC) transporters are molecular machines that are bacterial ABC transporter MsbA (PDB code 3B60). ATP molecules and Mg 2 driven by the chemo-mechanical coupling of ATP binding and hydrolysis in are docked into the NBDs of the OF state based on the crystal structure of the the nucleotide-binding domains (NBDs) to large-scale conformational changes structure Mg-ATP-bound NBD of the of HlyB (PDB code 1XEF). To charac- in the transmembrane domains (TMDs), which ultimately leads to the translo- terize the functional properties of the PEPs, we perform non-equilibrium MD

BPJ 8578_8581 Sunday, February 18, 2018 149a simulations from the IF state to the OF state in the full-length protein as well as 1The University of Chicago, Chicago, IL, USA, 2University of Michigan, after removing the peptidase domains. These fully atomistic models along with Ann Arbor, MI, USA. non-equilibrium MD simulations employing different sets of collective vari- Genetic predisposition plays a profound impact on the incidence of atrial fibrilla- ables reveal structural elements and changes underlying the alternating access tion. The T-box transcription factor gene Tbx5 has been implicated in AF in family mechanism in this bacterial protein exporter. studies and by GWAS. We previously demonstrated that adult-specific Tbx5- knockout mice develop spontaneous primary AF two weeks after Tbx5 deletion. 746-Pos Board B516 Tbx5 deficient cardiomyocytes showed prolonged action potentials (APs) and Lipid Binding and Lipid-Uptake in P-Glycoprotein: Comparison of the triggered activity. Inclusion of heavy Ca2þ buffering in the pipette (using 5 mM Inward- and Outward-Facing Conformation BAPTA) normalized AP duration, leading us to hypothesize that electrophysio- Estefania Barreto-Ojeda, Abigael Gritter, Valentina Corradi, logical abnormalities and triggered activity were secondary to abnormal calcium Peter Tieleman. handling. We aimed to define the electrophysiological basis of AP prolongation Biological Sciences, University of Calgary, Calgary, AB, Canada. and the abnormal trigger in TBX5-knockout atrial myocytes. RNA-seq, qtPCR, P-glycoprotein (Pgp) belongs to the ABC transporters superfamily. The ATP and Western blots are consistent with reduced RyR2 and SERCA, but no decrease hydrolysis at its nucleotide binding domains (NBD) triggers conformational in L type calcium channels. In acutely isolated myocytes, peak L-type calcium changes of the transmembrane domains (TMD), enabling the translocation of current was increased from 1.88 5 0.2 A/F to 4.4 5 0.8 A/F at 5 mV without substrates to the extracellular medium. Pgp translocates a wide range of chem- any change steady state inactivation kinetics. Ca2þ sparks were significantly ically diverse substrates, ranging from toxins to drugs, lipids and lipid-like mol- diminished at all pacing frequencies between 0 and 2Hz. Consistent with reduced ecules, and prevents the accumulation of chemotherapy agents inside the cell. spark frequency we observed a 24 5 8% reduction in SR calcium load and a 31 5 This makes Pgp a key player in cancer multidrug resistance (MDR). Methods 9% reduction in the rate of SERCA mediated calcium uptake. Na-CaX mediated that allow the study of drug and lipid binding and uptake at the molecular level Ca2þ efflux activity increased 38 5 18%. Treating myocytes with L type Ca2þ will contribute, in the long term, to the development of new drugs to inhibit channel inhibitors normalized AP prolongation, consistent with observed cellular substrate translocation, and therefore improve chemotherapy treatments. In Ca2þ handling defects. Thus, loss of TBX5 in atrial cardiomyocytes leads to a this work, we perform coarse-grained (CG) molecular dynamics (MD) simula- 2þ decrease in the role of the SR in cellular Ca handling. Increased inward ICa tions of Pgp embedded in a symmetric POPC:POPE bilayer to investigate the 2þ and Ca extrusion from the cell via inward INa-CaX provide a molecular mecha- lipid-uptake of Pgp in the inward- (PgpIN) and outward-facing (PgpOUT) nism for the depolarizing currents responsible for AP prolongation and may ac- conformation. These 20ms long simulations allow the characterization of count for early and delayed afterdepolarizations. lipid-binding sites in the two conformations, and the description, at the molec- ular level, of lipid-uptake events. To accounts for the effects of cholesterol, we 749-Pos Board B519 consider a second set of simulations of PgpIN and PgpOUT in a symmetric Analyzing the Effects of Membrane Lipid Type on Transmembrane PC:PE:CHOL bilayer. Our results highlight how lipid-uptake is influenced Proteins (aHL and 5-HT3) using Molecular Dynamics Simulations by CHOL molecules located at the surface of the Pgp. Nicholas B. Guros1,2, Jeffery B. Klauda1, Arvind Balijepalli2. 1Chemical and Biomolecular Engineering, University of Maryland, College 747-Pos Board B517 Park, MD, USA, 2NIST, Gaithersburg, MD, USA. Lipids and Ions Traverse the Membrane by the Same Physical Pathway in Alpha-hemolysin (aHL) is a transporter protein that is used to measure the size the nhTMEM16 Scramblase and charge of macromolecules such as DNA across a membrane through an Tao Jiang1,2, Kuai Yu3, H Criss Hartzell3, Emad Tajkhorshid1,2. intensive analysis of channel conductance. Experimentally, 1,2-diphytanoyl- 1Department of Biochemistry, Center for Biophysics and Computational sn-glycero-3-phosphocholine (DPhPC) membranes are used, however, most Biology, University of Illinois at Urbana Champaign, Urbana, IL, USA, molecular dynamics (MD) simulations of aHL use either 1-palmitoyl-2- 2Beckman Institute for Advanced Science and Technology, University of oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dipalmitoyl-sn-glycero-3- Illinois at Urbana-Champaign, Urbana, IL, USA, 3Department of Cell phosphocholine (DPPC) membranes. While DPhPC has been studied in silico Biology, Emory University School of Medicine, Atlanta, GA, USA. for pure membranes and with DNA-origami channels, its role as the primary From bacteria to mammals, different phospholipid species are segregated be- lipid used to study aHL experimentally has not been investigated with simula- tween the inner and outer leaflets of the plasma membrane by ATP-dependent tions. This work uses DPhPC membranes to study the transporter protein aHL lipid transporters. Disruption of this asymmetry by ATP-independent phospho- with MD to demonstrate the importance of lipid type on transporter function, lipid scrambling is important in cellular signaling, but its mechanism remains specifically its effect on channel conductance which is critical to probing the incompletely understood. Although the recently crystalized structure of þ function of this channel. Additionally, this work examines the effects of two nhTMEM16, a Ca2 -activated phospholipid scramblase and non-selective ion lipid systems on the ion channel 5-HT3, namely a simple POPC membrane channel, provides insight into the site of catalysis, the absence of phospholipids versus a POPC/1-stearoyl-2-docosahexaenoyl-sn-glyerco-3-phosphocholine in the structure leaves unanswered the question of how both lipids and ions are (SDPC)/cholesterol membrane which is more representative of that found in conducted. Using MD simulations coupled with experimental assays, we show neural membranes. Using the Anton2 supercomputer, we generated several that the surface hydrophilic transmembrane cavity exposed to the lipid bilayer multi-microsecond trajectories to examine ion channel function with respect on nhTMEM16 serves as the pathway for both lipid translocation and ion con- to membrane lipid type on a biologically relevant time scale. duction across the membrane. In the simulations, ion conduction through the aqueous pore formed between the protein and the lipid headgroups developed 750-Pos Board B520 in parallel with phospholipid transport and became more robust as lipids became Desmopressin Causes Different Effects on Water and Urea Permeability of more tightly packed and organized in the aqueduct as scrambling proceeded. In Principal Cells in OMCD and IMCD this flexible pore structure, lipids play a structural role by lining the hydrophilic Evgeniy I. Solenov. ion conduction pathway with their headgroups. Ca2þ binding stimulates the open Group Molecular Physiology of Cell, Institute of Cytology and Genetics The conformation of the aqueduct by altering the structure of transmembrane helices Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russian that line the cavity. Furthermore, we have identified key amino acids necessary Federation. for phospholipid scrambling and validated the idea that ions permeate TMEM16 Changes in the water and urea permeability (Pf,Pu) of principal cells along the Cl- channels via a structurally homologous pathway by showing that mutation of collecting duct (CD) could be linked to the countercurrent mechanism of the two residues in the pore region of the TMEM16A Ca2þ-activated Cl-channel kidney. To date, the regulation of water and urea permeability along the CD convert it into a robust scramblase. Our study makes a number of predictions has not been fully evaluated. about amino acids implicated in channel gating, lipid permeation, and ion trans- The aim of our study was to assess the effect of vasopressin V2 receptor port that can be tested experimentally, which provides crucial insight into evolu- signaling on the water and urea permeability of principal cells in OMCD and tionary relationships of the TMEM16 family members. IMCD. The V2 receptor agonist desmopressin (dDAVP) was used in experiments. For 748-Pos Board B518 measurement of the volume kinetics, cells in fragments of OMCD and IMCD Mechanisms of AP Prolongation and Triggered Activity in a TBX5 Model were balanced in hyper-osmotic PBS containing 300 mM of mannitol. Assess- of Atrial Fibrillation ment of Pf was based on the kinetics of cell volume changes as result of the os- Leonid Tyan1, Wenli Dai1, Rajiv Nadadur1, Yitang Wang1, Stefan Mazurek1, motic challenge. Urea permeability assay was based on the kinetics of cell Jenna Bekeny1, Kaitlyn Shen1, Margaret Gadek1, Brigitte Laforest1, swelling in hyper-osmotic PBS when 300 mM of mannitol were substituted Francisco Alvarado2, Hector Valdivia2, Michael Broman1, Le Shen1, with 300 mM of urea. Changes in the cell volume were measured with the Cal- Ivan Moskowitz1, Christopher Weber1. cein quenching method. For statistical analysis t-test was used.

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The initial rate of volume changing was proportional to the apparent water to Asn 66 and Trp 50. Simulation of the docked structure reveal that distance permeability. dDAVP (109M), caused significant increase of apparent water between intracellular gate residues are increasing and are thus indicative of and urea permeability in OMCD cells (Pf: control 0.01350.003; dDAVP transition towards inward-open conformation. Comparative simulations of sub- 0.02350.012; Pu: control 0.00350.001; dDAVP 0,005350.001 cm/s; strate bound and unbound forms, for at least 2ms, suggests that substrate facil- p<0.05; n=12). There was no effect of dDAVP on the apparent water and urea itates relatively rapid conformational transitions. Detailed study of sequence of permeability in IMCD cells (Pf: control 0.01950.001; dDAVP 0.0250.006; events suggest that there exist more than one intermediate occluded conforma- Pu: control 0.00450.002; dDAVP 0.00550.001;cm/s; NS; n=54). tions. We have also simulated the inward-open to outward-open for elucidating We have shown that V2 receptor signaling in OMCD and IMCD causes induc- the transition through reversible pathway. tion of different responses in water and urea transport in principal cells. The exact molecular mechanisms of the transmembrane water transport in principal 754-Pos Board B524 cells from OMCD and IMCD require further study. Structural Determinants in the IF-OF Transition in Human Glucose Transporters 751-Pos Board B521 Mrinal Shekhar, Emad Tajkhorshid. Droplet Interface Bilayers on a Petri Dish - Formation Methods and University of Illinois Urbana-Champaign, Urbana, IL, USA. Characterization GLUTs are the predominant sugar transporter, that is central to the regulation of Sanhita Dixit, Alexandra Pincus, Bin Guo, Gregory W. Faris. glucose supply and metabolism in multitude of organs and tissues including SRI International, Menlo Park, CA, USA. brain. Characterizing the large-scale structural transitions of membrane trans- Lipid bilayers are ubiquitous as biomimetic assemblies for modeling cell mem- porters using conventional simulation technologies is limited due to the long branes. Specifically, lipid bilayers formed as an adhesive interface between two time-scales involved (on the order of tens of milliseconds to seconds). Recog- lipid monolayer coated droplets are currently routinely used to study and model nizing this issue, we have employed a knowledge-based computational approach cell membrane transport functions. In the report, we discuss how laser based towards describing large-scale conformational transitions using a combination of convection methods are well suited to form such bilayers in an open Petri several distinct enhanced sampling techniques. Our approach has enabled us to dish format in an oil matrix. We also present results from our laboratory obtain previously uncharacterized stable GLUT OF state, and characterize mul- demonstrating water permeability and proton transport across such bilayers. tiple intermediate states connecting the IF and OF states. Furthermore, we were able to identify important interhelical interactions and the gating helices suggest- 752-Pos Board B522 ing a local gating mechanism apart from global large scale transition. Interest- Anion-Transport Mechanism of A Triazole-Bearing Derivative of ingly, our free energy calculations show the global minimum to be an Prodigiosine intermediate state (termed as InT state) that lies between the IF and OF confor- Claudia Cossu1, Michele Fiore1, Valeria Capurro2, Emanuela Caci2, mations. This is further substantiated by our multiple long and short equilibrium Roberto Quesada3, Oscar Moran1. simulations which tend to preferentially sample the aforementioned intermediate 1Istituto di Biofisica, CNR, Genoa, Italy, 2U.O.C. Genetica Medica, Istituto state over the end states. As a consequence, we propose a unique mechanism for Giannina Gaslini, Genoa, Italy, 3Departamento de Quı´mica, Facultad de GLUT uniporters, wherein the transporter optimally rests in the InT state and Ciencias, Universidad de Burgos, Burgos, Spain. periodically opens and closes the gate to allow for substrate egress and entry. Anion selective ionophores, anionophores, are molecules capable of facilitating the transmembrane anion transport.These moleculesare proposed asreplacement ther- 755-Pos Board B525 apy of anion channels in genetic diseases, like cystic fibrosis. A small molecule con- Transport of Lignin-Breakdown Products by Genetically Engineered Non- taining a triazole heterocycle, EH160, inspired in the natural product prodigiosin, Lignolytic Bacteria and Fungi proved highly efficient anion exchanger in phospholipid bilayers. Anionophore- Meghan C. Barnhart-Dailey, Dulce Hayes, Dongmei Ye, Danae Maes, driven chloride efflux from liposomes is consistent with a carrier that facilitates Leah Appelhans, Michael Kent, Jerilyn Timlin. the transport of anions through bilayers down the electrochemical gradient, Sandia National Laboratories, Albuquerque, NM, USA. exchanging permeable anions from both sides of the bilayer. The active form of Lignin is typically 15-28% of the total dry weight of biomass, making it a signif- the compound is the protonated fraction, depending on the pH in the aqueous phase. icant potential source of revenue, yet it is currently a waste stream in biofuel pro- However, the transport is not coupled with proton (or hydroxide) translocations. duction that is usually burned to generate power. Efficient, controllable The selectivity sequence of EH160 is formate-acetate-nitrate-chloride- conversion of lignin to useful molecular building blocks has been elusive. While bicarbonate. Sulphate, phosphate, aspartate, isothionate and gluconate are virtually much lignin breakdown occurs extracellularly, the breakdown products, a sub- not transported by these anionophores. This molecule also transport anions in living stantial fraction of which are mono- and di- aromatics, are known to be taken cells at doses that do not compromise cellular viability. The capacity of EH160 to up by lignolytic fungi and bacteria and metabolized intracellularly. Assimilation transport chloride and bicarbonate with efficiencies close to the natural anion chan- of these breakdown products is an important part of lignin conversion, yet very nel CFTR take shape as a promising starting point for the development of CF- little is currently known about the transporters that shuttle these compounds in therapy drug candidates. Work supported by the European Union’s Horizon lignolytic organisms. Additionally, the substrate range and kinetics of transport 2020 research and innovation programme under grant agreement No 667079. into microbes are also not known. We have genetically engineered the non- 753-Pos Board B523 lignolytic organisms, Escherichia coli and Saccharomyces cerevisiae, to express Structural Events in a Bacterial Uniporter Leading to Translocation of putative lignin transporters from lignolytic bacteria and fungi, including Phaner- Glucose Inside the Cytosol ochaete chrysosporium and Enterobacter lignolyticus SCF-1. Building upon a Indrani Bera, Jeffery B. Klauda. previous study of native transport in these organisms, we express these potential Department of Chemical and Biomolecular Engineering, University of lignin transporters in cultures grown in minimal media supplemented with indi- Maryland, College Park, MD, USA. vidual lignin breakdown products of interest. Molecular uptake was determined Among other classes of sugar transporters, there exist a comparatively new via LC-MS of the cell lysate. Additionally, click-chemistry enabled fluorescence family of transporters named SWEET transporters. SWEETs and semi- microscopy was performed for select lignin breakdown products to determine SWEETS (in bacteria) are uniport transmembrane proteins which translocate single cell uptake efficiency following transporter expression. Our results sugar inside the cell across the concentration gradient. Recently, outward show differential uptake and kinetics across the various transporters as well as open and inward open crystallographic structures of E.coli semi-SWEET between lignin breakdown products. We conclude that transport of lignin break- were deciphered. It is hypothesized that sugar is transported from the extracel- down products can be enhanced and tailored through expression of exogenous lular side (via outward-open state) to intracellular side (inward-open state) transporters. This work will contribute to increasing efficiency of lignin conver- through intermediate occluded state (both extracellular and intracellular gates sion in biofuel production pathways. closed). The conformational transition, often a spontaneous process, is a revers- ible one. In this study, we have carried out unbiased all-atom molecular dy- Posters: Genetic Regulatory Systems namics simulations with the outward-open conformation to study this transition mechanism with and without substrate. We find that at around 756-Pos Board B526 200ns the outward-open structure changes to occluded form leading to closure Epigenetic Regulations by Set-Domain Containing Proteins in Fission of extracellular gates stabilized by electrostatic and hydrophobic interactions. Yeast Identification of SET7, a Novel & First Histone Methyltransferase Further simulations suggest transition towards the inward open form. We Targeting H3K37 also study the glucose docked structure to identify the substrate bound translo- Jihyeon Kim. cation pathway. Glucose binds to the semi-SWEET with strong hydrogen bonds Kyungpook National University, Daegu, Republic of Korea.

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Chromatin dynamics and gene expression are mediated by epigenetic modifica- Cells maintain a cell size homeostasis through coordinating cell growth and di- tions on DNA and histones. Abnormal patterns of epigenetics could be linked to vision determined at the point of the commitment to cell division, termed Re- various diseases such as diabetes, metabolic diseases, and various cancers. One striction Point in metazoans, and Start in the model organism, S. cereviseae. of the key histone modifications is histone methylation catalyzed by histone Start depends on size-dependent expression of the G1/S regulon (SCB/MCB methyltransferase (HMTases). Histone methylation plays important roles in target sites), which includes G1 cyclins Cln1/2, as controlled by the trigger numerous biological processes including DNA damage repair, DNA replica- (Cln3), the transcription complexes SBF (Swi4-Swi6) and MBF (Mbp1- tion, cell division, gametogenesis, aging, and longevity. However, the mecha- Swi6), and the transcriptional repressor Whi5. SBF and MBF functionally over- nisms and pathways of histone methylation aren’t fully understood. lap, and are both thought to contribute to G1 cyclin expression; however, it is Fission yeast, Schizosaccharomyces pombe (S.pombe), is an ideal model organ- not known to what extent, and how these contributions are affected by environ- ism to understand the basic pathways of epigenetics. Since S.pombe has only 13 mental conditions. Specifically, SBF controls Cln2 expression via high affinity set genes that contain evolutionally conserved catalytic SET domain and only 4 SCB sites and SBF/MBF controls Cln1 via MCB sites. To dissect how endoge- Set proteins have been identified as HMTases versus 69 in human. Here we neous G1/S cyclins (Cln1/2/3) levels evolve as a function of cell size and time, show the characterization of all 13 S.pombeset deletion mutants and observed we used 2-photon scanning Number and Brightness (sN&B) and FLIM/Phasor, that some set mutants exhibit remarkable defects under several environmental coupled to time-lapse microfluidics, to quantify G1 cyclins in live G1-phase conditions. Furthermore, we identified Set7 as the first and novel HMTase tar- single cells. Cln2 expression, via SBF, was necessary for the positive feed- geting H3K37, a novel histone lysine mark in S.pombe, in eukaryotic HMTases. back loop responsible for robust passage through Start in glucose, where Cln2 accumulated rapidly. Deletion of Swi4 resulted in large cells which did 757-Pos Board B527 not exhibit a strong nuclear peak of Cln expression. Cln1 levels were elevated Absolute Quantification Reveals Growth and Nutrient-Dependent Control in glycerol, and deletion of Mbp1 or Cln1 showed a significant large size of G1/S Transcription Factor Abundance as a Determinant of Start phenotype in glycerol but not glucose, consistent with increased roles for 1 2 1 2 Sylvain Tollis , Savanna Dorsey , Michael D. Tyers , Catherine A. Royer . Cln1 and Mbp1 in glycerol. Consistently low concentrations of the trigger cy- 1 2 IRIC-Universite de Montreal, Montreal, QC, Canada, Rensselaer clin, Cln3, were observed in the nucleus at the beginning of G1 in both mother Polytechnic Institute, Troy, NY, USA. and daughter cells. Further quantification of the interactions between the How cell size control is achieved remains a fundamental unsolved problem, different Start regulators may reveal further details on the molecular mecha- largely due to a lack of reliable quantitative information. Start, the commitment nisms of cell size control. to division in S. cerevisiae, is associated with an MBF (Mbp1/Swi6) and SBF (Swi4/Swi6) controlled transcription burst. We have used scanning Number 760-Pos Board B530 and Brightness microscopy to reliably measure Mbp1, Swi4/6 and Whi5 (SBF A Systematic and Scalable Approach for Dissecting the Molecular Mech- repressor) protein levels in single live yeast cells. We find that these factors exhibit anisms of Transcriptional Regulation in Bacteria low nuclear concentrations (50-200nM) and form dimers. While Swi4 and Mbp1 Nathan M. Belliveau1, Stephanie L. Barnes1, William T. Ireland1, compete for Swi6, SBF/MBF do not saturate target DNA sites. Strikingly, [Swi4] Suzannah M. Beeler1, Justin B. Kinney2, Rob Phillips1. increases with cell size in G1. Mathematical modeling positions the [Swi4]/ 1California Institute of Technology, Pasadena, CA, USA, 2Cold Spring [Whi5] ratio and the fraction of SBF/MBF-bound DNA sites as new key determi- Harbor Laboratory, Cold Spring Harbor, NY, USA. þ nants of Start. Moreover, we find that MBF components are upregulated ( 50%) Organisms across all domains of life must make regulatory decisions in in cells grown in glycerol. In agreement with our model predictions, unlike wild- response to changing environments. The decision about when and where to type the mbp1 deletion mutant is only slightly smaller on poor compared to rich turn on transcription in bacteria is mainly controlled through the binding of medium, reflecting a leading role for MBF in driving premature Start and small transcription factors to promoter regions of the DNA. However, while the cat- cell size on poor nutrients. These results reveal that metabolically-driven changes alog of bacterial genomes continues to expand rapidly, we remain ignorant in Start factor expression ratios exercise significant control over the balance be- about how almost all of the genes in these genomes are regulated. Even for tween growth and division in budding yeast. More generally, we propose that Start the organism Escherichia coli, whose regulation is arguably best understood, factor expression levels constitute a hub that integrates various metabolic and we still have no indication if or how more than half of the genes are regulated. environmental signals to couple the cell cycle to the cellular environment. Here we show how a combination of massively-parallel reporter assays, mass 758-Pos Board B528 spectrometry, and information-theoretic modeling can be used to dissect bacte- The Role of mRNA Localization in Membrane Protein Trafficking rial promoters. We use the approach to recover nucleotide-resolution models of Specificity promoter mechanism for a variety of well-characterized and previously unan- Shankar Mukherji. notated promoters in E. coli. We believe this approach opens up the possibility Physics, Washington University, Saint Louis, MO, USA. of quantitatively dissecting the mechanisms of promoter function across the Organelle identity and function depend on accurate trafficking of proteins to genome of E. coli and other bacteria as well. organelle membranes. From the endoplasmic reticulum to mitochondria to per- oxisomes among others, however, the biophysical mechanisms underlying spe- 761-Pos Board B531 cific trafficking of membrane proteins to their target organelle membranes often Effects of Gene Duplication on the Non-Equilibrium Dynamics of remain obscure. Focusing on the mitochondrial receptors Tom70 and Tom20 as Probability Mass in Toggle-Switch: Cellular States, Sources and Sinks, a model system, we quantitatively determined the role of subcellular localiza- Oscillations tion of mRNAs in achieving protein targeting specificity to the mitochondrial Anna Terebus, Jie Liang. outer membrane. By dissecting the sequence determinants of mitochondrial re- Bioengineering, University of Illinois at Chicago, Chicago, IL, USA. ceptor mRNA trafficking specificity, we find that mRNA localization to the A toggle switch is a highly abundant network motif formed by two genes mutu- mitochondria is required for accurate receptor targeting to the mitochondria; ally inhibiting each other. Understanding the response of toggle switch under in the absence of high mRNA localization to the mitochondria, protein accumu- evolutionary selection pressure is of great importance, as it often serves as a lates in both the ER and cytoplasm. Our data is consistent with a reaction- memory device controlling switching between different cellular phenotypes, diffusion based mathematical model in which mitochondrial receptor protein which are essential during cell differentiation and cell cycle. Changes in targeting specificity is dependent on both translation-independent mRNA local- gene copy number are among the most frequent evolutionary events, however, ization to the mitochondria as well as evasion from ER trafficking pathways their effects on the toggle switch behavior are unknown. Here we examine how such as the signal recognition particle pathway. We expect that our mathemat- gene duplication in the toggle switch affects the non-equilibrium dynamics of ical framework will aid future studies of unraveling the function of RNA local- transitions between different cellular states. While there are many stochastic ization in cellular spatial organization. models of toggle switch, majority of them combine transcription and translation into single protein synthesis process, with the mRNA transcription process ne- 759-Pos Board B529 glected. Furthermore, existing studies rely on sampling of reaction trajectories Absolute Quantification of G1/S Start Network Machinery Demonstrates and may not provide a full description of the probability landscape. Here we Nutrient and Growth Dependent Transcription Factor Complexes Affect employ a detailed toggle switch stochastic model in which the reactions of tran- Cln1/2 Expression Differentially scription and translation are both explicitly modeled. We use the recently Savanna B. Dorsey1, Sylvain Tollis2, Michael Cook3, Jing Cheng4, developed ACME method to compute the exact probability landscape. We Labe Black1, Stephen Notley1, Michael D. Tyers4, Catherine A. Royer1. further employ our previously developed model of discrete probability velocity 1Rensselaer Polytechnic Institute, Troy, NY, USA, 2Universite de Montreal, and flux fields in the direction of each of the modeled species, and compute the Montreal, QC, Canada, 3Mount Sinai Hospital, Toronto, ON, Canada, probability fluxes of protein concentration in the steady state, with and without 4Universite de Montreal, Montreal, QC, Canada. gene duplication. We discuss changes in the dynamics of non-equilibrium

BPJ 8578_8581 152a Sunday, February 18, 2018 probability landscape and the appearance of new cellular states, sources and models with fixed and codon-dependent elongation rates. Additionally, we sinks, as well as changes in their locations. We also discuss the existence simulate the effect of chemical perturbations, puromycin and harringtonine, and the nature of oscillations in the systems upon gene duplication. These re- which inhibit elongation and initiation steps, respectively. Because codon us- sults can help us to gain insight into the effect of gene duplication on the age and chemical environments both affect translation mechanisms, the ki- switching between different cellular phenotypes. netics of protein production can be decoded to extract new information about cellular states and mRNA sequences. 762-Pos Board B532 Using Green Fluorescence Protein as Probe to Screen Compound Rescuing 765-Pos Board B535 Non-Sense Mutation of the Tumor Suppressor p53 Gene Stochasticity Promotes Synchronized Gene Expression between Cells in Jingjing Zhou, Zhengding Su. Somite Segmentation Hubei University of Technology, Wuhan, China. Hikaru Nozoe, Tatsuya Yamada, Yuichi Sakumura, Yasumasa Bessho, Many diseases are caused by premature termination codons (PTCs) including Kazushi Ikeda. the cancers which contain premature terminated p53. Aminoglycoside antibi- Nara Institute of Science and Technology, Nara, Japan. otics have been proved to enable to induce the readthrough of PTCs and restore In developing vertebrates, a spatial repetitive pattern of somite is formed along the production of functional full-length p53. Currently, there is lack of a fast the head-to-tail axis in the presomitic mesoderm. Because somite segmentation and efficient high-throughput screening method. In this study, we demonstrated is closely related to body structure, it is important to elucidate how the somite is a simple and efficient screening method using E. coli cells harboring fusion formed to understand vertebrate body formation. The timing of somite segmen- gene containing a p53-PTC gene followed by green fluorescent protein tation in the mammals is regulated by Hes7 expression and the neighboring (GFP) gene. Our data showed that the cells treated with aminoglycosides could cells synchronize Hes7 expression to spatially aggregate cells in the same so- reproduce full-length p53 protein, monitored by the fluorescence of GFP pro- mite. This expression synchronization shows a strong robustness to distur- tein. Our work suggested that the system reported here can be extended to bance. In our preliminary experiment, Hes7 expressions in different cells screening aminoglycoside compounds for rescuing premature termination were desynchronized and the somite cells dispersed when we applied distur- codons. bance to mouse embryo by heat shock or drug treatment. In the next somite for- mation, however, the cells recovered the synchronization. This rapid recovery 763-Pos Board B533 in Hes7 synchronization demonstrates the robustness of vertebrate develop- Pressure Induced SOS Response in Escherichia coli Involves Mrr Restric- ment in obtaining stability from destabilization. However, the mathematical tion Endonuclease Dissociation mechanism is still unknown. There are time delays in the intranuclear transport Anais Bourges1,2, Oscar E. Torres M.3, Anirban Ghosh3, Wubishet Tadesse3, of Notch signal and extranuclear transport of mRNA. Many previous studies Gilles Labesse4, Nathalie Declerck4, Abram Aertsen3, Catherine Royer2. introduced the fixed time delay and described Hes7 expression process as a 1 2 Universite Montpellier, Montpellier, France, Renssealer Polytechnic time-delay system using delay differential equations. If using this deterministic 3 Institute, Troy, NY, USA, Laboratory of Food Microbiology, Leuven, equation, because the fixed time delay makes it difficult to adjust the expression 4 Belgium, Centre de Biochimie Structural, Montpellier, France. time, the strong Delta-Notch interaction should be required to show the rapid Subjecting the mesophilic Escherichia coli bacterium to a pressure of 100 recovery. However, Hes7 expression is a stochastic event and the interaction MPa elicits an SOS response, despite the fact that pressure itself cannot between the somite cells are not strong in the mammals. compromise DNA covalent integrity. Screens for pressure shock resistance re- In this work, we introduced stochasticity in transcription and translation times vealed the constitutive presence in several strains of E. coli of a cryptic, endog- into the mathematical model to examine whether the somite cells can rapidly enous type IV restriction endonuclease, Mrr (Methylated adenine Recognition synchronize in Hes7 expression without the strong interaction between cells. and Restriction), that cleaves methylated DNA. Since the enzyme is present and By numerical simulation, our stochastic model showed the synchronized oscil- non-toxic in the absence of pressure treatment, activation must be pressure lation of Hes7 expression and the rapid recovery of the synchronization after dependent. Although types IV REases do not have a cognate methyltransferase desynchronization by the disturbance. (MTase), HhaII has been shown to activate Mrr and induce an SOS response through methylation of high affinity sites, recognized by Mrr. Our previous Posters: Neuroscience in vivo fluorescence results using scanning Number and Brightness suggest that pressure shock ‘‘pushes’’ the equilibrium from the inactive tetrameric 766-Pos Board B536 form of Mrr toward the active dimer which can bind cryptic sites on E. coli Molecular Mechanisms of Synaptic Vesicle Priming by Munc13 and chromosome and cleave DNA. In contrast, HhaII induction appears to Munc18 ‘‘pull’’ the equilibrium toward the dimeric Mrr active forms bind to DNA. Ying Lai1, Axel T. Brunger2. To test this model, we used the same approach to evaluate Mrr stoichiometry 1The Department of Molecular and Cellular Physiology, Stanford University, in both catalytically defective mutants as well as a constitutive mutant which Palo Alto, CA, USA, 2The Department of Molecular and Cellular Physiology, is lethal for E. coli. Some of the mutants were found to respond only to pressure Stanford University, Palo Alto, CA, USA. and not to HhaII MTase induction, or vice versa. Homology modeling of the Munc13 catalyzes the transit of syntaxin from a closed complex with Munc18 tetrameric Mrr catalytic domain supports the proposed mechanism of action into the ternary SNARE complex. Here we report a new function of Munc13, drawn from our in vivo results of the mutants. sN&B confirmed the role of independent of Munc18: it promotes the proper syntaxin / synaptobrevin sub- the tetramer-dimer equilibrium in modulating Mrr activity. We are now at- configuration during assembly of the ternary SNARE complex. In cooperation tempting to study association/dissociation process of purified Mrr under pres- with Munc18, Munc13 additionally ensures the proper syntaxin / SNAP-25 sure by fluorescence correlation spectroscopy (FCS) thanks to a high- subconfiguration. In a reconstituted fusion assay with SNAREs, complexin, pressure set-up we recently implemented on our 2-photon microscope. and synaptotagmin, inclusion of both Munc13 and Munc18 quadruples the Ca2þ-triggered amplitude and achieves Ca2þ-sensitivity near physiological 764-Pos Board B534 concentrations. In Munc13-1/2 double-knockout neurons, expression of a Stochastic Modeling of Single RNA Translation Dynamics constitutively open mutant of syntaxin could only minimally restore neuro- Luis U. Aguilera1, Tatsuya Morisaki2, Timothy J. Stasevich2, transmitter release relative to Munc13-1 rescue. Together, the physiological Brian Munsky1. 1 functions of Munc13 may be related to regulation of proper SNARE complex Department of Chemical and Biological Engineering, Colorado State assembly. University, Fort Collins, CO, USA, 2Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA. 767-Pos Board B537 Translation is an essential step in which ribosomes decipher mRNA sequences Mobile Calcium Channels Contribute to Variability of Pre-synaptic Trans- to manufacture proteins. Recent advances in single-molecule imaging allow mitter Release live-cell quantification of the kinetics of ribosome initiation and elongation. Martin Heine1, Jennifer Heck1, Pierre Parutto2, Anna Ciuraszkiewicz1, Here, we integrate single-molecule data and stochastic models to investigate Arthur Bikbaev1, Romy Freund1, Anna Fejtova3, David Holcman2. how elongation rates vary among different gene transcripts. Our computational 1Leibniz Institute for Neurobiology, Magdeburg, Germany, 2Ecole Normale method automatically generates discrete translation models to match any Superieure, Paris, France, 3Universit€atsklinikum Erlangen, Erlangen, mRNA sequence. The models are then solved using stochastic dynamics; the Germany. results are quantified in terms of translation spot intensity; and we fit these to The temporal coding within neuronal networks is dominated by the release single-mRNA translating spots observed under the microscope. We compare properties of individual synapses between neurons. The initial trigger for

BPJ 8578_8581 Sunday, February 18, 2018 153a synaptic transmission is the opening of calcium channels to initiate fusion of a 11 5 1 mM for Fl-GluBP, at physiological ionic strength, pH 7.5 and 3 oC. transmitter vesicle. Beside the need to have a fusion competent vesicle at the Investigating glutamate association kinetics by stopped-flow fluorimetry, synaptic membrane, the positioning between voltage gated calcium channels biphasic fluorescence increase was detected. 80% of the fluorescence enhance- (VGCC) and the vesicle is critical for the success of transmitter release. Here ment was too fast to measure. Rates in the range of 200 to 800 s1, showing we used sptPALM experiments of mEOS-tagged calcium channels to monitor saturation, were measured for the second phase, interpreted as an isomerisation. the organization of CaV2.1-channels in the presynaptic membrane. A major Data for the hyperbolic concentration dependence of isomerisation were best fit determinant of the presynaptic release probability is the stabilisation of to a two-step mechanism in which rapid, diffusion-limited binding is followed 4 1 VGCC by presynaptic scaffold proteins. Many interactions are described to by isomerisation: equilibrium constant K1 of 4.6 x 10 M for step 1, consis- 9 1 1 1 1 depend on the interaction between the C-terminal tail of the channel and tent with kþ1 R 10 M s and k-1 R 216000 s ; kþ2 of 2220 s and k-2 of 1 different domains of scaffold proteins. Alternative splicing of exon 47 result 229 s for step 2 (Kd(overall) 27 mM). We propose that a similar mechanism of in the expression of either CaV2.1-channels that lack a significant proportion diffusion-limited glutamate binding exists for and explains the rapid gating of (200 as) or have the full length of the C-terminus. We used both splice var- AMPAR. iants to explore their presynaptic dynamics and impact in synaptic transmis- 1Li et al., 2003 Biochemistry 42, 12358-12366. sion. Both channel splice variants are temporal confined in nanodomains, 2Miller et al., 1983 J Biol Chem 258, 13665-13672. which themselves can travel within the presynaptic membrane. Using optoge- 3Helassa et al., submitted. netic tools to interfere with the mobility of VGCC in the neuronal membrane 4Okumoto et al., 2005 PNAS 102, 8740. enabled us to uncover a contribution of mobile VGCC to synaptic transmission. This work is funded by BBSRC grant BB/M02556X/1 to K.T. These findings suggest a flexible organization of VGCC in the presynaptic membrane and offer a very flexible modulation of temporal synaptic transmis- 770-Pos Board B540 sion. We hypothesis that this molecular flexibility contribute to the temporal Single Molecule Translation Imaging of Local Protein Synthesis and RNA coding of information within neuronal networks. Docking Reveals the Regulation of Site Specific Axon Remodeling In Vivo Clemens F. Kaminski1, Hovy Ho-Wai Wong2, Florian Strohl1, 768-Pos Board B538 Julie Quiaojin Lin2, Christine E. Holt2. Novel iGluSnFR Variants Optimised for Rapid Glutamate Imaging 1Dept. Chem Engineering and Biotech, Cambridge University, Cambridge, Nordine Helassa1, Celine Durst€ 2, Catherine Coates1, Urwa Arif1, United Kingdom, 2Physiology Development and Neuroscience, Cambridge Christian Schulze2, Simon Wiegert2, Michael Greeves3, Thomas Oertner2, University, Cambridge, United Kingdom. Katalin To¨ro¨k1. Localised protein synthesis (LPS) is known to take place at precise subcellular 1Molecular and Clinical Sciences Research Institute, St. George’s University locations to guide the growth response in neurones, for example in the presence of London, London, United Kingdom, 2Institute for Synaptic Physiology, of chemical guidance cues. We show here that LPS is also a major determinant Center for Molecular Neurobiology Hamburg, Hamburg, Germany, 3School of axon branching in developing neurones. Axon branching is key to maximise of Biosciences, University of Kent, Canterbury, United Kingdom. the opportunity for post synaptic connections to be made and therefore for the Intensity-based glutamate-sensing fluorescent reporter iGlu-‘sniffer’ development of neuronal circuitry. We show the use of high resolution live im- (iGluSnFR) is a useful tool for neuroscience that has enabled detection of gluta- aging to determine the spatial and temporal dynamics of local protein synthesis mate release from single presynaptic terminals. However the probe’s fluores- in Xenopus retinal ganglion cells [1, 2]. We see that LPS and axon branching is cence rise and decay kinetics appeared too slow to give an accurate readout tightly regulated by spatial patterns of endogenous RNA granules as they are of glutamate dynamics at the synapse during high frequency bursts. We thus being trafficked through the neurones. We see that RNA granules dock at sites generated novel variants with faster glutamate binding kinetics by mutation of branch emergence and that prolonged docking of RNA granules leads to the of amino acid residues coordinating glutamate at the binding site. Fast variants development of axon branches. Branches can form and retract, depending on iGluf and iGluu have comparable brightness and fluorescence dynamic range to granule movement, and branches that remain stable are invaded by RNA gran- iGluSnFR. The Kd for glutamate measured by equilibrium binding titration at ules over prolonged periods. Our results reveal a close relationship between ar- 20 C is increased from 33 mM (iGluSnFR) to 137 mM and 600 mM (iGluf bor dynamics and RNA trafficking and show that RNA docking predicts sites of and iGluu, respectively). At 34 C, in vitro dissociation rate measured by branch emergence. Live visualization of b-actin synthesis reveals the rapid stopped-flow fluorimetry are increased up to 6-fold from 233 s1 for iGluSnFR accumulation of nascent b-actin in discrete ‘hotspots’ in branches and at branch 1 (toff=4.3 ms) to 1481 s for iGluu (toff=0.7 ms), making iGluu the fastest gluta- points. The demonstration here that axonal arborization is disrupted by a loss of mate fluorescent reporter to-date. At single presynaptic terminals stimulated at b-actin translation implicates RNA localization and local translation broadly in 100 Hz in hippocampal slice culture, iGluu has 5-fold faster ‘‘off’’ rate (toff=2.6 wiring the nervous system and raises the possibility that axonal, as well as den- ms) than iGluSnFR, with the signal returning to baseline between each stim- dritic, arborization defects underlie some neurodevelopmental disorders. [1] ulus, revealing complete clearing of synaptic glutamate between high fre- Wong HH et al, Neuron 95 (4), 852-868 (2017) [2] Strohl, F et al, Scientific quency release events. Glutamate neurotransmission shows pronounced reports 7, 709 (2017) depression during high frequency bursts that can be attributed to a depletion of presynaptic resources or desensitization of postsynaptic receptors. By 771-Pos Board B541 Physiochemical Principles of AMPAR Insertion in Dendritic Spines comparing iGluu signals and AMPA receptor currents, we show that synaptic Miriam Bell1, Daniel Tartakovsky2, Padmini Rangamani1. depression during 100 Hz trains is entirely due to reduced glutamate release 1 2 while the recovery after 500 ms has a postsynaptic component. UC San Diego, La Jolla, CA, USA, Stanford University, Stanford, CA, This work was funded by the Wellcome Trust 094385/Z/10/Z and BBSRC BB/ USA. M02556X/1 to K.T; German Research Foundation to T.G.O. (SPP 1665, SFB Dendritic spines are small signaling compartments in neurons and house impor- tant signaling networks, receptors, and molecules associated with learning and 936, FOR 2419) and J.S.W. (SPP 1926, FOR 2419); European Research Coun- 2þ cil to J.S.W. (ERC-2016-StG 714762). memory. In particular, Ca /calmodulin-dependent protein kinase II (CaMKII), protein phosphatase 1 (PP1), and a-amino-3-hydroxy-5-methyl-4-isoxazolepro- 769-Pos Board B539 pionic acid receptor (AMPAR) have been identified as molecular markers of Diffusion-Limited Glutamate Binding to GluBP is Revealed by Novel Fluo- memory, suggesting that their spatio-temporal dynamics play an important rescent Probe role in structural plasticity. Furthermore, dendritic spines have characteristic Catherine Coates, Nordine Helassa, Katalin To¨ro¨k. shapes that have been linked to healthy state, disease, aging, and other factors; Molecular and Clinical Sciences Research Institute, St. George’s, University however, the exact relationship between shape and function remains unknown. of London, London, United Kingdom. To elucidate this relationship, we developed a spatial model of Ca2þ-influx AMPAR-s are the fastest responding ligand-gated ion channels with channel through N-methyl-D-aspartate receptor (NMDAR), CaMKII/PP1 activation, opening times in the ms range1, requiring rapid glutamate binding. Bacterial and AMPAR insertion in a realistic spine geometry. Using this model, we periplasmic glutamate/aspartate binding protein (GluBP) has structural homol- show that i) variables in membrane voltage mediated Ca2þ-influx, particularly ogy to AMPAR, thus the glutamate binding mechanisms of the two protein the number of active NMDAR and extracellular Ca2þ levels, primarily regulate families are expected to be similar. However, current indicators of glutamate cytosolic protein dynamics through their impact on Ca2þ dynamics. ii) AM- kinetics record association rate constants in the order of 107 M1s1(2,3), thus PAR dynamics depend on a combination of membrane curvature effects, endo- may be too slow for the detection of the rapid AMPAR response kinetics. plasmic reticulum (ER) spatial distribution, cytosolic protein concentrations, We have developed a novel glutamate sensor based on GluBP (Fl-GluBP), and stargazin binding. iii) AMPAR levels depend on both exocytosis from cyto- labelled with an environmentally sensitive fluorophore at a Cys residue near solic stores and, more significantly, diffusion of AMPAR from extrasynaptic 4 the binding site. The Kd of 600 nM measured for GluBP , is increased to membrane (ESM) regions on the dendrite.

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772-Pos Board B542 775-Pos Board B545 Illuminating Post-synaptic Scaffolding and Kinase Regulation Mecha- Family-Wide Biophysical Analysis of Dpr-DIP Interactions nisms of Pyk2 by Hydrogen/Deuterium Exchange Mass Spectrometry Filip Cosmanescu1, Phinikoula S. Katsamba2, Alina P. Sergeeva2, Eric S. Underbakke, Hanna Wokpetah. Goran Ahlsen2, Saurabh Patel1, Joshua Brewer1, Liming Tan3, Shuwa Xu3, Biochemistry, Biophysics, Mol. Biol, Iowa State University, Ames, IA, USA. Qi Xiao3, S. Lawrence Zipursky3, Barry Honig1,2, Lawrence Shapiro1. The post-synaptic density (PSD) is a collection of scaffolded signaling proteins 1Department of Biochemistry and Molecular Biophysics, Columbia that govern synaptic plasticity. Pyk2 is a non-receptor tyrosine kinase enriched University, New York, NY, USA, 2Howard Hughes Medical Institute, at the PSD of glutamatergic synapses. Activated by Ca2þ influx, Pyk2 partici- Columbia University, New York, NY, USA, 3Department of Biological pates in the translation of synaptic activity into signaling cascades that tune Chemistry, HHMI, David Geffen School of Medicine, University of synaptic strength. Pyk2 exerts its signaling effects by recruiting Src kinase California, Los Angeles, Los Angeles, CA, USA. into a Ca2þ-induced signaling complex. Although Pyk2 serves as the critical Two subfamilies of Drosophila immunoglobulin superfamily (IgSF) cell sur- link between Ca2þ signaling and the Src pathway, the molecular mechanisms face proteins, Dprs (defective proboscis response) and DIPs (Dpr interacting underlying Pyk2 activation remain ambiguous. We integrated hydrogen/deute- proteins) are broadly expressed in the nervous system and involved in the rium exchange mass spectrometry (HDX-MS), kinase assays, and mutagenesis development of neural circuits. Neurons with distinct synaptic specificities to dissect regulatory protein interactions and conformational changes in the each express unique combinations of Dprs while a subset of their synaptic part- Pyk2 activation complex. HDX-MS was performed to interrogate interactions ners express the complementary DIPs. A qualitative interactome developed and conformational changes responsible for Pyk2 activation. Pyk2 surfaces from high-throughput experiments has shown that each DIP interacts with a implicated in activity regulation via HDX-MS were further probed by mutagen- unique set of Dpr proteins. Using surface plasmon resonance (SPR), we have esis. Ultimately, HDX-MS revealed a Pyk2 activation mechanism involving generated a quantitative Dpr and DIP interactome, which contained several higher-order clustering via scaffolding protein interactions and relief of autoin- novel features. We determined the binding affinities of the majority of Dpr- hibitory inter-domain interactions. DIP heterophilic interactions, revealing novel binding groups that span a range of affinities. Crystal structures of Dpr-DIP heterocomplexes were determined 773-Pos Board B543 and used to design site-specific mutants that, along with SPR experiments, Mechanochemistry of Calcium Induced Neurite Retraction and Rupture reveal the major determinants of Dpr-DIP binding specificity. Using analytical 1 2 2 1 Katherine Pearce , Miriam Bell , Padmini Rangamani , Suzanne Scarlata . ultracentrifugation (AUC), we show that some Dpr and DIP family members 1Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, 2 form homophilic dimers as well. Multiple crystal structures of DIP homodimers MA, USA, University of California San Diego, San Diego, CA, USA. reveal the molecular determinants of homophilic binding and structure-guided In mammalian cells, Gaq/PLCb /PIP2 signal pathways generate calcium mutants along with AUC experiments further validated their mechanism of signals after activation with acetylcholine. This is important for neuronal interaction. The existence of DIP and Dpr homodimers suggests the possibility differentiation studies and neurodegenerative diseases. We studied the mech- of still-unknown mechanisms of Dprs and DIPs in neural circuit formation. anochemical response of calcium-induced neurite retraction using a rat neuronal model. We stimulated PC12 cells with acetylcholine and observed 776-Pos Board B546 partial or full neurite retraction under extended stimulation using live cell Increase in Activity of L-Type Calcium Channel CaV1.2 by Amyloid Beta imaging. During retraction, we see a drastic recombination and redistribution Peptide 1-42 (Ab42) via Beta 2 Adrenergic Receptor (b2AR) Signaling of actin in cells transfected with mCherry-actin. As the retraction occurs we Boram Lee, Jennifer L. Price, Johannes W. Hell. also see a spike in actin signal intensity in the membranous region at the Pharmacology, University of California, Davis, Davis, CA, USA. base of the neurite that we believe is related to the increasing amount of Alzheimer’s disease (AD) is a progressive neurodegenerative disease. Accumu- cell membrane being pulled toward the stoma of the cell as the actin cytoskel- lation of plaques consisting predominantly of Ab42 peptide is a hallmark of eton breaks down. This increase in signal intensity was also seen using AD. This is formed when beta amyloid precursor protein (bAPP) is cleaved fluorescent PIP2 sensor. The PIP2 signal is seen not only at the base of the by beta-secretase 1 (BACE1), ultimately resulting in pathological plaques neurite but was also seen migrating from the cytosol to the membrane and damaged neurons. 2þ 2þ during extended periods of stimulation. In order to elucidate the mechanisms CaV1.2 is the most prevalent L-type Ca channel in the brain. Ca influx into underlying the neurite response, we developed a computational model for neurons is escalated in animal models of AD, and Ab42 peptide stimulates ac- the spatio-temporal dynamics of calcium-induced neurite response. The tivity of CaV1.2 in hippocampal neurons and the mouse model. model parameters are informed by the experiments and we are currently The mechanism how Ab42 upregulate the activity of L-type calcium channels exploring the role of actin severing by ADF/Cofilin and active contraction associated with AD is unclear. Ab42 can also activate the b2 adrenergic recep- by myosin. tor (b2AR). Since CaV1.2 forms a complex with the b2AR, and Cav1.2 activity is upregulated by the b2AR activation, we investigated the hypothesis that 774-Pos Board B544 Ab42 increases activity of CaV1.2 by activating b2AR. Structural Studies of C1QL-Mediated Complexes Single-channel recordings from hippocampal neurons were conducted with Perla Arianna Pen˜a Palomino, Susanne Ressl. Ab42 and inverse Ab42. Unlike inverse Ab42, Ab42 augmented the open prob- Molecular and Cellular Biochemistry, Indiana University Bloomington, ability of CaV1.2. In order to test whether the effect of Ab42 is mediated by Bloomington, IN, USA. b2AR, hippocampal neurons were pretreated with the b2AR-specific antagonist Proper brain function is based on neuronal networks, which are based on syn- ICI118,551 or the b1AR-specific antagonist CGP20712. Only ICI118,551 in- apses, the fundamental unit of neuronal communication. Synaptic adhesion hibited the stimulation of CaV1.2 by Ab42. In addition, it was examined proteins bind across the synaptic cleft to form complexes tethered to both whether Ab42 induces phosphorylation of CaV1.2 at S1928 and S1700 by pre- and post-synaptic membranes. Dysfunction of synaptic adhesion proteins immunoblotting. Phosphorylation of S1928 and S1700 was increased by cause brain disorders, ‘synaptopathies’. Members of the family of complement Ab42 but not by inverse Ab42, and the Ab42 effect was blocked by component 1, q subcomponent-like proteins (C1QL1-4) promote synapse orga- ICI118,551. Accordingly, Ab42 enhances activity of CaV1.2 and increases nization (1, 2). The underlying mechanism is unknown. Genetic analysis on phosphorylation of S1928 and S1700 through b2AR signaling. C1ql3 revealed its expression in the limbic system with phenotypes of hyper- activity, sleeping disturbances, and a deficit in forming emotional memories 777-Pos Board B547 (3)(4). C1QLs are secreted into the synaptic cleft and bind to a post- Reduced Cooperativity of Voltage-Gated Sodium Channels in the Hippo- synaptically localized G protein-coupled receptor (GPCR) called adhesion campal Interneurons of Aged Mouse Model of Alzheimer’s Disease GPCR B3 (ADGRB3) (1, 2). This raises the question whether C1QL has poten- Carlos M. Perez, Ghanim Ullah. tial to bidirectionally coordinate a trans-synaptic complex to mediate a ternary Physics, University of South Florida, Tampa, FL, USA. complex that influences synapse homeostasis. Moreover, C1QLs’ electrostatic Amyloid beta (Ab) associated with Alzheimer’s disease (AD) leads to surface is distinct from each other and they can form higher oligomer species abnormal behavior in inhibitory neurons, resulting in hyperactive neuronal net- (5), which together with calcium specificity dictate the nature of binding and works, epileptiform behavior, disrupted gamma rhythms, and aberrant synaptic stoichiometry, resulting in a novel mechanism of how trans-synaptic adhesion plasticity. Previously, we used dual modeling-experimental approach to is achieved. We have conducted an unbiased screen for novel C1QL3 binding explain several observations, including failure to reliably produce action poten- partners and have identified multiple candidates that have potential to be in vivo tials (APs), smaller AP amplitudes, higher resting membrane potential, and binding partners of C1QL3. Here we present structural characterization of higher membrane depolarization in response to a range of stimuli in hippocam- C1QL3 alone and together with its complex proteins using electron microscopy pal inhibitory neurons from 12-16 month old female APPswe/PSEN1DeltaE9 and X-ray crystallography. (APdE9) AD mice as compared to age-matched non-transgenic (NTG) mice.

BPJ 8578_8581 Sunday, February 18, 2018 155a

Our experimental results also showed that AP initiation in interneurons from tein), a key mediator of neuronal excitatory pathways, increased in dehydrated APdE9 mice is significantly different from that of NTG mice. AP in interneu- hippocampus after subthreshold stimulation.These results show that brain rons from NTG mice is characterized by abrupt onset and an upstroke that is dehydration levels comparable to those measured following short-term water much steeper and occurs with larger variability as compared to cells from deprivation in mice increase neuronal excitability. Neurons appear very sen- APdE9 mice. The phase plot (the rate of change of membrane potential versus sitive to water-activity changes, suggesting local dehydration in the interstitial the instantaneous membrane potential) of AP produced by interneurons from matrix contributes to the cognitive deficits seen in dehydrated humans. As a APdE9 mice shows a biphasic behavior whereas those from NTG mice shows plausible mechanism, we suggest that lower water activity in dehydrated a monophasic behavior. Here we show that using the classic Hodgkin-Huxley brains increases the effective concentration of all solutes, modifying the (HH) model for the gating of voltage gated sodium channel (VGSC) cannot timing, diffusivities, and probabilities of neurotransmitters binding to their reproduce these features and a model that takes into account a cooperative acti- cognate receptors. vation of VGSCs is needed. We further show that VGSCs in interneurons from APdE9 mice exhibit significantly lower cooperativity in their activation as 780-Pos Board B550 compared to those form NTG mice. Protective Role of Olesoxime in Alpha-Synuclein-Induced Mitochondrial Grant Information: National Institute of Health on Aging (R01AG053988). Dysfunction Amandine M.F. Rovini, Maria Queralt-Martin, Philip Gurnev, 778-Pos Board B548 Sergey M. Bezrukov, Tatiana K. Rostovtseva. Prefrontal Cortical Neurons are Recruited as Secondary Associative Molecular Transport, NIH / NICHD, Bethesda, MD, USA. Memory Cells for Associative Memory and Cognition Context Jin-Hui Wang1, Jing Feng2, Huajuan Xiao1, Wei Lu2. Parkinson’s disease (PD) is a neurodegenerative disease associated with loss of 1Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, dopaminergic neurons and presence of Lewy bodies, which main protein 2Qingdao University, Qingdao, China. component is alpha-synuclein (a-syn). The molecular determinants underlying Associative memory cell is recruited for the integrative storage of associated a-syn secretion, aggregation, and intracellular toxicity are still unclear. signals. In learning exogenous associated signals afferent from different mo- Recently, using a channel reconstitution assay, our lab showed that a-syn inter- dalities, the sensory cortices are mutually innervated to recruit primary asso- acts with the outer mitochondrial membrane voltage-dependent anion channel ciative memory cells. In associative thinking and logical reasoning based on (VDAC). Moreover, under certain conditions a-syn can either partially block associative memory cells, secondary associative memory cells are hypotheti- this channel or translocate through it. Olesoxime, a cholesterol-like neuropro- cally recruited in cognition- and emotion-related brain areas. This hypothesis tective drug, has been shown to provide significant pro-survival benefits in a was examined in the prefrontal cortices of associative learning mice by behav- human neuronal model of a-syn-mediated toxicity. ioral task, two-photon cell imaging, electrophysiology and AAV-mediated Aim neural tracing in vivo. Paired whisker and olfactory stimulations lead to The dual goal of this work is 1) to address a tentative mechanism of olesoxime odorant-induced whisker motion and whisker-induced olfactory response. Af- protection against a-syn-induced mitochondrial dysfunctions and 2) to clarify ter this cross-modal associative memory forms, barrel and piriform cortical the role of VDAC in a-syn toxicity and olesoxime protection, using different neurons become to encode whisker and olfactory signals as well as to be cell-based and in vitro methods. mutually innervated, i.e., primary associative memory cells are recruited in Experimental approaches these sensory cortices. In the meantime, certain neurons in the prefrontal cor- We first showed that olesoxime reverses the effects of a-syn–mediated mito- tex are able to encode whisker and olfactory signals, which are recorded by chondrial toxicity by monitoring mitochondrial membrane potential and local field potentials and two-photon cell imaging. Moreover, individual neu- ROS production in a dopaminergic model of cells overexpressing a-syn. rons in the prefrontal cortex receive synapse innervations from barrel and piri- Given that a-syn and olesoxime have a putatively common target, VDAC, form cortices, where are injected by GFP- and RFP-tagged AAVs, we explored their effects at the molecular level. Using multi-channel respectively. Our studies provide morphological and functional evidences VDAC voltage-gating protocol, we observed that olesoxime, by itself, pro- for the recruitment of secondary associative memory cells in the prefrontal motes VDAC closure. In single-channel experiments we found that olesox- cortex, which may be used for cognitions. In the neural circuit of primary ime prevents a-syn translocation through VDAC. To verify this finding in a associative memory cells, secondary associative memory cells and memory cellular model, we employed a proximity ligation assay to look specifically presentation cells, these memory cells determine the specificity of associa- at a-syn-VDAC and a-syn -complex IV and to evaluate the impact of olesox- tively stored signals, the number of associative memory cells influences mem- ime. We found that a-syn binds to VDAC at the outer membrane and to com- ory strength and maintenance, as well as the functional plasticity of neurons plex IV at the inner membrane and that olesoxime reduces these interactions. and synapses in this circuit influences whether these memory cells are able Thus, our results support the pro-survival benefits of olesoxime in a human to integrate signals, to facilitate cognitions, to trigger memory presentation neuronal model of a-syn-mediated toxicity where VDAC plays a crucial cells for information recall and retrieval. role. 779-Pos Board B549 781-Pos Board B551 Brain Interstitial Matrix Hydration Inversely Correlates with Neuronal Patch-SEQ Provides Insight into the Etiology of Hyperexcitable Neurons Excytability in Patients with Temporal Lobe Epilepsy Anirudh Vashisht1, Michael Morykwas1, Ashok Hegde2, Louis Argenta1, Victoria Wolseley1, Tade Souaiaia2, Robert Chow1. Maria P. McGee1. 1Physiology and Biophysics, USC, Los Angeles, CA, USA, 2Cell Biology, 1Plastic and Reconstructive Surgery, Wake Forest University Medical Center, Suny Downstate Medical Center, Brooklyn, NY, USA. Winston-Salem, NC, USA, 2Biological and Environmental Sciences, Georgia Combining single-cell electrophysiology and transcriptome profiling offers College and State University, Milledgeville, GA, USA. insight into the etiology of mesial temporal lobe epilepsy. We have developed Interstitial flux of cerebrospinal fluid (CSF) mediates optimal transfer, clear- a novel approach to study the genetic/molecular/electrophysiological mecha- ance, and exchange of molecules in the brain. To determine whether distur- nisms of mesial temporal lobe epilepsy. This approach involves therapeutic sur- bances during dehydration or edema lead to neuronal dysfunction, we gical resection of brain tissue from patients suffering from medically refractory quantified hydration in the brains of mice subjected to mild water deprivation mesial temporal lobe epilepsy – which eliminates or reduces seizure severity in and measured neuronal excitability in the dehydrated and rehydrated hippo- 80% of patients. Multi-electrode-array (MEA) recordings are conducted to campus. Specifically, we titrated water activity ex vivo using artificial CSF so- map brain slices into interictal and non-interictal regions and to guide cell se- lutions of nonpenetrating inert polymers adjusted to give colloidosmotic lection for single-cell patch-clamp recording; electrophysiological recording is pressures in the 0-200 mmHg range. Compared to nondeprived controls, the followed by cytoplasm extraction and subsequent single-cell transcriptomics. brains of water-deprived mice had hydration potentials of 94.5 5 5.4 and This technique was applied to over 250 single cells from 11 surgical patients 103.8 5 3.5 mmHg after 12 and 24 hours, respectively, corresponding to identifying both differentially expressed genes and significant differences in 23 and 28 mmHg increases in potential, or 20% and 27% decreases in hydra- electrophysiological characteristics (interspike interval, adaptation, rebound), tion. The brain-matrix hydraulic conductance, 0.051 5 0.0019 ml/min/g/ specific to the interictal region. Our findings demonstrate the utility of mmHg, did not change significantly (n = 6 hydrated/dehydrated pairs). The combining single-cell patch clamp recording with transcriptome profiling, field excitatory postsynaptic potentials (fEPSP) in the hippocampus, adjusted and suggest that regional partitioning based on the MEA data is a proxy for to hydrations 25 and 125 mmHg below controls, increased 47% and 66%, the aberrant firing that constitutes the epileptic phenotype. Currently we are respectively, but did not change at hydrations up to 75 mmHg above control. able to demonstrate a distribution level electrophysiological change across a In addition, the level of phosphorylated CREB (cAMP response-binding pro- range of stimulated events describes the phenotype most accurately; our ability

BPJ 8578_8581 156a Sunday, February 18, 2018 to show the transcriptomics that underlie this distribution will allow us to eluci- roidogenesis but it is also implicated in immunomodulation, apoptosis and mi- date a molecular description of the disease. tophagy. TSPO is expressed at particularly low level in the healthy brain, however, there is a marked upregulation following brain injury, inflammation 782-Pos Board B552 and neurodegeneration. The molecular mechanism underlying the involvement Electrophysiological Alteration of Gene Expression in Human Embryonic of TSPO in neuroinflammation remains ill-defined even though targeting of the Brain Neurons using Single Cell RNA seq protein is exploited to diagnose inflammatory states of the brain through posi- Jae Mun Kim. tron emission topography (PET) scanning of TSPO specific radioligands. Neuroscience, University of Southern California, Los Angeles, CA, USA. To address this, we made use of the CRISPR/Cas9 genome editing system to Patch clamp recording is commonly used to investigate neuronal electrophys- target TSPO for knockout (KO) in mouse microglial cell line (BV2) to function iological properties. Following electrophysiological recording, it is possible to as a negative control for TSPO function in vitro. We successfully established a extract the cytoplasm of the neuron and to apply single-cell RNA sequence stable TSPO knockout BV2 cell line, validated via biochemical read-out and analysis (RNAseq). Patch clamp aRNA in vitro Amplification (PAIA) com- sequence scrutiny. bines the method of patch clamp with linear amplification of RNA. We per- Hitherto, our data suggest that the TSPO KO correlate with a reduced inflam- formed the PAIA protocol on 226 motor neurons from human embryonic matory phenotype, while parental BV2 show marked upregulation in TSPO spinal cord of gestational age 9-16 weeks, and recorded 3 distinct groups of signal, confirming a role of TSPO in neuroinflammation. As a confirmation firing patterns in these cells: single, repetitive adaptive, repetitive non- of the above, the newly synthesized TSPO ligand, GE-180, was also able to adaptive. With Differential gene expression analysis with DESeq, we discerned reduce the neuroinflammatory phenotype, mimicking the absence of the marker genes differentially expressed between neurons according to distinct protein. electrophysiological patterns. Lastly we explored if TSPO is implicated in neuroinflammation through the 783-Pos Board B553 NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflamma- Information Processing Mechanism Underlying a Perceptual Change by a some pathway. We are able to show that ligands for TSPO are able to increase Neuroglobin the expression of NLRP3 considerably, further hinting at the pro-inflammatory Shigekazu Oda1, Yu Toyoshima2, Mario de Bono3. role that TSPO may possess. 1Quantitative Bology, National Institute for Basic Biology, Okazaki, Japan, Our findings, in so far, highlight the importance of much needed further char- 2The University of Tokyo, Tokyo, Japan, 3Cell Biology, MRC Laboratory of acterization of TSPO and of the potential effects mediated by GE-180 as well as Molecular Biology, Cambridge, United Kingdom. the other TSPO ligands used in diagnostic. Neural circuits encode environmental information that is represented by neural 786-Pos Board B556 response patterns, and animals can use this neural coding for their behavior. Dynamic Regulation of P2X7 Receptors in Human Microglia by Bacterial Therefore, the modulation of neural coding is the basis of a behavioural change. Phagocytosis We found that neuroglobin (GLB-5), one of O -binding haem proteins ex- 2 Laura Janks1, Ligia Subitioni Antonio1, Jeroen R. Coppens2, pressed in neural circuits, alters the worms to prefer broader range of oxygen Terrance M. Egan1. concentrations. Quantitative imaging experiments indicated that GLB-5 con- 1Pharmacology and Physiology, Saint Louis University, St. Louis, MO, USA, fers a sigmoidal O response curve in a major O sensory neuron (URX). 2 2 2Neurological Surgery, Saint Louis University, St. Louis, MO, USA. The expression of GLB-5 in a CO sensory neuron transforms the neuron to 2 Microglia are the resident innate immune cells of the central nervous system be able to sense O concentration changes, suggesting that GLB-5 could act 2 (CNS) and play a crucial role in the pathogenesis of several nervous system as a second O receptor. Our computational model based on experimental 2 disorders. Following CNS injury, damaged cells release ATP into the extracel- data suggested that the relationship between URX and reversal behavior is suf- lular milieu which promotes neuroinflammation by activating ligand-gated ion ficient to explain worms’ preference behavior and its alteration by GLB-5. channels called P2X receptors located on neuroglia. Of the seven P2X recep- Thus, our results demonstrated the information processing mechanism underly- tor subtypes, the P2X7 receptor (P2X7R) has the most well-established role in ing the change in a worm’s preference behavior. mediating neuroinflammation. A wealth of previous studies in rodents demon- 784-Pos Board B554 strate that the P2X7R drives neuroinflammation by stimulating release of pro- Gabaergic Innervation of the Salivary Gland inflammatory mediators (IL-1b, IL-6, TNFa, COX-2), microglia proliferation, Joseph S. Lee, David R. Giovannucci. migration of neuroglia via MMP-9 activation, and by limiting the phagocytic Neurosciences, University of Toledo, Toledo, OH, USA. capacity of microglia cells. In view of these prominent roles attributed to the Autonomic neural activity closely regulates the production of salivary compo- P2X7 receptor in the rodent CNS, we investigated whether the P2X7R medi- nents critical for oral health. This process can be disrupted in patients treated ates diverse innate immune functions in primary human microglia. Using mo- with benzodiazepines, leading to the severe oral condition of xerostomia, or lecular, electrophysiological, immunochemical, and pharmacological dry mouth. Acini (secretory unit of salivary glands) are primarily innervated techniques, our data demonstrate that human microglia express functional by cholinergic and noradrenergic inputs, but the discovery of gamma- P2X7Rs which show time-dependent changes after repetitive activation or aminobutyric acid (GABA) and its receptors in the salivary glands hints at bacterial exposure, and help to signal many of the key components of the im- an unexplored mechanism for benzodiazepine-induced xerostomia. In the cur- mune response. rent study, biochemical, live-cell imaging, immunofluorescence and electro- physiological methods were used to elucidate the GABAergic system in the 787-Pos Board B557 mouse parotid gland and define its effect on acinar regulation. RT-PCR iden- The Magnetocaloric Effect as a Mechanism for Natural Magnetosensation tified mRNA transcripts for multiple GABA receptor subunits in enzymati- A. Martin Bell, Jacob T. Robinson. cally dispersed acinar cells and immunofluorescence microscopy indicated Electrical and Computer Engineering, Rice University, Houston, TX, USA. the expression of GABA receptors in mouse parotid tissue. However, both Many animals demonstrate the ability to detect and respond to magnetic fields. live cell calcium imaging and whole-cell recordings of acinar cells revealed This magnetic sense is used to navigate and orient with respect to the earth’s no acute impact of GABA on cellular physiology. In contrast, immunohisto- magnetic field by detecting a combination of field strength, orientation, inclina- chemical analysis of retrogradely traced parotid postganglionic neurons aligns tion, and polarity. While the existence of a magnetic sense is well-established, with the identification and characterization of GABA-evoked chloride cur- the mechanism or mechanisms underlying magnetosensation are not known, rents in a subset of neurons recorded from primary ganglionic cultures. Our though models have been proposed that depend on radical pair generation or data suggest GABA has no direct effect on the parotid acini but may down- on mechanical detection of magnetic force. Here we propose a mechanism of regulate acinar function by inhibiting neurotransmitter release from autonomic magnetosensation based on the magnetocaloric effect, where thermally sensi- neural inputs. tive ion channels respond to energy generated by the entropy changes resulting from rotation of a magnetically anisotropic material in the presence of a mag- 785-Pos Board B555 netic field. We explore how the magnetocaloric effect could be used as a natural Focusing on the Mitochondrial Expression of TSPO as a Marker and Pro- sense and identify paths to experimentally test this model. We establish a moter of Neuroinflammation parameter space for candidate magnetic materials, and outline experiments to Aarti Singh1, Kenneth Smith2, Michelangelo Campanella1. distinguish magnetic senses resulting from the magnetocaloric mechanism 1Royal Veterinary College, London, United Kingdom, 2University College from those resulting from chemical or magnetomechanical mechanisms. The London, London, United Kingdom. magnetocaloric hypothesis provides a new hypothesis for a magnetosensation Translocator Protein (TSPO) is an 18kDa mitochondrial protein, its primary mechanism that may be able to resolve some of the unanswered questions about function is reported to bind strongly to cholesterol molecules and initiate ste- how animals sense the earth’s magnetic field.

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788-Pos Board B558 790-Pos Board B560 Characterization of DEGT-1: A DEG/ENaC/ASIC Ion Channel Subunit Synthetic Peptides Deried from Spider Toxin, GsMTx4, Reduce Mechan- Involved in Touch Sensation ical and Neuropathic Pain Sylvia Fechner1, Frederic Loizeau2, Adam L. Nekimken2, Shao-Xi Ke1, Ping Dong1, Zhi-Gang Zhong1, Jie Xu1, Yan-Hong Xing1, Isabel D’Alessandro3, Beth L. Pruitt2, Miriam B. Goodman1. Kai-Qin Chen1, Mingxi Tang2, Zhe Zhang1,3, Qiongyao Tang1,3. 1Molecular and Cellular Physiology, Stanford University, School of 1Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical Medicine, Stanford, CA, USA, 2Bioengineering and Mechanical University, Xuzhou, China, 2Pathology, The Affiliated Hosp. of Southwest Engineering, Stanford University, Stanford, CA, USA, 3Wellesley College, Med. Univ., Zhoulu, China, 3Jiangsu Province Key Laboratory of Anesthesia Wellesley, MA, USA. and Analgesia Application Technology, Xuzhou Medical University, The touch receptor neurons (TRNs) of C. elegans are used as a system to un- Xuzhou, China. derstand the molecular events responsible for touch sensation. Key actors in The peptide GsMTx-4, extracted from the venom of Grammostola spatulata, this process include members of the DEG/ENaC/ASIC ion channel family. has been widely used to identify mechanosensitive (MS) channels in sensory These proteins form a mechano-electrical transduction (MeT) channel: MEC- neuron, it was reported having effect on reducing the mechanical hyperalgesia 4 is an essential, pore-forming subunit, MEC-10 is a non-essential, regulatory and neuropathic pain in rats recently. GsMTx4 belongs to inhibitor cysteine subunit. These two proteins are co-expressed with DEGT-1, another homolo- knots (ICK) peptides and has six cysteines that forms 4 loops. It also possess gous, potential subunit. Given that DEG/ENaC/ASIC proteins assemble into a hydrophobic face that is proposed to promote interfacial adsorption into the trimeric ion channels, the presence of a third, homologous protein re-opens lipid bilayer for performing its function. To investigate the role of the loops the question of which DEG/ENaC/ASIC proteins co-assemble to form the as well as identify core amino acid in the loop, we synthetized a variety of pep- MeT channels in vivo. Thus, we are investigating the role of DEGT-1 in touch tide mimetics of GsMTx4 to test their inhibitory effects on hyperalgesia. We sensation and MeT channel formation. showed that intraperitoneal injection of the synthesized mimetics which con- Using a classical touch assay, we detected a more severe defect in touch sensa- tains the main amino acids in loop2 and loop3 in GsMTx4 significantly tion if both DEGT-1 and MEC-10 were removed simultaneously vs. individu- increased the mechanical threshold for paw withdrawal in Randall Sellito ally. With in-vivo patch clamp recordings of native MeT currents activated by test, eliciting significant analgesic responses to inflammation-induced mechan- feedback-controlled mechanical stimulation, we identified a similar genetic ical hyperalgesia in the similar way as full length of GsMTx4 does. These syn- enhancement: the maximal amplitude of the MeT currents was further reduced thetic mimetics also reduced mechanical allodynia induced by inflammation if both DEGT-1 and MEC-10 were removed simultaneously. Thus, DEGT-1 is and by sciatic nerve injury. However, these synthetic peptides were ineffective likely a subunit of the native MeT-channel complex. at changing the withdrawal latency of hot plate and tail-flick tests. Furthermore, To characterize DEGT-1-containing channels independent of the native tissue, substitution of cysteines in the mimetics reduces or even eliminates the effects we introduced the well-known d mutation and expressed DEGT-1d alone and in on hyperalgesia. In a similar way, mutation of the hydrophobic residues also combination with MEC-4d in Xenopus oocytes. We found that DEGT-1d forms abolishes the analgesic effect on rats. These results suggest that the mimetics homomeric channels whose properties differ from those of MEC-4d: it is less selectively alleviate mechanical hyperalgesia. In addition, both the cysteines sensitive to amiloride, blocked by ibuprofen, and is pH-sensitive. As found for and the hydrophobic residues in the peptides play essential roles for inhibitory MEC-10d, co-expressing DEGT-1d with MEC-4d decreased current amplitude function on pain. compared to MEC-4d alone. Collectively, these results support the conclusion that DEGT-1, like MEC-10, plays a regulatory role in MeT channel formation 791-Pos Board B561 and function. NAV, AChR and Na/K PUMP Densities as a Function of EOD Frequency: Work supported by DFG (SF), SNSF (FL), F31NS100318 (ALN), SSRP (ID), Predictions for and Observations from the Weakly Electric Fish R01NS047715 (MBG), R01EB006745 (MBG/BLP). Eigenmannia Bela Joos1, Yue Ban2, John E. Lewis3, Michael R. Markham2, 789-Pos Board B559 Catherine E. Morris4. Single Molecule Colocalization Analysis of TRPV1 Interactions in Un- 1Physics, University of Ottawa, Ottawa, ON, Canada, 2Biology, University of roofed HEK293T/17 Cells Oklahoma, Norman, OK, USA, 3Biology, University of Ottawa, Ottawa, ON, Eric Senning, Sharona E. Gordon. Canada, 4Ottawa Hospital Research Institute, Ottawa, ON, Canada. Department of Physiology and Biophysics, University of Washington, Eigenmannia virescens individuals, to sense and communicate, continually Seattle, WA, USA. generate an electric organ discharge (EOD) at some frequency within the range Cellular fluorescence colocalization techniques are useful first steps in observing 200-600Hz. The skeletal muscle-derived EOs are comprised of syncytial elec- formation of a complex between two membrane bound proteins. However, fluo- trocytes, each of whose innervated excitable posterior membranes continually rescence signals from other cellular compartments, including cytoplasm and ER, fires action potentials in response to cholinergic stimuli. EOD-related whole an- can make it difficult to assign signals to the plasma membrane definitively. To imal O2-consumption reveals a nonlinear increase in the cost of EODs with circumvent this signal contamination, we studied the interaction properties of increasing frequency (Lewis et al 2014 J Neurosci 34:197). We modeled the two fluorescently labeled, integral membranes proteins in plasma membrane excitable membrane firing at 200-600Hz. Naþ entry through Nav and acetyl- sheets. The sheets were prepared from intact HEK293T/17 cells using ultrasonic choline channels (AChRs) was tallied. A dynamic steady-state of continual unroofing, which disrupts all organelles and leaves the plasma membrane lipids firing implies near-perfect ion homeostasis achieved by Na,K-ATPase and proteins intact. Our initial experiments with TRPV1-GFP and TRPV1- (pump) activity. Modeling showed that Nav channel density, but not AChR SNAP (fluorophore: SNAP-Surface 549) revealed a broad range of the Pearson’s density, would need to increase as firing frequency increased. To counter the coefficients (0.38-0.25), prompting us to consider alternative ways to evaluate influx through higher density Nav channels, pump density would also need colocalization of the two constructs. Because the expression levels of our two to increase. Modeling, which assumes synaptic transmission operating at proteins per unit area did not differ by more than an order of magnitude and very high speed (200 Hz is already extraordinary), predicts frequency- because we expected the colocalization of fluorescent features to be within the independent AChR density for electrocytes; this will need to be tested. Testing diffraction limit, we relied on a statistical comparison between the number of is underway, however, for Nav channel and the pump densities. E.virescens overlapping features in our experiment compared to the number of overlapping have two Nav1.4 isoforms (Nav1.4a, Nav1.4b); they are expressed in electro- features in a randomly-generated sample (Lagache et al., 2015). We found that, cytes only on the innervated posterior membrane. Electrocytes have one even when the Pearson’s coefficient is not able to reliably indicate colocalization Na,K-ATPase alpha subunit isoform, expressed both anteriorly and posteriorly. of TRPV1-GFP expressed alongside TRPV1-SNAP in unroofed cells, using the Nav1.4a is expressed exclusively in the EO, while Nav1.4b is expressed in both Jacop plug-in of ImageJ enabled us to establish the assembly of TRPV1 fluores- skeletal muscle and EO. We used quantitative RT-PCR to measure transcrip- cent subunits into higher order oligomers (Bolte and Cordelieres, 2006). We tion levels of genes encoding Nav1.4a, Nav1.4b and the Na,K-ATPase. We þ þ extend the single molecule colocalization method to unroofed cells expressing found that transcription levels of Nav1.4a, and Na /K ATPase have a strong TRPV1-GFP and SNAP-labeled p75 or TrkA to evaluate the statistical signifi- positive correlation with EOD frequency, while the transcription levels of cance of their respective interactions. Nav1.4b showed no correlation with EOD frequency.

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Posters: EPR and NMR: Spectroscopy and chemical and atomic-level structural parameters across different strains and conditions, particularly to examine the modes of action of old and new antibi- Imaging otics. We have introduced a new approach to measure the relative balance of teichoic acids and peptidoglycan within the cell wall structure. We extend 792-Pos Board B562 this analysis to whole cells, where using spectral signatures of the cell wall, Whole Cell 13C Solid-State NMR of a Fully Labelled Micro-organism: we identify distinct resonances to measure teichoic acid levels in intact bacte- How Far Can We Go? rial cells. The distinguishing chemical shifts of the sugars and peptides in pepti- Alexandre Arnold1, Jean-Philippe Bourgouin1, Bertrand Genard2, doglycan, along with the phosphoribitol backbone and D-alanine ester Dror Warschawski3, Francesca Zito3,Rejean Tremblay2, Isabelle Marcotte1. decorations of teichoic acids provide insight into peptidoglycan and teichoic 1 2 UniversiteduQuebec a` Montreal, Montreal, QC, Canada, Institut des acid levels, respectively, in whole cell NMR samples. Sciences de la Mer de Rimouski, UniversiteduQuebec a` Rimouski, This analysis did not require isotopic labeling and can be performed with any 3 Rimouski, QC, Canada, CNRS, Laboratoire de Biologie Physico-Chimique bacterial strain. We do often couple such measurements with isotopic labeling des Proteines Membranaires, Paris, France. and selective NMR detection schemes to fully dissect the atomic-level structure In vivo and whole-cell NMR is currently being developed to circumvent the ca- of the cell wall. We will also report on a focused set of results regarding the veats of working with in vitro model systems. However, the complexity and influence of growth state and the influence of inhibitors of teichoic acid synthe- heavy spectral overlap of whole-cell NMR spectra is tremendous and specific sis on overall cell wall composition. labelling of a single biological target is not always possible for all molecules of interest. 795-Pos Board B565 Herein we will describe our recent results on the NMR study of an intact fully Laser-assisted NMR in the Presence of a Cryogenic Probe Enables Multi- 13C labelled micro-organism: the green microalgae Chlamydomonas reinhard- dimensional Data Collection on Amino Acids and Proteins at Unprece- tii. Whole cell-spectra are simplified by applying different polarization transfer dented Sensitivity excitations (ultrashort CP, NOE and INEPT) which differentially excite rigid, Miranda Mecha, Yusuke Okuno, Hanming Yang, Silvia Cavagnero. semi-rigid and mobile species in the algae. Resolution is further enhanced by Chemistry, UW - Madison, Madison, WI, USA. dipolar (DAR or PDSD) or J-mediated 2D polarization transfers. This approach Nuclear magnetic resonance spectroscopy (NMR) provides atomic-level res- allowed us to discriminate and characterize the highly crystalline starch re- olution of biomolecular structure and dynamics in physiologically relevant serves in situ and to assign the highly mobile and abundant galactolipid head- environments. Yet, NMR can typically only be performed on highly concen- groups. The complex cell walls of this microalgae appears to have considerable trated samples (e.g. 100 mM to 1 mM). Biological samples are often intrin- dynamic complexity since both rigid and mobile saccharides are found in the sically dilute and available in the low mM range and below. Sometimes the cell wall glycoproteins. low sample concentration can be overcome by employing more material per The NMR strategies herein described enable a simplification of spectra and unit volume. However, this strategy may be unfeasible in sample-limited identification of main constituents in a micro-organism. The advantages and projects, or it may lead to sample loss due to aggregation or other degrada- limits of the possible extension of this approach to other micro-organisms - bac- tive processes. Here, we overcame the inherent low sensitivity of NMR spec- teria in particular - will be discussed. troscopy by exploiting photochemically induced dynamic nuclear polarization (photo-CIDNP) in combination with a cryogenic probe on a 793-Pos Board B563 600 MHz NMR spectrometer. The photo-CIDNP sensitizer fluorescein, Discovery of Phosphoethanolamine Cellulose and the Genetic Basis for its tailored to low sample concentrations, was employed and experiments Biosynthesis in E. coli Biofilms were carried out in an oxygen-free environment. Laser irradiation caused Wiriya Thongsomboon1, Alexandra Possling2, Regine Hengge2, several specific challenges, which were overcome in the context of the novel Lynette Cegelski1. pulse sequence 13C REPRINT. We successfully collected 1D and 2D 1H-13C 1Department of Chemistry, Stanford University, Stanford, CA, USA, heteronuclear correlation data on a model SH3-domain protein at 500 nano- 2Institute of Biology/Microbiology, Humboldt-Universit€at zu Berlin, Berlin, molar and 4 micromolar concentration in only a few minutes, respectively. In Germany. summary, photo-CIDNP heteronuclear correlation spectroscopy in the pres- E. coli integrates amyloid fibers termed curli and a modified form of cellulose ence of a cryogenic probe yields superior signal-to-noise ratio relative to to assemble a mechanically robust extracellular matrix (ECM) during the for- the constant-time SOFAST HMQC and SE-HSQC pulse sequences, down mation of bacterial communities termed biofilms. In our solid-state NMR anal- to the low-micromolar and nanomolar range for both free amino acids and ysis of the intact matrix material produced by uropathogenic E. coli, the most proteins. This advance enables the atomic-resolution biophysical investiga- common causative agent of urinary tract infections, we made the unanticipated tion of protein structure and dynamics in solution at unprecedentedly low discovery that the polysaccharide portion of the matrix consists of a modified concentration. form of cellulose — phosphoethanolamine (pEtN) cellulose. We have identi- fied the genetic basis for the installation of the modification, involving genes 796-Pos Board B566 and their respective proteins, which did not previously have defined roles in cel- Identifying the Optimal Level of Regularization in Deer Data Analysis lulose production. The pEtN functionality on the cellulose is derived from Thomas H. Edwards, Stefan Stoll. serine and we employ an isotopic labeling strategy using labeled serine to Chemistry, University of Washington, Seattle, WA, USA. detect the presence of pEtN cellulose in other biofilm formers. Functionally, Double Electron-Electron Resonance (DEER) spectroscopy is an Electron the pEtN cellulose modification is essential for E.coli biofilm formation. An Paramagnetic Resonance (EPR) experiment that accurately reports the distribu- E. coli mutant incapable of modifying cellulose cannot form well-integrated tion of distances, P(r), between two or more unpaired electrons over the range biofilms on agar. Thus, approaches to inhibit the modification of cellulose in of about 10 to 160 a˚ngstro¨ms. DEER is becoming increasingly important as a E. coli could offer new opportunities to prevent biofilm formation. In addition, tool for structural biology due to its ability to elucidate protein conformational the identification of the biosynthetic production of this zwitterionic modified landscapes, as well as its amenability to complex samples such as membrane- cellulose inspires a new generation of engineered systems to produce pEtN bound proteins and large protein complexes. The spin-spin distance distribu- and alternately modified cellulose materials. tion, P(r), is most often inferred from the time-domain experimental data with a regularized least-squares method known as Tikhonov regularization. 794-Pos Board B564 Although this method typically provides reliable estimates of P(r), it requires Dissecting the Bacterial Cell Wall with Solid-State NMR the input of a regularization parameter, a, which scales the magnitude of a pen- Joseph A. Romaniuk, Lynette Cegelski. alty term with respect to the fitting error. This regularization parameter signif- Chemistry, Stanford University, Stanford, CA, USA. icantly affects the estimated P(r), so selecting its optimal value is crucial for Gram-positive bacteria surround themselves with a macromolecular cell wall accurate analysis of DEER data. The most popular procedure for selecting a that is essential to cell survival and growth and serves as a major target for an- in DEER data analysis is a graphical method called the L-curve criterion, which tibiotics. While the cell wall of S. aureus is comprised of only two major struc- has empirical justification. However, to date, a systematic exploration of the tural components, peptidoglycan and teichoic acids, it is a thick and many extant regularization parameter selection methods has not been carried heterogeneous matrix that is difficult to characterize with traditional biophysi- out in the context of DEER data analysis. Here, we evaluate 16 methods, cal techniques like x-ray crystallography and mass spectrometry. including the L-curve criterion, against a large, physically-derived test set of We use solid-state NMR spectroscopy to study the composition of isolated, but simulated DEER data. The test set of P(r)s was generated using a side-chain intact cell walls and also whole cells to measure and quantitatively compare rotamer library in conjunction with a crystal structure (2LZM). This test set

BPJ 8582_8585 Sunday, February 18, 2018 159a was then used to generate a large set of experimentally realistic simulated time- Removal of Ca increased the distance between the spin labels, indicating greater domain data. lobe separation. Intermolecular distances measured between BSL (on CaM) and TOAC (on RyRp) revealed that in the absence of Ca, the complex is structurally 797-Pos Board B567 heterogeneous, as evidenced by multiple populations in the distance distribution. Methodological Development to Study Lipid Membranes of Intact Bacte- 2 Addition of calcium induced a shift toward a single compact structure. This effect ria and Microalgae by H Solid-State NMR was more significant with BSL on the N- lobe than on the C-lobe, suggesting that 1 1 2 Jean-Philippe Bourgouin , Alexandre Poulhazan , Francesca Zito , the N-lobe of CaM functions as the main Ca-sensor in regulating RyR. These re- 1 1,2 1 Alexandre A. Arnold , Dror E. Warschawski , Isabelle Marcotte . sults provide insight into the structural dynamics of the Ca-dependent regulation 1 2 UniversiteduQuebec a` Montreal, Montreal, QC, Canada, Centre National of RyR by CaM. These studies are currently being extended to investigate the ef- de la Recherche Scientifique, Paris, France. fect of CaM mutation and oxidation. This work was supported by NIH grants to In addition to providing structural integrity to cells and organelles, lipid mem- DDT (R37 AG26160, T32 AR007612). branes are the hub of important processes such as ion transport, signalling and trafficking, and constitute one of the first barriers to the external environment. 800-Pos Board B570 Our laboratory has thus developed a range of experiments designed to study bio- Design and NMR Structural Studies of New Antimicrobial Peptides with logical cell membranes in vivo by solid-state NMR. In this work, we describe a Higher Activity methodology to study membrane lipids of intact bacteria (Escherichia coli) and Yongae Kim, Ji-Sun Kim, Ji-Ho Jeong. microalgae (Chlamydomonas reinhardtii)by2H SS-NMR with magic-angle Chemistry, Hankuk University of Foreign Studies, Yong-In, Republic of spinning. We have developed a new strategy to selectively label the cell mem- Korea. brane with deuterated endogenous lipids. The method consists in feeding a The availability of antibiotics has allowed for the successful treatment of many cold culture batch with the complete fatty acid profile obtained from fully deuter- bacterial infections as well as the ability to perform invasive medical proced- ated bacteria or microalgae. As a consequence, membranes are specifically ures including surgery and chemotherapy. However, their wide use has led to labelled with their own lipids. These ‘‘native’’ lipids ensure a natural satu- pathogens’ increased drug resistance and the need to find novel classes of anti- rated/unsaturated lipid ratio, thus, preserving the biophysical properties of the microbial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a membrane. This approach is in addition more cost-effective than exogenous syn- cationic amphipathic peptide consists of 23-mer peptide, corresponds to the thetic deuterated lipids. This strategy is further demonstrated with microalgae carboxy terminal 113-135 region of component-3 of proteose-peptone and is using total lipid extracts. We characterize the membranes of these labelled or- a good candidate as a peptide antibiotic because it has an antibacterial activity ganisms by assessing membrane fluidity using in vivo2H SS-NMR. The endog- but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which enous and exogenous membrane labeling is compared and cell viability has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant analyzed. Elaboration of a simple and robust methodology to probe bacterial amino acids are recently developed by using peptide engineering techniques membranes is essential for the study of the effect of antimicrobial peptides on in our laboratory and show better antibioitic activities than wild-type LPcin live bacteria. Our work paves the way for the use of 2H SS-MAS NMR as a diag- and no toxicity at all. nostic tool for the health of bacteria, microalgae and other microorganisms. Recently, we designed and selected more antimicrobial peptides LPcin-YK5, LPcub-YK8 and LPcin-YK11 based on LPcin-YK3. Designed all analogues 798-Pos Board B568 were expressed and purified using several biophysical techniques and charac- Antifreeze-Mechanistic Study through VT-EPR Spectral Analysis and ICE terized using antimicrobial activity tests and various spectroscopic methods Growth Inhibition of Spin Labeled ICE Binding Proteins like MALDI-TOF MS and CD spectrometry, as well as 1D/2D solution Adiel Perez, Antonia Flores, Justin Quon, Yong Ba. NMR and solid-state NMR techniques in membrane environments. Chemistry and Biochemistry, California State University, Los Angeles, Los In here, we will present the optimizing processes with high-yield expression Angeles, CA, USA. and purification of new LPcin analogs and solid- state NMR structural studies Species that inhabit extreme cold climates, survive by producing Ice Binding to figure out antibacterial killing mechanisms. Proteins (IBPs). Through protein-binding to the surface of ice crystal nucleus, inhibition to the ice crystal growth is achieved which prevents cell damage. 801-Pos Board B571 This study focused on type 1 IBPs from the winter flounder. The mechanism Trajectory-Based Simulations of Electron Paramagnetic Resonance of ice growth inhibition was studied by variable temperature dependent elec- Spectra tron paramagnetic resonance (VT-EPR) techniques on spin-labeled type I Peter Martin1, Stefan Stoll2, David Thomas1. IBPs at different sites along its a-helical structure. The spin-labeled IBPs 1University of Minnesota, Minneapolis, MN, USA, 2University of were formed through labeling the cysteine substituted IBPs. The EPR spectral Washington, Seattle, WA, USA. lineshapes were analyzed by multicomponent EPR lineshape simulation to The combination of EPR spectroscopy and site-directed spin labeling (SDSL) is obtain the correlation times of the spin labels in the surrounding environments. a powerful tool for probing structure and dynamics in biological systems. How- Cryo-microscopy was used to observe the ice crystals. The changes in anti- ever, the corresponding spectra can be complex and difficult to interpret due to freeze activities and shapes of the ice crystals by the spins labeled IBPs were the required use of spin labels as spectroscopic probes. To accurately model compared with the wild type IBP. We will discuss the results including obser- experimental data, user-friendly programs have been developed to simulate vations of the spin labeled IBP binding to ice seed crystals, EPR spectra, and spectra, especially for continuous-wave (CW) EPR in the slow-motion regime simulations. Categorization of molecular motion and correlation times sur- (dynamical time scales of z10-100 ns for nitroxides at 9-10 GHz). The standard rounding the spin labeled groups were observed in anisotropic, slow, interme- method is to simulate spectra in the frequency domain by numerically solving the diate, and fast tumbling conditions, which revealed the water/ice surroundings stochastic Liouville equation. Such programs are very fast, but are often along the different sites of the spin labeled IBPs at various temperatures. restricted to specific simplified rotational diffusion models and limited to spe- cific spin labels. When more complex models are needed, trajectory-based 799-Pos Board B569 time domain simulation methods provide a promising alternative. Here we Probing the Calcium-Dependent Structural States of Calmodulin-RyR demonstrate our implementation of time domain methods in EasySpin, which al- using Bifunctional Spin Labels and Deer lows us to simulate spectra using motional models that are difficult to implement Cheng Her, Andrew R. Thompson, Christine B. Karim, David D. Thomas. using frequency domain methods. As a starting point, the program uses trajec- Biochemistry, Molecular Biology and Biophysics, University of Minnesota, tories that are calculated either internally using stochastic dynamics, with an Minneapolis, MN, USA. arbitrary orienting potential, or externally by molecular dynamics. The latter We have used a combination of site-directed spin labeling and EPR spectroscopy feature allows for detailed studies of how both spin label and protein dynamics to probe the interaction of calmodulin (CaM) with a peptide (RyRp) correspond- contribute to EPR spectra, and the program will be made publicly available. ing to the CaM-binding site on the ryanodine receptor (RYR), the muscle calcium release channel. A bifunctional spin label (BSL) attached stereospecifically at Cys 802-Pos Board B572 sites i and iþ4 was used to label CaM at positions 34 and 38 in the N-lobe and 106 Protein-Protein Interactions of Co-reconstituted SERCA, PLB and and 110 in the C-lobe. TOAC, an unnatural paramagnetic amino acid, was used to DWORF Investigated by Electron Paramagnetic Resonance label RyRp at position 5. Interspin CaM-CaM distances measured by DEER indi- Mark D. Rustad, Peter D. Martin, Daniel R. Stroik, Christine B. Karim, cated that CaM, in the absence of peptide and Ca, exists predominantly in a closed David D. Thomas. conformation (short interspin distance), while Ca induces a shift in equilibrium Biochemistry, Molecular Biology and Biophysics, University of Minnesota, toward two additional conformations, one more open and another more closed Minneapolis, MN, USA. (‘‘compact’’). Addition of unlabeled RyRp decreased the distance, indicating We have used electron paramagnetic resonance (EPR) spectroscopy to investi- that the two lobes of CaM collapse toward each other (compact conformation). gate the protein-protein interaction of a recently discovered micropeptide,

BPJ 8582_8585 160a Sunday, February 18, 2018 dwarf open reading frame (DWORF), with the sarco/endoplasmic reticulum Posters: Electron Microscopy calcium ATPase (SERCA) in the presence and absence of phospholamban (PLB) in lipid bilayer vesicles. Heart failure is one of the leading causes of 805-Pos Board B575 death in developed countries. A characteristic pathology of cardiac disease is þ Effects of cryo-EM Freezing on the Structural Ensemble inadequate activity of SERCA, the integral transmembrane Ca2 pump. For € 2þ Lars V. Bock, Helmut Grubmuller. every ATP hydrolyzed, SERCA transports two Ca ions from the cytosol to Theoretical and Computational Biophysics, MPI for Biophysical Chemistry, the sarcoplasmic reticulum (SR), enabling muscle relaxation. A primary regu- Go¨ttingen, Germany. lator of SERCA is PLB, an additional integral transmembrane protein of SR. 2þ The recent revolution in cryo electron microscopy (cryo-EM) allows to deter- PLB allosterically affects SERCA by reducing its apparent Ca affinity. mine structures of macromolecular complexes at atomic resolution. Specif- Recently, the 34-codon micropeptide DWORF was discovered, and when co- ically, cryo-EM provides information on structural heterogeneity and expressed with PLB and SERCA, it was found to restore the activity of ensembles of macromolecules. To obtain cryo-EM images of macromolecules, PLB-inhibited SERCA to that of the uninhibited values. However, the struc- the samples are first rapidly cooled down to liquid nitrogen temperatures. The tural basis for this observation has not yet been determined. We have investi- rapid cooling preserves some information of the room temperature ensemble. gated the protein-protein interactions of co-reconstituted SERCA, DWORF However, to what extent the the structural ensemble is perturbed, is currently and PLB in lipid vesicles using electron paramagnetic resonance (EPR) spec- unknown. To quantify the effects of cooling, we started all-atom explicit-sol- troscopy and associated kinetics assays. The sensitivity of EPR to structural dy- vent molecular dynamics simulations of the ribosome from snapshots taken namics, using stereospecifically attached spin labels, allows us to develop a from a room temperature ensemble with linearly decreasing temperature at model explaining the observed effects on SERCA activity, which will aid in various cooling rates. The cooling leads to a marked decrease in the structural the development of novel therapeutic remedies for cardiac pathologies. heterogeneity and the slower the cooling, the more pronounced these effects are. This suggests that with longer cooling times, the system is more likely 803-Pos Board B573 to locally equilibrate into a free-energy minimum, resulting in more homoge- Exploration of Eukaryotic Cells and Organelle NMR Signatures neous conformations. To estimate the cooling effects on the time scales of Sabrina H. Werby, Lynette Cegelski. the experiment, we use a statistical model of the cooling process with param- Chemistry, Stanford University, Stanford, CA, USA. eters obtained from the simulations. Finally, this model allows us to qualita- Whole-cell protein profiling, spatial localization, and quantification of activ- tively reconstruct the biologically more relevant structural ensemble before ities such as gene transcription and even protein translation are possible with cooling. modern biochemical and biophysical techniques. Yet, addressing questions of overall compositional changes within an intact cell – capturing the relative amounts of protein and ribosomal RNA levels and lipid content simul- 806-Pos Board B576 taneously - pose a challenge to many analytical methods and would require Laser-Based Zernike Plate for Phase Contrast Transmission Electron numerous extractions and purifications with caveats due to isolation yields Microscopy € and detection methods. A holistic view of cellular composition could aid in Osip Schwartz, Jeremy J. Axelrod, Robert M. Glaeser, Holger Muller. the study of cellular function and misfunction. Here, solid state NMR is used University of California, Berkeley, Berkeley, CA, USA. to identify 13C NMR signatures for cellular organelles in HeLa cells, without Recent technological advances in transmission electron microscopy (TEM) the use of any isotopic labeling. These signatures were characterized both in enabled the reconstruction of biological macromolecules with near-atomic isolated organelles and in the context of whole HeLa cells. The results reveal resolution. The macromolecules embedded in vitreous ice are transparent to that a cell can be deconstructed into its cellular machinery on a spectroscopic the electron beam, but impart a phase to the transmitted electron wave. level. We additionally performed 13C NMR on intact cells from additional This phase is conventionally detected by defocusing the imaging system, mammalian cell lines, demonstrating biological variability and similarities be- which, however, offers only limited contrast at lower spatial frequencies. tween them. Many diseases are known to effect specific organelles, such as This drawback makes it challenging to reconstruct particles smaller than mitochondrial diseases. An ability to measure parameters of composition and 100 kDa, or those exhibiting significant structural variability. The phase of to provide quantitative comparisons among samples, without ambiguities the electron wave function can be detected without defocusing using a Zernike possible from cellular digests and extractions, is an attractive approach to guide phase plate to retard the transmitted electron wave by a quarter wave relative analysis of biochemical changes in a cell or tissue. Fundamentally, it is also to the scattered wave. However, building an electron retarder has proven diffi- intriguing to catalog and examine the different ratios of biomolecule types in cult due to charging and gradual destruction of material objects exposed to the cells with single-sample spectroscopic snapshots. We believe that this explora- electron beam. tion will serve as a discovery tool to reveal changes in cellular pools to help We report our progress towards building a Zernike phase plate for TEM using dissect the modes of action of molecules that influence cellular processes and the ponderomotive potential of an intense laser field. With no material objects to understand changes in mis-functioning cells. inserted in the electron stream, this approach offers a stable, controllable phase shift and negligible electron loss. For practical use in life science 804-Pos Board B574 TEM, the phase plate must operate continuously, which requires reaching Characterization of Ligand- and Ion-Dependent Dynamics of F. Nuclea- an intensity of a few hundred GW/cm2 with a continuous-wave (CW) laser tum Glycine Riboswitch Aptamer II via Site-Directed Spin Labeling EPR system. We experimentally demonstrate that CW laser field can be amplified Michelle Ehrenberger. to the intensity of more than 40 GW/cm2 in a high finesse, high numerical University of Florida, Gainesville, FL, USA. aperture near-concentric Fabry-Perot optical resonator. Our numerical model Riboswitches are functional, non-coding RNA elements consisting of a ligand- shows that the phase shift profile created by the fundamental mode of such binding aptamer domain and a gene expression-regulating expression platform resonator can function as a nearly ideal Zernike phase plate for protein struc- domain (EPD). The glycine riboswitch is unique in that it consists of two ap- ture studies. tamer domains—which each bind one molecule of glycine—linked to a single EPD, but the purpose of this tandem aptamer is yet unknown. Ruff and Strobel 807-Pos Board B577 [1] have demonstrated that glycine binding enhances and is enhanced by inter- Dual-Energy Serial Block Face SEM Imaging of Biological Structures at aptamer interaction as the two aptamers dimerize. Here, we began to test their Near Isotropic Spatial Resolution model by probing dynamics in aptamer II alone. Site-directed spin labeling Qianping He1, David C. Joy2,3, Guofeng Zhang1, Richard D. Leapman1. (SDSL) with continuous wave electron paramagnetic resonance (cw-EPR) 1National Institute of Biomedical Imaging and Bioengineering, Bethesda, spectroscopy was utilized to probe site-specific changes in backbone dynamics MD, USA, 2Department of Materials Science and Engineering, Knoxville, of the aptamer II domain of the Fusobacterium nucleatum (FN) riboswitch in MD, USA, 3Center for Nanophase Materials Sciences, Oak Ridge National the wild-type and a glycine binding mutant (U141A). Results show differential Laboratory, Oak Ridge, TN, USA. dynamics between two spin-labeled sites, one adjacent to the glycine binding Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale site and one near the 3’ terminus. For both sites, addition of Mgþþ to a Kþ 3D ultrastructure of entire cells and tissue volumes. In SBF-SEM, an in- environment tightens the folding state, as seen by a reduction in backbone dy- chamber ultramicrotome successively removes thin sections from a plastic- namics. The binding site mutation did not alter backbone dynamics at the 3’ embedded, heavy metal-stained specimen. After each cut, the freshly exposed terminal site, but it did mitigate the magnesium- and glycine-induced reduction block face is imaged using a backscattered electron detector. The x-y resolution in dynamics near the glycine binding site. This work adds to our growing un- in the plane of the block face is approximately 5 nm while the resolution along derstanding of how splinted-ligation SDSL can be utilized to interrogate differ- the z-axis is limited by the minimum slice thickness of around 25 nm. We have ential dynamics in large dynamic RNAs. explored the feasibility of improving the z-resolution in SBF-SEM by recording

BPJ 8582_8585 Sunday, February 18, 2018 161a images at two primary beam energies, thus sampling different depths below the vealed that the 1/4.7 A˚ -1 layer line is not a meridional but arises from a 1-start block surface. helix. This shows that the two strands in the filament are staggered, with a rise Monte Carlo simulations reveal a linear relationship between the depth of test per subunit of 4.7 A˚ within each protofilament. We have confirmed this sym- structures and the ratio of backscattered image intensities recorded at different metry using the Iterative Helical Real Space Reconstruction (IHRSR) method primary beam energies. We found that the electron probe penetrates 13 nm to generate near-atomic resolution density maps of these amyloid filaments. and 25 nm into the heavy-atom stained sample block at primary beam en- Understanding the molecular mechanism in which ThT binds to amyloids rep- ergies of 1.0 keV and 1.4 keV, respectively. This has enabled us to determine resents a first step for structure-guided design of small molecules that may act the 3D structure within a 25-nm surface layer at a z-resolution of around 12.5 as biomarkers for early PD detection. nm based on a physical model of electron scattering. The model obtained from the simulation was tested experimentally using a Zeiss Sigma-VP SEM equip- 810-Pos Board B580 ped with a Gatan 3View SBF system. Experiments performed on embedded Fully Automated Correlation-Based Refinement of Atomic Models into blocks of mouse liver demonstrated that 12.5-nm isotropic spatial resolution High Resolution Cryo-EM Density Maps could be achieved for large sample volumes. Andrea C. Vaiana, Maxim Igaev, Carsten Kutzner, Helmut Grubmueller. Theoretical and Computational Biophysics, Max Planck Institute for 808-Pos Board B578 Biophysical Chemistry, Go¨ttingen, Germany. Human and Bacterial CTP Synthase Filament Structure and Function Modern cryo-electron microscopy (cryo-EM) can resolve large biomolecular Diverge complexes in different functional states, producing near-atomic resolution Eric M. Lynch1, Derrick R. Hicks1, Matthew Shepherd2, James A. Endrizzi3, density maps previously attainable only by X-ray crystallography. However, Allison Maker1, Jesse M. Hansen1, Rachael M. Barry4, Zemer Gitai4, advances in refinement of atomistic models into maps lag behind advances Enoch P. Baldwin3, Justin M. Kollman1. in generation of high resolution density maps. Here, we present a method 1Department of Biochemistry, University of Washington, Seattle, WA, USA, for fully automated refinement of atomic models into high-resolution cryo- 2Department of Biology, McGill University, Montreal, QC, Canada, EM maps. The method involves correlation-based molecular dynamics fitting 3Department of Molecular and Cellular Biology, University of California, in real space via a continuous series of simulated maps of increasing resolu- Davis, Davis, CA, USA, 4Department of Molecular Biology, Princeton tion and a final step of simulated annealing. An efficient, parallel version of University, Princeton, NJ, USA. the correlation-based fitting algorithm is implemented within the GROMACS CTP synthase (CTPS) is one of several metabolic enzymes recently found to simulation package. Refinements of large molecular complexes can be per- form micron-scale filaments in prokaryotes and eukaryotes. CTPS is an essen- formed in parallel with no manual intervention and no ad hoc restraints. We tial, universally conserved enzyme responsible for catalyzing the rate-limiting test our method on available structures of a rabbit muscle aldolase, two 20S step in CTP biosynthesis – conversion of UTP to CTP in an ATP-dependent proteasome complexes, and a bacterial 70S ribosome complex. Resolutions process. CTP is necessary for cellular function, being a component of DNA of the test systems are between 2.5A˚ and 3.4A˚ . We demonstrate that the and RNA, and also critical in phospholipid synthesis. Previously, we showed method yields higher quality models than those produced by combining auto- that E. coli CTPS (eCTPS) polymerizes into catalytically inactive filaments mated de novo chain building and reciprocal space refinement tools at high concentrations of CTP product, providing a novel mode of feedback in- commonly used in crystallography and cryo-EM. Cross-validation against in- hibition. Here, we present the high-resolution cryoEM structure of the CTP- dependent cryo-EM reconstructions confirms that the improved quality is not bound eCTPS filament, revealing the mechanism for its polymerization- due to overfitting. Full refinement of a system requires setting up only one mo- based inhibition. Further, we characterize the structural and biochemical prop- lecular dynamics simulation in GROMACS. The current implementation is erties of human CTPS (hCTPS). Remarkably, we find that hCTPS filaments optimized to run on medium-sized GPU clusters typically used for cryo-EM assemble in the presence of substrates, but not products, and have increased cat- image processing. This method will contribute to drastically reducing the alytic activity relative to free enzyme, precisely the opposite of eCTPS. We also time and effort currently needed to produce accurate atomic models from solve the cryoEM structure of the hCTPS filament. The human and E. coli high resolution data. CTPS filaments differ significantly in overall architecture and the conformation of individual CTPS protomers, with hCTPS locked in a novel active conforma- 811-Pos Board B581 tion in the filament. We demonstrate that this active conformation is not unique ‘Fixing’ the Gateway between Electron Microscopy and BSL3 Viruses 1 2 2 1 to hCTPS, and is also observed in free, substrate-bound eCTPS. We propose a Amar D. Parvate , Evan Willaims , Colleen B. Jonsson , Jason K. Lanman . 1Biological Sciences, Purdue University, West Lafayette, IN, USA, model whereby human and bacterial CTPS undergo a conserved conforma- 2 tional cycle controlled by substrate and product binding, but have opposite de- University of Tennessee Health Center, Memphis, TN, USA. terminants for polymerization; the inactive product-bound conformation is Scientific investigations of biosafety level 3 (BSL3) agents require special stabilized in the bacterial filament, while the active substrate-bound conforma- containment facilities and highly trained staff. The risks posed by BSL3 vi- tion is stabilized in the human filament. We also show that polymerization- rus handling restrict their investigations, especially in structural biology as based regulation differs between hCTPS isoforms, and that hCTPS polymeriza- large quantities of the pathogens are required for high resolution studies. tion is regulated by phosphorylation. Overall, our work demonstrates how Cryo-electron microscopes (cryoEM) inside a BSL3 facility incur very higher-order structural organization of metabolic enzymes acts to regulate high costs and require an extensive and cumbersome protocol for decontam- enzymatic activity. ination. An alternative strategy is to chemically inactivate pathogenic viruses to facilitate electron microscopy of BSL3 viruses in a BSL1 environment. 809-Pos Board B579 We have developed inactivation protocol for BSL2 viruses and evaluated Structural Studies of Alpha-Synuclein Amyloid Filaments the integrity of the inactivated samples by EM analysis. Orthobunyavirus Guilherme A.P. de Oliveira, Weili Zheng, Edward H. Egelman. (BSL2) was used as a model organism and the method was later replicated Biochemistry and Molecular Genetics, University of Virginia, on Orthohantaviruses (BSL3). Inactivation was performed by chemical fixa- Charlottesville, VA, USA. tion with 1% glutaraldehyde. Cryo-tomography studies were performed on Amyloid filaments of the alpha-synuclein (alpha-Syn) protein are considered the purified and inactivated Orthohantaviruses with a target resolution of the hallmark of Parkinson’s disease (PD) and no biomarkers for early diagnosis 2 nm by sub-volume averaging. The protocol was extended to whole cells have yet been developed. Thioflavin T (ThT) is the most frequently used dye to infected by pathogenic viruses. Hantaan virus infected cells were inactivated detect amyloids. We are using cryo-EM of ThT-bound alpha-Syn fibrils of PD using 1% glutaraldehyde. Previously reported cell-inactivation protocols familial variants (e.g., A30P, E46K and, A53T) to understand its mechanism of based on conventional fixation and alcohol dehydration collapse cell mem- binding. The heterogeneity of amyloid filaments has been one of the greatest branes and extract cellular contents to disrupt cellular integrity. We used obstacles to structural studies. We used the fluorescence of ThT to follow the high pressure freezing on the inactivated cellular samples, which preserved kinetics of alpha-Syn fibril formation at different ionic strengths to control fibril organelles and membranes with minimal extraction of cellular contents. It length and sample homogeneity for cryo-EM studies. The results show we can was observed that Hantaan virus infected cells generate plasma membrane modulate secondary nucleation events involved in filament growth (i.e., the projections that sequester virus egress and direct progeny virus particles fragmentation rate) using sodium chloride concentrations below 1mM. towards neighboring cells. Using the cell-inactivation method Hantaan Controlled proteinase-K digestion coupled with electron-spray ionization virus-infected mammalian cells will be prepared for cellular tomography mass spectrometry of the fibril variants revealed distinctly different filament to investigate the progress of Hantaan viruses infection in mammalian cells. structures. Additionally, the power spectra of A30P and A53T filaments The inactivation step can be included most of the virus purification protocols showed the 1/4.7 A˚ -1 layer line typical of cross-beta amyloids but the two and used to facilitate EM studies of BSL3 virus samples in a BSL1 have distinctly different helical organization. The A53T power spectrum re- environment.

BPJ 8582_8585 162a Sunday, February 18, 2018

812-Pos Board B582 Ying Liu, Sonoko Ishino, Yoshizumi Ishino, Gerard Pehau-Arnaudet, Mart PH-Gating of GAP Junction Channels: Visualization of a ‘‘Ball-and- Krupovic, David Prangishvili. (2017). A novel type of polyhedral viruses in- Chain’’ by Cryo-EM fecting hyperthermophilic archaea. J. Virol. Ali K. Khan, Maciej J. Jagielnicki, Michael D. Purdy, Mark Yeager. Molecular Physiology and Biological Physics, University of Virginia, 815-Pos Board B585 Charlottesville, VA, USA. Structural Insights into the Regulation Mechanism of HSP90 by Co- Gap junction channels (GJCs) mediate intercellular communication between chaperone AHA1 adjacent cells and are formed by the end-to-end docking of two hexameric Yanxin Liu, David A. Agard. hemichannels comprised of connexin (Cx) subunits. The central, axial pore Biochemistry and Biophysics, University of California, San Francisco, San enables the exchange of hydrated ions, second messengers and metabolites Francisco, CA, USA. up to 15 A˚ in diameter. A variety of physiological processes and patholog- Heat Shock Protein 90 (Hsp90) is a ubiquitous molecular chaperone that facil- ical states affect channel activity, including intracellular Ca2þ, pH, membrane itates the folding and maturation of hundreds of ‘‘client’’ proteins, especially potential, and trans-junctional voltage (Vj). Here we used single-particle elec- those involved in signaling and regulatory processes. This Hsp90 chaperone tron cryo-microscopy (cryoEM) to examine the conformational changes asso- machinery is also essential for maintaining cellular protein homeostasis and ciated with pH-mediated regulation of GJCs. Recombinant human Cx26 was for dealing with proteotoxic stress. The client maturation cycle is regulated purified as dodecameric GJCs, which remained homogenous and monodis- by a large number of co-chaperones that sequentially interact with Hsp90 perse when examined at pH 7.5 and 6.4. In general, the structures of Cx26 throughout its ATPase cycle. One of the most important, but poorly understood GJCs reconstituted in amphipol recapitulated the authentic Cx43 GJC in co-chaperones in eukaryotes is Activator of Hsp90 ATPase homolog 1 (Aha1). native membranes at 5.7 A˚ resolution and our previous Cx26 X-ray crystal As a potent stimulator of the Hsp90 ATPase activity, Aha1 is thought to accel- structure at 3.8 A˚ resolution. Within the transmembrane and extracellular do- erate client protein remodeling. A knockout of Aha1 in yeast severely compro- mains, the map resolution was 3.5 A˚ , whereas the cytoplasmic loop and mises the maturation of different Hsp90 client proteins. However, Aha1 cytoplasmic tail were less well-resolved, likely due to conformational flexi- overexpression inhibits the rate of Hsp90-dependent refolding of denatured bility. 3D classification of images recorded from particles at pH 6.4 yielded luciferase, as well as cystic fibrosis associated protein CFTR. How Aha1, as two conformations, one resembling the open physiological pH structure and an Hsp90 activator, facilitates ATP hydrolysis while inhibiting client matura- another with a pore-occluding density. Modeling of the polypeptide into the tion remains enigmatic. To better understand this question, we have determined cryoEM maps suggested that acidic pH elicits a helix-coil transition and asso- the structure of the full-length 200-kDa Hsp90/Aha1 complex at 3.8 A˚ resolu- ciation of the amino-termini to form the gating particle. To our knowledge, tion using cryo-electron microscopy. When bound to Aha1, the Hsp90 was this is the first instance in which a complete, oligomeric ‘‘ball-and-chain’’ captured in the closed conformation, similar to the X-ray crystal structure of has been visualized. Hsp90 bound with inhibitory co-chaperone p23. However, the binding interface is completely different from that used by p23 as well as that observed by crys- 813-Pos Board B583 tallography of a fragment of Aha bound to a fragment of Hsp90. In our struc- Reliability of Electron Microscopy Atomic Model Refinement ture, the C-terminal domain of Aha1 stabilizes the Hsp90 closed dimer by Lyman Monroe, Genki Terashi, Daisuke Kihara. bridging the C-terminal domain of one protomer and the middle domain/N- Biology, Purdue, West Lafayette, IN, USA. terminal domain of the other protomer. This structure begins to explain the An increasing number of biomolecular structures are solved by electron mi- Aha1 inhibitory effect on Hsp90 and also provides a basis for designing co- croscopy (EM). However, the quality of structure models determined from chaperone specific inhibitors to regulate Hsp90 related diseases. EM maps vary substantially. To understand to what extent structure models are supported by information embedded in EM maps, we used two computa- 816-Pos Board B586 tional structure refinement methods to examine how much structures can be Rapid Screening of FABS from Phage Display Libraries for Structural refined using a dataset of 49 maps with accompanying structure models. Studies The extent of structure modification as well as the disagreement between Evan Green1, Natalia Sevillano2, Nancy Li2, Yifan Cheng1, Charles Craik2. refinement models produced by the two computational methods scaled 1Department of Biochemistry and Biophysics, University of California, San inversely with the global and the local map resolutions. A general quantitative Francisco, San Francisco, CA, USA, 2Department of Pharmaceutical estimation of deviations of structures for particular map resolutions are pro- Chemistry, University of California, San Francisco, San Francisco, CA, USA. vided. Our results indicate that the observed discrepancy between the depos- The antigen binding fragments (Fabs) of an antibody are useful tools for cry- ited map and the refined models is due to the lack of structural information oEM because they increase particle contrast, break pseudosymmetry, and can present in EM maps and thus these annotations must be used with caution stabilize conformational states. Generation of recombinant Fabs from phage for further applications.This work was published in Structure 25:592-602, display libraries can take as little as one week but the screening and validation (2017). of Fabs often takes several months. Here we present a new method in which Fabs can be screened from small scale E. coli cultures using magnetic bead cap- 814-Pos Board B584 ture and microgram quantities of antigen. Each Fab can be visualized using Cryo-Electron Microscopy of a Polyhedral Virus Infecting Hyperthermo- negative stain EM to determine whether the Fabs form a complex with the an- philic Archaea tigen, if they are conformationally specific, and which epitope the Fabs bind. Fengbin Wang1, Ying Liu2, Thomas Edwards3, Ulrich Baxa3, Quantitative image analysis makes it possible to determine the relative affin- Mart Krupovic2, David Prangishvili2, Edward H. Egelman1. ities of each Fab. Insights from this method can help identify the most useful 1 2 University of Virginia, Charlottesville, VA, USA, Institut Pasteur, Paris, Fabs for further structural work reducing the time of this screen step to one 3 France, Frederick National Laboratory for Cancer Research, Frederick, MD, week or less. This method has been applied to screen Fabs against the USA. ESCRT-I core complex, urokinase-type plasminogen activator (uPA), and The origin and evolution of archaeal viruses have been poorly understood, the ABC transporter TmrAB. partially due to the limited sampling from a variety of archaeal species. Recently, a novel type of polyhedral virus (Sulfolobus polyhedral virus 1, 817-Pos Board B587 SPV1) infecting hyperthermophilic archaea has been isolated and character- A Novel Filamentous Virus Infects Hyperthermophilic Acidophiles that ized (Ying et al., 2017). The SPV1 genome encodes 45 open reading frames, Live in Nearly Boiling Acid but only nine of them can be annotated and none of the major virion proteins Tomasz Osinski1, Fengbin Wang1, Ying Liu2, Mart Krupovic2, show sequence similarity to other known viruses. In addition, SPV1 displays a David Prangishvili2, Edward H. Egelman1. very interesting feature: it condenses its circular double-stranded DNA 1Biochemistry and Molecular Genetics, University of Virginia, genome into solenoidal shells that have not been previously seen. We have Charlottesville, VA, USA, 2Department of Microbiology, Institut Pasteur, been using cryo-EM to study the structure of the SPV1 virion. We can Paris, France. show that the capsid has icosahedral symmetry with a novel T=43 surface lat- Sulfolobus filamentous virus (SFV) was isolated from very hot, highly acidic tice, not previously observed in other viruses. Under the capsid is a mem- springs in Beppu, Japan. It is membrane enveloped, and contains a 37 kbp dou- brane, with proteins linking the outer capsid to the membrane so that the ble stranded DNA genome which encodes for two major capsid proteins. A membrane itself has icosahedral symmetry. Details that emerge from this BLAST search using either of these proteins fails to recognize any sequence structure should give new insights into the divergence of archaeal viruses, similarity with any other protein in the database, suggesting that archaeal viral as well as provide clues on how structures such as this can be stable in nearly proteins are grossly undersampled in existing databases. Further, there is no boiling acid. apparent sequence similarity between these two proteins. We have generated

BPJ 8582_8585 Sunday, February 18, 2018 163a a cryo-EM reconstruction at better than 3.7 A˚ resolution, and show that there standard to verify sample preservation and image quality; and (3) the strep- are 17.14 asymmetric units per 46 A˚ turn of the solenoid. The asymmetric tavidin ‘‘background’’ in the image can be removed, whenever desired, by unit contains the two proteins, which form a pseudo-symmetrical heterodimer, Fourier filtering. wrapping around 12 bp of DNA. In SIRV2 (DiMaio et al., Science, 2015) iden- tical capsid proteins form symmetrical homodimers, while in AFV1 (Kasson et 820-Pos Board B590 al., eLife, 2017) two different capsid proteins form a pseudo-symmetrical het- Novel Methods for Biodistribution Analysis of Magnetic Nanoparticles erodimer. Surprisingly, the pseudo-symmetrical heterodimer in SPV is more In Vivo similar to the homdimer in SIRV2 than it is to the heterodimer in AFV1. As Alicia Cascella1, Ali Hadjikhani2, Joseph Favata2, Sina Shahbazmohamadi1. with AFV1 and SIRV2, where we showed that the DNA is found in A-form, 1Biomedical Engineering, University of Connecticut, Storrs, CT, USA, it is in A-form in SFV although the supercoiling is at a much larger radius. 2Mechanical Engineering, University of Connecticut, Storrs, CT, USA. This is consistent with our previous prediction that DNA will be maintained Since the original fabrication of nanoparticles for medicine in the 1990s, ap- in A-form by both bacterial spores and viruses that infect extremophiles. As plications have been researched in drug development, drug delivery, drug with AFV1, the membrane enveloping SFV is anomalously thin. These studies release, and more. Magnetic nanoparticles were first developed for their provide new insights into how both proteins and DNA can be stable in the most high contrast in MRI images, but recent advances have shown their value aggressive environments. in drug delivery systems and cancer therapies. In this research, electron micro- scopy methods are explored in the quantification of electromagnetic nanopar- 818-Pos Board B588 ticle (MEN) distribution within mouse organs in the weeks following Ultrastructural Study of Keratinocytes and Melanocytes in Seborrheic treatment. These MENs were developed as drug carriers, and injected into Keratosis mouse organs and analyzed over time. The efficiency of MEN as drug carrier Seulgi Noh1, Hyosun Choi1, Il Whan Kim2, Ji Young Mun1,3. 1 stems from the electrical interactions they have with cancer cell membranes BK21 Plus Program, Department of Senior Healthcare, Graduate School, and their ability to release drugs when exposed to an external magnetic field. Eulji University, Daejeon, Republic of Korea, 2Department of Dermatology, 3 Typically, the procedure to measure the distribution of nanoparticles would be Korea University Ansan Hospital, Ansan, Republic of Korea, Department of fluorescence imaging, which has downfalls including its resolution of the Biomedical Laboratory Science, College of Health Sciences, Eulji nanoparticles. By using scanning electron microscopy in conjunction with University, Seongnam, Republic of Korea. backscatter electron microscopy, composite mosaic images with sub-micron Melanin is responsible for skin, hair and eyes pigmentation, which is protect- resolution can be generated for analysis, and then further used in studies as ing skin from UV damage and skin cancer. Melanin is synthesized and a measure of cell penetration or dilution of MENs over time. Additionally, secreted from the melanocytes in the basal layer of the epidermis via organ- correlation with 3D X-Ray Tomography for volumetric evaluation of distribu- elles called melanosomes. Melanocyte distributes melanin by transferring me- tion will also be investigated. As the trend of nanoparticles in medical lanosomes at a ratio of approximately 1:36 to the surrounding basal research continues, these methods show how microscopy tools can be an keratinocytes. However, the exact mechanism that melanin is transferred to effective in vivo measuring tool for future developments in the nanoparticle keratinocytes in melanocytes is currently unknown. When melanin is consis- space. tently increasing or accumulating, it caused dermatological diseases called hyper-pigmentary diseases such as seborrheic keratosis, melanoma. Sebor- rheic keratosis is a common benign skin tumor which is developed from 821-Pos Board B591 the proliferation of keratinocytes and accumulated pigment in epidermis. High Resolution cryo-EM Structure of a HIV Nef-Inhibited AP-1 Clathrin The cause of seborrheic keratosis is unknown, but associated with photodam- Adaptor Complex age and aging. Previous studies are mostly histopathologic analysis, and detail Kyle L. Morris, Cosmo Buffalo, Xuefeng Ren, James H. Hurley. structural study using electron microscopy was not enough. The understanding Department of Molecular and Cell Biology and California Institute for of melanosome transfer is necessary for studying treatment of hyper- Quantitative Biosciences, University of California Berkeley, Berkeley, CA, pigmentary disorders particularly seborrheic keratosis. In this study, we inves- USA. tigated ultrastructure of melanocytes and surrounding keratinocytes in perile- The lentiviral protein Nef promotes infectivity and subverts immune surveil- sional seborrheic keratosis and normal skin using transmission electron lance of infected cells by downregulating cell surface proteins such as SER- microscopy. This will help to study the mechanisms behind a wide range of INC5 and MHC-I. The Nef proteins of HIV-1 group O, HIV-2, and SIV also human hyper-pigmentary diseases. This research was supported the Basic Sci- target the restriction factor tetherin. These downregulatory activities are medi- ence Research Program through the National Research Foundation of Korea ated by Nef hijacking the clathrin adaptor proteins AP-1 and AP-2 so as to (NRF) funded by the Ministry of Science, ICT & Future Planning [NRF- redirect cargoes to the lysosome. Nef hijacks AP-1 in part by enhancing its 2015R1C1A1A02037153]. Arf1-dependent trimerization. Nef and Arf1 in combination promote structur- ally and functionally distinct closed (trapped) and open (active) trimeric con- 819-Pos Board B589 formations. A new 3.5 A˚ cryo-EM structure of the AP-1:Arf-1:NL4-3 Streptavidin Affinity Grids for cryo-EM Nef:tetherin closed trimer reveals novel contacts that stabilize this hijacked Robert M. Glaeser1, Bong-Gyoon Han1, Zoe Watson2, Fred Ward2, complex. These data are interpreted in terms of a mechanism for trapping Jamie H.D. Cate2. non-preferred cargoes such as tetherin with respect to HIV-1 M and N-group 1Lawrence Berkeley National Lab, Berkeley, CA, USA, 2University of Nefs. California, Berkeley, CA, USA. The preparation of specimen grids for cryo-EM often proves to be difficult 822-Pos Board B592 because of unwanted factors such as preferential orientation of particles, Standardizing the Focused Ion Beam-SEM Workflow as a Tool for Versa- too few particles being seen within holes, particle disruption occurring within tile Imaging of Cellular Structures thin aqueous films, and unexpected aggregation of sample material. In in- Ashleigh M. Raczkowski, Edward T. Eng, William J. Rice, stances where it is suspected that those difficulties might be caused by inter- Sargis Dallakyan, Carl Negro, Laura Y. Kim, Kelsey D. Jordan, action with the air-water interface, a possible solution is to immobilize Bridget Carragher, Clinton S. Potter. particles on a structure-friendly support film, thus preventing diffusion to Simons Electron Microscopy Center, New York Structural Biology Center, the air-water interface of the thin aqueous film that remains after blotting New York, NY, USA. away excess sample. We have developed a form of EM grid on which Focused Ion Beam-SEM (FIB-SEM) methods cover a large variety of appli- streptavidin-monolayer crystals serve as an affinity support-film. A thin cations at different resolution scales that can visualize cellular structure of covering of trehalose is used to embed the streptavidin monolayers during larger mammalian tissue compartments or can provide information about storage, thereby preserving the crystalline structure and biotin-binding func- intracellular structures in single cells. An advantage of these applications is tion of the streptavidin. These affinity grids can thus be prepared in any that internal tissue architecture, as well as intracellular structures can be desired quantity and stored for future use. Many strategies are possible for directly quantified and measured. We have worked on standardizing the immobilizing particles by affinity-binding to the streptavidin monolayer after data collection and processing workflow by incorporating our FIB-SEM image washing off the protective layer of trehalose. One of the simplest is to stacks into Appion. This allows for project management and automated pro- randomly biotinylate one or two surface-lysine residues on particles of inter- cessing in the Appion pipeline. We demonstrate these applications using ex- est. Advantages of these grids include: (1) a good surface density of immo- amples from research projects at the Simons Electron Microscopy Center bilized particles is obtained rapidly at sample concentrations as low as where we are applying 3D microscopic techniques to a wide variety of 50 nM; (2) the streptavidin component of the image serves as an internal research problems.

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823-Pos Board B593 (crRNA) biogenesis, and target interference. CRISPR-Cas systems have been Structure Determination of Amyloid-b Fibrils by Cryo-EM classified into 2 classes, 6 types and 19 subtypes. There are six subtypes in Carla T. Schenk. Class 1 type I system, I-A through I-F. They employ a multi-protein-crRNA ICS-6, FZ Juelich, Juelich, Germany. complex Cascade to recognize foreign dsDNA. We have used cryo-EM to un- Amyloid fibrils are associated with many diseases; Amyloid-b in particular is derstand target recognition and inhibition in the type I-F Csy surveillance com- related to Alzheimer’s Disease. We present the structure of an Ab(1-42) fibril plex. Reconstructions with anti-CRISPR inhibitors, such as AcrF2, elucidate an determined by cryo-EM to a resolution of 4.0 A˚ . The fibril consists of two in- inhibition mechanism by the blockage of target dsDNA binding. tertwined protofilaments in which individual subunits form a ‘LS’-shaped to- pology revealing a previously unknown dimer interface, different from 827-Pos Board B597 previous solid-state NMR models. All 42 aminoacids are well resolved in the Electron Tomography to Visualize Individual IGM and IGG Molecule EM density map, including the N-terminal part. The high resolution structure using Cryo-EM is in agreement with solid-state NMR and X-ray diffraction experiments. Takeshi Mise1, Ayumi Maegawa1, Ruby May Andales1, Ulf Skoglund2, Akira Kamei1. 824-Pos Board B594 1Rsearch and Development, Okinawa Protein Tomography Ltd., Onna-son, Accounting for Microtubule Distortions in Cryo-EM Structures using Japan, 2Structural Cellular Biology Unit, Okinawa Institute of Science and Patch Refinements Technology Graduate University, Onna-son, Japan. Garrett Debs, Xueqi Liu, Hyo Keun Cha, Charles Sindelar. Determining the Three-dimensional (3D) structures of flexible proteins or pro- Yale University, New Haven, CT, USA. tein complexes by conventional approaches such as crystallography, NMR, and Microtubules are dynamic, filamentous structures that are essential for many single-particle analysis is in most cases limited to obtaining the 3D structure of different cellular processes. These processes often rely on a number of micro- a single molecule from averaging the structures of millions of molecules. On tubule associated proteins (MAPs) that either regulate or carry out these func- the other hand, electron tomography provides the unique possibility of visual- tions. Characterizing interactions between MAPs and microtubules is crucial to izing individual molecules of flexible proteins or protein complexes in their understanding of how these processes are carried out. Recent advances in cryo- different configurations. Due to not averaging, the downside of electron tomog- electron microscopy have made observing interactions between microtubules raphy is that it generates lower resolution structures compared to the averaging and several different MAPs at near atomic resolution possible. However, the methods such as crystallography, NMR, and single particle analysis. Electron resolution of these structures is variable, depending on the MAP. Recent studies tomography is an established method for the 3D visualization of individual have shown that the microtubule lattice is flexible and different stabilizing macromolecules such as proteins in biological samples using Cryo-EM. In drugs and MAPs affects this flexibility. These results suggest that distortions this presentation, we show examples of electron tomography using IgM and in the microtubule lattice are limiting overall resolution. Rather than aligning IgG (anti- EGFR antibody) with and without bound antigen. Successful devel- whole segments of microtubules (like in conventional microtubule reconstruc- opment and commercialization of therapeutic antibodies requires in-depth un- tions), we have developed a technique that aligns patches of tubulin. By sub- derstanding of their mechanism of action. We believe that electron tomography tracting the surrounding microtubule density and leaving a patch of tubulin may aid in the preparation of antibodies and may contribute to the field of drug dimers, each patch can be aligned independently from the bulk of the microtu- design at an early stage. bule. We have tested this ‘patch refinement’ technique on a synthetically, dis- torted dataset. Using traditional reconstruction techniques yielded a low 828-Pos Board B598 resolution structure, but upon application of the patch refinement technique, 3D-Structual Modeling of Myogenic Differentiation of C2C12 Myoblasts the resulting structure was nearly indistinguishable from an equivalent undis- by Advanced Electron Microscopy and Light Microscopy torted, synthetic dataset. The patch refinement method has also been applied Takako M. Ichinose1,2, Sei Saitoh3, Atsuko H. Iwane1,2. to an experimental dataset that was resolved to >5A˚ using traditional methods. 1Riken QBiC, Suita, Japan, 2Osaka Univ., Suita, Japan, 3NIPS, Okazaki, Following patch refinement, the resolution increased to <3.6A˚ . Using the Japan. alignment parameters following patch refinement, we were able to trace the Technological development in the field of regenerative medicine has advanced path of individual protofilaments and observe distortions in the lattice. Further dramatically in recent years. The process of differentiation to a cell that can be use and development of this technique will allow us to analyze microtubule dis- the basis to a functioning tissue is a part of an important life phenomenon and tortions and work towards obtaining an atomic structure of a microtubule. its clarification allows us important regenerative medicine developments. Although there were many reports on what kind of the target molecules will 825-Pos Board B595 be involved at that time in the own process, the relation between the process Pushing Size and Resolution Limits of Single Particle Cryo-EM at 200 keV of obtaining the function and the structure information is not yet clear. We Mengyu Wu, Mark A. Herzik Jr., Gabriel C. Lander. are trying to clarify the relationship between the important functions and the Integrated Structural and Computational Biology, The Scripps Research structural model that has obtained using the advanced electron microscope, Institute, La Jolla, CA, USA. FIB-SEM/SBF-SEM, and the 3D reconstruction techniques. They contain Technical and methodological advances in single-particle cryo-electron mi- many cell field information that is a little bit difficult to obtain by an optical croscopy (cryo-EM) have expanded the technique into a resolution regime microscope. At this annual meeting we will apply that technology of intracel- that was previously only attainable by X-ray crystallography. Although lular 3D microscopic processes to the solution of myogenic differentiation single-particle cryo-EM has proven to be a useful technique for determining process. the structures of biomedically relevant molecules at near-atomic resolution, ˚ C2C12 cell line as a model for stem cell differentiation, was maintained in growth nearly 98% of the structures resolved to better than 4 A resolution have been media consisting of DMEM containing 10% FBS by keeping 50% cell confluence determined using 300 keV transmission electron microscopes (TEMs). We or less. For differentiation of the cells were growth into reach 100 % confluence, demonstrate that it is possible to obtain cryo-EM reconstructions of sub- ˚ and then changed into the differentiation culture medium containing the concen- 100 kDa macromolecular complexes to better than 3 A resolution using a tration of 2% horse serum. The cells, 5 days, 8 days after differentiation induction 200 keV TEM. These structures are of sufficient quality to unambiguously were observed their dynamic morphological changes compared to before differ- assign amino acid rotameric conformations and identify ordered water mol- entiation. On day 5 after differentiation they formed thick and columnar multinu- ecules and bound co-factors, features previously thought only to be resolv- cleated body. As the differentiation process progressed, the number and the size of able using TEMs operating at 300 keV. mitochondria increased and formed thicker cell body. We would also like to discuss about mitochondrial remodeling and regarding the role of phagosome 826-Pos Board B596 associated with during the differentiation process of C2C12 cells. Cryo-EM Studies on the Mechanism and Inhibition of Target DNA Bind- ing in the Type I-F Csy Surveillance Complex Posters: Optical Microscopy and Superresolution Edward T. Eng1, Hui Yang2, Ashleigh M. Raczkowski1, Dinshaw Patel2, Clint S. Potter1, Bridget Carragher1. Imaging: Novel Approaches and Analysis I 1Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA, 2Structural Biology Program, Memorial Sloan 829-Pos Board B599 Kettering Cancer Center, New York, NY, USA. Non Radiative Excitation Fluorescence Microscopy: A New Method for Bacteria and archaea have evolved RNA-guided adaptive immune systems to Studying Membrane Adhesion at the Nanoscale target foreign nucleic acids, which are known as CRISPR-Cas systems. Lina Riachy, Dali El Arawi, Rodolphe Jaffiol, Cyrille Vezy. CRISPR immunity contains three stages: spacer acquisition, CRISPR RNA LNIO, UTT Institut Charles Delaunay CNRS, Troyes, France.

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Non-radiative Excitation Fluorescence Microscopy (NEFM) is a promising missed if the membrane density is not considered. We conclude that topog- technique allowing the observation of biological samples beyond the diffrac- raphy must be considered before declaring whether molecules to show clus- tion limit. By coating a substrate with an homogenous monolayer of quantum tering and our findings imply that clustering may not be as common as the dots (QDs), Fo¨rster Resonance Energy Transfer (FRET) could be achieved current literature suggests. from QDs (donors) to dye molecules located in the sample (acceptors). There- fore, the excitation depth of the sample is then given by the Fo¨rster radius, 832-Pos Board B602 which corresponds to few nanometers above the surface. Here, we present ‘Forbidden’ States Boost Fluorescence Resonance Energy Transfer this original method to probe the adhesion of Giant Unilamellar Vesicles (FRET) Studies of Membrane Receptors in Live Cells 1 2 1 3 (GUVs), negatively charged, in strong interaction with a positively charged sur- Benjamin Schreiber , Michael Kauk , Hannah Heil , Carsten Hoffmann , Katrin G. Heinze1. face ( QDs layer is coated with Poly-L-Lysine ). Distances between the surface 1 and GUVs are lower than 5 nm. We used the QD-quenching level to calculate Molecular Microscopy, Rudolf Virchow Center of the University of € € 2 and map the absolute distance between the membrane and the surface with a Wurzburg, Wurzburg, Germany, Department of Pharmacology and Toxicology, Julius Maximilian University of Wurzburg,€ Wurzburg,€ nanometer resolution. By tuning the electrostatic interactions between the sur- 3 face and the membrane, we have been able to measure a displacement of about Germany, Institute of Molecular Cell Biology, University Hospital Jena, 1 nm of the lipid membrane height. Jena, Germany. For probing biomolecular interactions or conformational changes in a live-cell 830-Pos Board B600 setting the distance depending Fluorescence resonance energy transfer (FRET) Evaluating the Permeability across the Actin-Based Compartment Barrier is often the method of choice. The G-protein-coupled receptor (GPCR) super- in the Plasma Membrane from Single-Molecule Trajectories family mediates cellular responses and communication across cellular mem- Alexey Yudin1, Takahiro K. Fujiwara2, Taka A. Tsunoyama1, branes, and is the largest known class of molecular targets with proven Akihiro Kusumi1,2. therapeutic value. On the one hand, real-time monitoring of membrane recep- 1OIST, Onna-son, Japan, 2WPI-iCeMS, Kyoto, Japan. tors and their interactions is vital to understand how they act in concert. On the Previously, by analyzing single-molecule trajectories of several lipids and pro- other hand, the design of GPCR FRET probes is crucial to ensure unhampered teins at a 25-ms resolution, our group showed that all the diffusing molecules in functionality and binding kinetics of the molecular complex. Thus, such FRET the plasma membrane of all the eukaryotic cells examined thus far undergo probes usually require labeling compromises concerning donor-acceptor posi- short-term confined diffusion within 100-nm compartments delimited by tions, distances and orientations, which limit the detectability of emitter signals actin-based membrane-skeleton mesh (fence) and transmembrane proteins in general, and the dynamic range of the FRET changes in particular. anchored to the fence (pickets) and long-term intercompartmental hop move- Here, we present an approach to optimize the energy transfer without changing ments, which was termed ‘‘hop diffusion’’. Hop diffusion was found for all the design of the FRET probe. We show that gold coated glass cover slips allow the raftophilic molecules examined thus far, showing that the plasma mem- reinforcing the otherwise forbidden donor-acceptor energy transfer by virtual brane compartmentalization by the actin-based membrane skeleton dominates optimization of the dipole orientation. First, we confirm the resulting enhanced the diffusion properties of all the membrane molecules. The membrane- FRET on our nano-coatings for the inactive M1 muscarinic acetylcholine re- skeleton mesh sizes observed by 3D electron microscopy agreed with the ceptors (mAChRs) labeled with a CFP-FlAsH pair in classical bleaching exper- compartment sizes determined from the diffusion measurements. Here, we iments. Second, we demonstrate the advantage of this enhanced FRET developed a method for evaluating the permeability of the compartment barrier, technique for ligand binding studies in live cells by the increased dynamic a critical parameter for hop diffusion, establishing its time scale. The method is FRET response between the inactive and active state of the M1 mAChR recep- based on the analytical solution in the Laplace space of diffusion equation in tor. We believe that our techniques has particular potential for pharmaceutical the presence of equally-spaced equipotential barriers. When the method was drug screening to boost non-ideal FRET probes, and thus amplify signal re- applied to single-molecule trajectories obtained at a 25-ms resolution, we sponses without interfering with the well-characterized molecular assay. were able to obtain compartment sizes and macroscopic diffusion coefficients 833-Pos Board B603 that agreed with our previous results (as well as permeability), showing the Detection of Small Molecular Complexes at the Plasma Membrane via method works well. Furthermore, the intercompartmental permeability in the Temporal Accumulation Analysis cell lines examined thus far could be evaluated even at worse time resolutions Florian Baumgart, Andreas M. Arnold, Gerhard J. Schutz€ . < 1 ms, allowing easier permeability determinations. In addition, we developed Institute of Applied Physics, TU Wien, Vienna, Austria. new software that detects the moments at which a membrane molecule transits Single molecule techniques have been frequently used to determine subunit to adjacent compartments (undergoing intercompartmental hops) at much stoichiometry of molecular oligomers in cells. Most current approaches, how- higher probabilities. The experimental probability density of the molecule ever, require extremely low expression levels of the complexes, rendering those sojourn time in a compartment obtained with this software well agreed with experimental strategies not generally applicable. In principle, single-molecule our analytical results. We generalize our findings and propose the canonical localization-based superresolution microscopy data contain quantitative stoi- hop diffusion model. chiometry information. Unfortunately, unknown blinking characteristics of the fluorophores make it rather difficult to extract it directly. In this work, 831-Pos Board B601 we extend our previously introduced approach for cluster detection at the Membrane Topography can Cause Apparent Clustering - Identification cellular plasma membrane (1) and its variant (2) to show, that - using appro- and Differentiation from Genuine Clustering priate calibrations - the method is able to identify small oligomeric complexes. Ingela Parmryd1, Jeremy Adler1, Kristoffer Bernhem2. 1 2 We evaluated the technique using Monte Carlo simulations, and validated the Medical Cell Biology, Uppsala University, Uppsala, Sweden, Department results with experiments on cells, where constitutive n-mers were expressed of Applied Physics, The Royal Institiute of Technology, Stockholm, Sweden. and fluorescently labelled. Quantitative data analysis indicates that even trimers Detailed knowledge on the organisation of plasma membrane components is can be robustly detected. (1) Baumgart et al. Nat Meth. 13:661; (2) Spahn et al. crucial to understand biological processes like cell adhesion, endocytosis and Nat Meth. 13:963. the initiation of cell signalling. Surprisingly, single-molecule localisation mi- croscopy (SMLM) has shown that virtually every plasma membrane protein 834-Pos Board B604 is organised in clusters even in unactivated cells. We establish that clusters Determinations of Orientation of Membrane-Associated Fluorescent could be a consequence of variations in topography like membrane folding Molecules Made Trivially Simple and undulations, protrusions like filopodia and invaginations like caveolae Josef Lazar1,2, Olga Rybakova1,2, Stepan Timr2. and clathrin coated pits, could also produce the appearance of clustering by 1Center for Nanobiology and Structural Biology, Institute of Microbiology increasing the local density of otherwise randomly distributed molecules. Using CAS, Prague, Czech Republic, 2Institute of Organic Chemistry and dual colour live cell SMLM, with the fluorophore DiI showing the membrane Biochemistry CAS, Prague, Czech Republic. density together with the transferrin receptor (TfR) or the GPI-anchored protein Two-photon polarization microscopy (2PPM) allows sensitive observations of CD59 we demonstrate that the topography-caused clustering can be identified. various molecular processes in living cells, such as activation of G-proteins, in- Nearest neighbour analysis reveals that DiI and CD59 have similar distribu- teractions between G-proteins and GPCRs, changes in cell membrane voltage, tions, whereas the TfR does not. Pair correlation (PC) analysis recognise or changes in intracellular calcium concentration. Apart from visualizing mo- mere membrane accumulation of randomly distributed molecules as clusters, lecular events, 2PPM also allows determining orientations of fluorescent mol- as does its derivative PC-PALM but neither method provide information on sin- ecules associated with lipid membranes. Such determinations can yield insights gle clusters. Image analysis, on the other hand, does not only reveal what clus- into protein structure, aid in development of genetically encoded probes, and ters are caused by mere topography variations but also clusters that would be allow quantitation of lipid membrane properties. We have now simplified the

BPJ 8582_8585 166a Sunday, February 18, 2018 process of determining the orientation of fluorescent molecules associated with 837-Pos Board B607 lipid membranes to the point of it becoming trivial. Quantitative information on Real-Time Optical Manipulation of Cardiac Conduction in Intact Hearts molecular orientations can now be obtained with a standard two-photon micro- Emilia Margoni1, Samantha Cannazzaro2, Claudia Crocini1, scope, or even a standard confocal microscope, with no polarization modula- Cecilia Ferrantini3, Raffaele Coppini3, Ping Yan4, Leslie M. Loew4, tion needed. Our microscopy procedure and software are likely to become a Marina Campione5, Leonardo Bocchi3, Danilo Giulietti6, Elisabetta Cerbai3, useful tool, allowing obtaining detailed structural information on various Corrado Poggesi3, Gil Bub7, Francesco S. Pavone1, Leonardo Sacconi1. membrane-associated molecules. 1European Laboratory for Non-linear Spectroscopy, Sesto Fiorentino (FI), Italy, 2Italian National Research Council, Sesto Fiorentino (FI), Italy, 835-Pos Board B605 3University of Florence, Florence, Italy, 4University of Connecticut Health Elucidating Invisible Barriers and Obstacles to Molecular Diffusion in Center, Farmington, CT, USA, 5Italian National Research Council, Padova, Live Cells by the Spatial Pair-Correlation Function: A Connectivity Italy, 6University of Pisa, Pisa, Italy, 7McGill University, Montreal, QC, View of the Cell Canada. Leonel S. Malacrida1,2, Per Niklas Hedde1, Suman Ranjit1, Optogenetics has provided new insights into cardiovascular research, leading to Francesco Cardarelli3, Enrico Gratton1. 1 new methods for cardiac pacing, resynchronization therapy and cardioversion. Laboratory for Fluorescence Dynamics, University of California, Irvine, Although these interventions have clearly demonstrated the feasibility of cardiac CA, USA, 2Departamento de Fisiopatolgı´a, Hospital del Clı´nicas, Facultad de 3 manipulation, current optical stimulationstrategies donot take intoaccountcardiac Medicina, Universidad de la Republica, Montevideo, Uruguay, Center for wave dynamics in real-time. Here, we developed an all-optical platform comple- Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, mented by integrated, newly developed software to monitor and control electrical Italy. activityin intactmouse hearts. The system combinesa wide-fieldmesoscope witha Despite recent advances in optical super-resolution, we lack of methods to digital projector for optogenetic activation. Cardiac functionality can be manipu- visualize the path followed by diffusing molecules in the cell. Fluorescence lated either in free-run mode with sub-millisecond temporal resolution or in a correlation spectroscopy (FCS) provides molecular dynamics at the single closed-loop fashion: a tailored hardware and software platform allows real-time molecule level; however, the classical single point FCS lacks spatial resolu- intervention capable of reacting within 2 ms. The methodology has been applied tion. Methods based on image analysis like raster-scanning and spatiotem- to restore normal electrical activity after atrioventricular block, by triggering poral image correlation still needs spatial averaging over relatively large ventricle in response to optically mapped atrial activity with appropriate timing. areas; hence the spatial resolution is compromised. Here, we develop a Furthermore, real-time intra-ventricular manipulation of the propagating electrical new method based on the spatial pair-cross-correlation in two dimensions wavefront has been demonstrated opening the prospect for real-time resynchroni- (2D-pCF) to obtain relatively high-resolution images of molecular diffusion zation therapy and cardiac defibrillation. The development of this innovative opti- dynamics and transport in live cells. The 2D-pCF method measures the time cal methodology provides the first proof-of-concept that a real-time optical-based for a particle to go from one location to another by cross-correlating the in- stimulation can control cardiac rhythm in normal and abnormal conditions, prom- tensity fluctuations at specific points in an image. Hence, a visual map of ising a new approach for the investigation of the (patho)physiology of the heart. connectivity for the average path followed by molecules is created. To test our approach we use a nanometer channel simulation and a biological sample 838-Pos Board B608 where we can predict the path (GUV label with a dye). Next, we acquired Spectral Resolution in Fluorescence Microscopy in Strongly Scattering confocal stacks for free EGFP in MDA-MB-231 cells using the new side- Media selective plane illumination microscope. The analysis of the 2D-pCF was Enrico Gratton, Alexander Dvornikov. done using image processing methods to obtain parameters of the angular UCI, UCI, Irvine, CA, USA. distribution of the pCF. We calculate first and second central moments of Spectral phasors were introduced as alternative to spectral demixing, for the the spatial distribution. Then we produce a series of images with the anisot- determination of FRET and for the measurement of dipolar relaxation in mem- ropy, eccentricity, center of mass shift, anisotropy/eccentricity angle and the branes. The basic concept in the spectral phasor approach is that the entire spec- connectivity map. The application of our new method shows a unique sensi- trum is not needed for some analysis, but only some of the elements of the tivity to identify the average path in our simulation and test sample. The re- spectral distribution are sufficient. In the case in which the spectrum is domi- sults for the MDA-MB-231 cells were able to identify connected/ nated by few spectral bands, this approach is very efficient and provides fast disconnected regions in the cell with spatial and temporal resolution. and valid alternative to the analysis of the full spectrum. One example of a sys- NIH_P41-GM103540/NIH_P50-GM076516 grants. tem that could benefit from the phasor analysis is in tissue spectroscopy in which the light to be collected has undergone multiple scattering so that it 836-Pos Board B606 cannot be easily focused on the entrance slit of a dispersive element or trans- Adaptive Precision Real-Time 3D Single Particle Tracking Microscopy formed in a parallel beam for use with hyperspectral cameras. In a typical appli- Shangguo Hou, Kevin Welsher. cation of thick tissue spectroscopy, the light, after being transmitted through the Chemistry, Duke University, Durham, NC, USA. tissue, is emitted over a very large cone angle. All the schemes designed to cap- Real-time 3D single particle tracking (RT-3D-SPT) has emerged as a powerful ture this transmitted (or fluorescence) light use a relatively narrow collection tool for investigating dynamic biological interactions due to its high spatial and angle with severe reduction of the signal. In the microscopy field, we intro- temporal resolution. Generally, RT-3D-SPT involves tracking precision and duced in 2011 a microscope design (DIVER) that can capture a very large solid tracking speed. Most biological processes are heterogeneous, which means emission angle (close to 2pi) and we have shown that this microscope, depend- there are different tracking precision requirements at various stages depending ing on the tissue thickness, can capture several orders of magnitude more light on the diffusive behavior of the probe in question. However, the spatial resolu- than conventional microscopes using epi detection. A limitation of the DIVER tion of current RT-3D-SPT methods maintains constant during the tracking pro- design is that interference filters cannot be used in the emission path since the cess. Herein we have developed a real-time 3D single particle tracking method light is impinging on the surface of the filter in front of the detector with a very capable of adaptively adjusting the tacking precision with the particle’s diffu- large angular distribution. Here we propose a simple device to overcome this sive speed. This method is based on a recently developed RT-3D-SPT method problem (Supported by NIH P41-GM103540 and NIH P50-GM076516). known as 3D dynamic photon localization tracking (3D-DyPLoT). The 3D-Dy- PLoT employs a tunable acoustic gradient (TAG) lens and a 2D electro-optic 839-Pos Board B609 deflector (2D-EOD) to dynamically drive a single focused laser spot around Overcoming Blinking Artifacts in Nanocluster Detection with Two Color the focal volume in 3D. Utilizing an optimized position estimation algorithm, Storm 3D-DyPLoT can track single particles with high tracking speed (up to 20 mm2/ Andreas M. Arnold, Florian Baumgart, Magdalena Schneider, s) and high localization precision at photon count rates as low as 10kHz. The Gerhard J. Schutz.€ adaptive precision is achieved by modulating the size of the laser focus scan- Institute of Applied Physics - Biophysics, TU Wien, Vienna, Austria. ning pattern with the particle’s moving speed. While the number of scanning Observations using single molecule localization microscopy have led to the points is maintained, the change the size of scanning pattern changes the spatial belief that the majority of tested membrane proteins are organized in clusters sampling rate, which leads to a tunable spatial localization precision. To at sizes below the diffraction limit. These nanoclusters are thought to play an demonstrate the biological applicability of this adaptive precision tracking important role in cellular signaling. However, concerns about the existence method, lentiviral particles expressing a VSV-G-YFP fusion landing on live of nanoclusters have been fueled by the notion that virtually all fluorescent Hela cell membrane events were tracked. This adaptive precision RT-3D- probes show complex blinking behavior including long-lived dark states. SPT method will provide an ideal tool to investigate the heterogeneous interac- This results in artificial localization clusters due to the repeated observation tions of proteins and biological particles. of single molecules. Existing post-processing approaches commonly struggle

BPJ 8582_8585 Sunday, February 18, 2018 167a to reliably distinguish real molecular clustering from such blinking artifacts. glucorticoid receptor (GR). We aim to develop rNB assay as a general method Recently, we proposed an experimental approach relying on the deliberate titra- that can be applied to any protein of interest to identify whether subpopulations tion of fluorescent labels in a number of samples. with different kinetic behaviors are in different oligomerization states. Here, we present a supplementary approach using information from two-color STORM experiments. Targeting the same protein species with differently 842-Pos Board B612 labelled antibodies allows for the calculation of distance distributions between Characterization of Malaria Detection Based on Third Harmonic Genera- localizations from both color channels. Molecular clusters exhibit a character- tion Imaging of Hemozoin 1 2 3 2 istic bias towards short distances. The use of two-color information overcomes Alexei Kazarine , Fadi Baakdah , Wellington Oyibo , Elias Georges , Paul William Wiseman1,4. the detection of apparent clustering due to fluorophore blinking, which is prob- 1 lematic in other methods, such as Ripley’s K analysis. Furthermore, rotation of Department of Chemistry, McGill University, Montreal, QC, Canada, 2Institute of Parasitology, McGill University, Montreal, QC, Canada, one color channel provides intrinsic controls, thus allowing for statistical sig- 3 4 nificance tests without the necessity of additional calibration. We evaluate College of Medicine, University of Lagos, Lagos, Nigeria, Department of the limits of the method with Monte Carlo simulations and experiments on clus- Physics, McGill University, Montreal, QC, Canada. tered and randomly distributed membrane proteins. Malaria is a major infectious disease that affects millions of people worldwide. Despite many efforts, this parasitic infection currently remains without an 840-Pos Board B610 effective vaccine. To properly tackle this problem, it is important to develop Spontaneously Blinking Dyes for Localization Microscopy: Simplified Syn- accurate, sensitive and user-friendly tools for the rapid screening of bloods thesis and Single-Molecule Characterization samples for malaria infection. Patrick J. Macdonald, Richard A. Haack, Susan Gayda, Sergey Y. Tetin. The current gold standard technique for malaria detection relies on light micro- Abbott Laboratories, Abbott Park, IL, USA. scopy after staining the parasite’s DNA using Giemsa stain. While effective at Super-resolution microscopy enables imaging of structures smaller than the detecting parasites, this method requires both time and skilled personnel. Anti- diffraction limit, defined by Abbe’s law. Single-molecule localization methods, gen detection is another malaria detection method that relies on the capture of such as STORM, PALM and their derivations, reconstruct images by plotting the parasite antigens. However, this method demonstrates poorer detection limits centroids of fluorescent point sources from a series of images where only a few and a higher operating cost. We previously demonstrated a promising new tech- molecules are fluorescing at a time. These approaches require less complex nique for the early detection of malaria involving the use of third harmonic gen- instrumentation than methods that depend on point-spread function engineering, eration (THG) imaging to detect a metabolic byproduct of the malaria parasite, and thus have gained fast growing popularity among scientists of very diverse hemozoin, inside affected red blood cells. THG imaging relies on a nonlinear op- backgrounds. However, the relative simplicity of the instrumentation comes tical process in which the frequency of incoming photons is tripled by the sample. with a trade-off in probe requirements. These methods rely on the partial sup- This allows for selective imaging of samples having high third order nonlinear pression or activation of the detectable on-state to achieve sparsely activated flu- susceptibility, of which hemozoin ranks among the highest. orophores. Additionally, chemical additives, such as reducing reagents, oxygen We now present the next level of our method and apply it to both cultured and scavengers, complex illumination schemes, molecular scaffolds, high irradiation real samples. By combining THG imaging with motorized scanning and simple intensities, or a combination of the above are usually required to optimize the re- analysis software, we perform image cytometry to identify both red blood cells sulting images. We have previously developed a rhodamine derivative which and hemozoin crystals within a single detection channel in unlabeled samples, converts between a bright and a dark state in response to protonation. At pH 7, allowing easy determination of infection at single cell level. We apply this tech- only 0.5% of the dye population is fluorescent, as the molecules transition be- nique to parasites cultured in packed red blood cells and parasites in whole tween the two states at equilibrium. The resulting blinking occurs on timescales blood obtained from malaria patients, and compare the effectiveness of our of seconds to minutes and can therefore be used for imaging approaches (e.g. method to parasite counts obtained by classical techniques. STORM) without further sample treatment or complex illumination schemes. 843-Pos Board B613 We use a single-molecule approach to further characterize the blinking behavior Multicomponent Analysis of Phasor Plot to Decipher Changes in Meta- of the dye and its variants. In the interests of practical application, we investigate bolic Trajectory of Biological Systems the synthesis of blinking dye variants that can be made from commercially avail- Suman Ranjit1, Alexander Dvornikov1, Moshe Levi2, Enrico Gratton1. able rhodamine requiring only a few, simple chemical reactions. The ease-of-use 1Biomedical Engineering, University of California Irvine, Irvine, CA, USA, as well as the brightness of these dyes make them an excellent tool for super- 2Biochemistry and Molecular & Cellular Biology, Georgetown University, resolution imaging techniques based on stochastic read-out. Washington, DC, USA. Recent developments of phasor approach to Fluorescence Lifetime Imaging 841-Pos Board B611 (FLIM) in cells undergoing oxidative stress or cultured in oleic acid enriched Monitoring the Live-Cell Oligomerization State of Free and Chromatin media and in mice liver sections having high fat diet, have shown presence of Bound Transcription Factors by Resampled Number and Brightness (RNB) a third autofluorescent component indicative of lipid droplets besides the phasor Eugenia Cammarota1,2, Alessia Loffreda1,2, Moreno Zamai2, components from free and enzyme bound NADH in the 400-450 nm emission Valeria R. Caiolfa2,3, Carlo Tacchetti2, Davide Mazza1,2. wavelength. This third component changes the position and shape of the phasor 1 2 Centro Europeo Nanomedicina, Milan, Italy, Experimental Imaging Centre, distribution away from the metabolic trajectory, defined by the line joining the 3 S Raffaele Scientific Institute, Milan, Italy, National Centre for phasor position of free and enzyme bound NADH, and makes the study of Cardiovascular Recearch, Madrid, Spain. changes in metabolism using the ratio of free to bound NADH difficult. How- The oligomeric state of proteins play a fundamental role in determining their ever, the phasor rule of addition applies to any number of components and activity within their signaling network. The affinity of many transcription fac- this was exploited to create a multicomponent analysis. A change in phasor tors (TFs) for DNA and their capability of inducing transcriptional activation, distribution towards free or bound NADH can then be separated and analyzed for example, is controlled by tuning their oligomerization state. An effective independent of the third individual. Law of phasor addition also enables calcu- way to quantify oligomerization in living cells is numbers and brightness lation of the fraction of the third species and helps in quantifying the differential (N&B), a method that allows extracting the number and intensity of mobile flu- distribution and accumulation of lipids in different cell/ tissues. This approach orescently labeled molecules from a sequence of microscopy images. Unfortu- has been employed to study lipid deposition in mice liver tissues from mice un- nately, N&B does not provide stratified information about the oligomerization dergoing high fat diet. We envision the use of multicomponent phasor distribu- of molecules with different kinetic behaviors (i.e. ‘‘freely diffusing’’ TFs vs. tion in different scenarios, including multicomponent mixtures, similar to lipid ‘‘transcriptionally competent’’ TFs bound to DNA), but only an average index mixtures under cholesterol treatment/ depletion and binding of dyes to different of oligomerization can be obtained. We propose an extension of the N&B areas and components of cells. (Work supported by NIH-P41_GM103540 and method, dubbed rNB (resampled Number and Brightness) that allows to mea- NIH-P50_GM076516). sure independently the oligomeric state of free and transiently bounded mole- cules, by changing the image acquisition framerate. Montecarlo (MC) Posters: Single-Molecule Spectroscopy I simulations of TFs alternating between free and transiently bound states prove the effectiveness of the method, showing that the measured apparent brightness 844-Pos Board B614 is function of the acquisition frame-rate. Moreover, the brightness values Comparison of Organic Blue/Red Dye FRET Pairs via Ensemble and reached at very highrlow sampling rates are compatible with the ones theoret- Single-Molecule FRET Spectroscopy ically predicted and verified by preliminary experiments on MCF7 cells ex- Niels Vandenberk. pressing fluorescently labelled p53 tumor-suppressor transcription factor or Chemistry, KULeuven, Heverlee, Belgium.

BPJ 8582_8585 168a Sunday, February 18, 2018

Single-molecule Fo¨rster resonance energy transfer (sm-FRET) has become a divided into 40 and 80 substeps. At low [ATP], time constant of the dwell popular application in the structural biology field to accurately determine dis- before 40 substep was [ATP]-dependent. The ATP-binding rate constant was tances at the nanometer scale and conformational dynamics of biomolecules. estimated as 1.0107 M1s1. At high [ATP], two time constants (0.5, 0.7 Here, single-molecule multi-parameter fluorescence detection (MFD) is a use- ms) independent of [ATP] were obtained before 40 substep. Another time ful tool, and when combined with nanosecond alternating donor/acceptor exci- constant (2.5 ms) of the dwell, [ATP]-independent at any [ATP] tried, was ob- tation, it provides a useful and robust readout of both donor and acceptor dye tained before 80 substep. These [ATP]-independent time constants presum- performance. Methods such as photon distribution analysis (PDA) are typically ably correspond to ATP hydrolysis, ADP release or phosphate release. In used to convert shot-noise broadened sm-FRET histograms into distance distri- the rotation driven by high [ATPgS], time constant of the dwell before 40 butions of molecular ensembles. Sm-FRET is quite demanding in terms of dye substep was prolonged to 143 ms, indicating that ATP is hydrolyzed during performance. Dyes must be bright, photostable, possess a low blinking proba- this dwell. Furthermore, when ATPgS-driven rotation was observed in the bility, sufficiently inert with respect to the labeled molecule, and coupling to a presence of ADP, 120 backward steps were observed. The 120 back- molecule of interest should preserve the dye’s photophysical properties. In this ward steps were further divided into 80 and 40 substeps. Time constant work, we assessed the performance of blue (Atto488 and Alexa488) and far-red of the dwell before 80 substep was [ADP]-dependent, indicating that it is dyes (Atto647N, Alexa647, StarRed and Atto655). First, we assessed the prop- caused by ADP binding. Assuming that backward step is reverse reaction erties of the dyes attached to single-labeled dsDNA via ensemble time- of forward step, it was concluded that 80 substep is triggered by ADP correlation, lifetime and anisotropy analysis, and then we employed double- release. Finally, remaining time constant of the dwell before 40 substep labeled dsDNA molecules incorporating one blue and one red dye at different was considered to be phosphate release. In conclusion, we proposed a com- inter-dye spacing with sm-FRET. Finally, we compared the experimentally plete chemo-mechanical coupling scheme of EhV1. Our model is totally measured inter-dye distances with accessible volume simulations, and investi- different from those of bacterial and mitochondrial F1-ATPases. This differ- gated a possible bias related to acceptor dye blinking. From the ensemble spec- ence may be relevant to their physiological functions, ATP hydrolysis and troscopy experiments we learned about bad dye performances of Atto655 for synthesis. sm-FRET. The single-molecule experiments showed us for every FRET-pair the visibility of at least two populations for a single expected one and neither 847-Pos Board B617 the buffer nor the acceptor excitation are the cause of these distinct populations Direct Observation and Quantification of Protein Dynamics on Negatively- but are due to acceptor photophysics. Compared with average E estimation, Supercoiled DNA PDA resulted in the most accurate distances. We can conclude that the Graeme A. King, Federica Burla, Erwin J.G. Peterman, Gijs J.L. Wuite. FRET-pair Alexa488/Alexa647 showed the best performance for this Department of Physics and Astronomy, Vrije Universiteit Amsterdam, application. Amsterdam, Netherlands. Cellular DNA is regularly subject to torsional stress during genomic pro- 845-Pos Board B615 cesses, resulting in a range of supercoiled DNA structures. In order to under- Conformational Dynamics of a Single HSP90 Molecule Monitored for stand how these structures can influence protein binding and activity, single- 24Hrs at Video Rate molecule methods such as magnetic, micro-pipette and angular-optical twee- Carsten So¨nnichsen1, Weixiang Ye1, Laura Tuting€ 1, Sirin Celicsoy1, zers are often employed. While these techniques enable fine control and Ruben Ahijado-Guzma´n1, Markus Go¨tz2, Thorsten Hugel2. detection of DNA supercoiling, it is often technically challenging to combine 1Physical Chemistry, Johannes Gutenberg University, Mainz, Germany, DNA torque control with fluorescence microscopy. Additionally, surface- 2Institute of Physical Chemistry, University of Freiburg, Freiburg, Germany. tethering hinders rapid buffer exchange. Here, we present a method to pre- Photo-bleaching limits single-molecule Fo¨rster resonant energy transfer pare negatively-supercoiled DNA, using dual-trap optical tweezers, that (FRET) observations to some seconds or about 10^6 excitations. An alternative overcomes both of these hurdles. Our approach, which exploits the intrinsic are plasmon rulers, where the separation of two gold nanoparticles connected mechanical properties of DNA, enables controlled formation of supercoiled by a single molecule is monitored by plasmon coupling. We demonstrate the DNA with between 10% and 70% lower linking number than that of B- first study of the conformational dynamics of a protein, here the molecular DNA. The reduced linking number can be maintained for hours in the force chaperon Hsp90 (Heat Shock Protein 90), with a plasmon ruler. We follow range 1-40 pN. As proof-of-principle, we first report combined force- the conformational dynamics of a single Hsp90 molecule for days with video manipulation and fluorescence imaging studies that recapitulate and extend rate, thus covering a bandwidth of more than 7 orders of magnitude. These mea- our understanding of the structure of negatively-supercoiled DNA as a func- surements reveal a previously unknown conformational state with average tion of tension. Next, using fluorescence imaging, we quantify how negative dwell times around 150s that is weakly coupled to ATP hydrolysis, as well supercoiling can regulate the DNA-binding dynamics of the vital mitochon- as dynamics on the timescale of hours that might indicate pathways to protein drial protein, Mitochondrial Transcription Factor A (TFAM). We demon- denaturing. strate that TFAM mobility on DNA is hindered by local underwound This novel technique to study protein dynamics allows an observation band- structures (at 5 pN), resulting in highly confined diffusion. This finding width at least 3 orders of magnitudes better than FRET. The increased band- has important implications for the ability of TFAM to search for its promoter width makes the identification of both fast and slow processes in sites in the mitochondrial genome and suggests that supercoiling could regu- biomolecular dynamics possible. Faster data acquisition hardware and larger late this process. More generally, however, this work showcases the power of data storage space will easily extend the observation bandwidth by one or our method to quantify the interactions of proteins with negatively- two magnitudes in both fast and slow direction. It will allow the observation, supercoiled DNA in unprecedented detail. Our approach thus represents a on a single molecule level, of slow processes such as protein denaturation significant addition to the single-molecule toolkit. and fast conformational changes. Furthermore, it will be possible to study the dynamical heterogeneity within the population of ‘identical’ proteins, 848-Pos Board B618 caused for example by different folding states of otherwise identical molecules Testing Kinetic Identities using Measurements of Transition Paths in or by small differences in the atomic composition of proteins with the same Single-Molecule Folding Trajectories nominal amino acid sequence. Noel Q. Hoffer, Krishna Neupane, Michael T. Woodside. Physics, University of Alberta, Edmonton, AB, Canada. 846-Pos Board B616 Recent advances in single-molecule assays have allowed individual transition Chemo-Mechanical Coupling of Rotary Molecular Motor Enterococcus paths during the folding of single molecules to be observed directly. We Hirae V1-ATPase as Revealed by Single-Molecule Analysis used transition paths of several DNA hairpins having different sequences, Tatsuya Iida1, Yoshihiro Minagawa2, Hiroshi Ueno2, Fumihiro Kawai3, measured with high-resolution optical tweezers, to test theoretical relations be- Takeshi Murata4, Ryota Iino3. tween the properties of the transition paths and the folding kinetics. We found 1SOKENDAI (The Graduate University for Advanced Studies), Okazaki, that folding and unfolding rates were related to the average transition-path time Japan, 2The University of Tokyo, Tokyo, Japan, 3Institute for Molecular for all hairpins, as expected from theory. However, the rate predicted from the Science, Okazaki, Japan, 4Chiba University, Chiba, Japan. average transition-path velocity via classical transition-state theory was typi- V-ATPase is a rotary molecular motor which functions as an ion pump. The cally 2-3 orders of magnitude too fast, emphasising the importance of barrier hydrophilic portion V1-ATPase (V1) converts chemical energy of ATP hydro- re-crossing events during folding. The probability distribution of transition lysis into unidirectional mechanical rotation. To understand chemo- path occupancies was found to predict the profile of the average velocity along mechanical coupling mechanism of Enterococcus hirae V1 (EhV1), we con- the transition paths, as expected theoretically, providing an alternate approach ducted single-molecule rotation assay using 40-nm gold nanoparticle as a for determining the average shape of the transition path trajectory. Recent the- low-load probe. We found that 120 steps per ATP hydrolysis were further ory also predicts that for a 1D harmonic barrier, the diffusion coefficient, which

BPJ 8582_8585 Sunday, February 18, 2018 169a sets the time scale of the folding process, can be determined from the average ular size. To implement solution-WIFI, we used oblique illumination and velocity at the barrier peak. We found the diffusion coefficients calculated from ratiometric imaging of diffusing molecules on a sCMOS camera with a tempo- the velocity at the barrier peak agree well with values obtained previously from ral resolution down to 2.5 ms. We used custom software to localize and track independent methods. particles, extract their intensities and associated ratios (e.g., Fo¨rster energy resonance transfer (FRET), spectral signatures, molecular stoichiometries), 849-Pos Board B619 and characterize thousands of individual molecules per minute of imaging. Cryogenic Dissection of the Phycobilisome’s Electronic Structure For validation, we tracked doubly labelled double-stranded DNA that serve 1 1 1 2 Peter D. Dahlberg , Allison H. Squires , Annina M. Sartor , Haijun Liu , as FRET standards, and recovered FRET distributions similar to those for sur- Robert E. Blankenship2, W.E. Moerner1. 1 2 face measurements. Our system was capable of tracking tiny fluorescent mol- Chemistry, Stanford, Stanford, CA, USA, Chemistry and Biology, ecules, such as single Cy3B dyes (with characteristic size of 1 nm and Washington University, St. Louis, MO, USA. diffusion coefficients reaching 50 mm2/s in 50% glycerol). We also determined The phycobilisome antenna complex from cyanobacteria absorbs and concen- the size distribution of fluorescently labelled nanodiamonds, as well as the frac- trates diffuse solar energy for efficient photosynthetic light harvesting. The tion of nanodiamond population that contain fluorescent nitrogen vacancy (NV) electronic structure of the phycobilisome is the result of hundreds of coupled centers. Solution-WIFI is a general method for high-throughput analysis of pigments distributed throughout a protein scaffold. These pigments form an en- structure, dynamics, and diffusion of many biomolecules without ergetic funnel, transferring energy from blue-absorbing pigments to red- immobilization. absorbing pigments on the way to the reaction center. In order to interrogate the complex electronic structure of the phycobilisome, we have constructed a 852-Pos Board B622 microscope capable of acquiring many single-complex emission spectra in par- Minimizing ATP Depletion by Oxygen Scavengers for Single-Molecule allel from a cryogenic sample that has been plunge frozen in liquid ethane and Fluorescence Imaging in Live Cells trapped in its native hydrated state. The observed phycobilisomes exhibit com- Seung-Ryoung Jung1, Yi Deng1, Christopher Kushmerick2, plex and heterogeneous photophysical changes in both brightness and emission Charles L. Asbury1, Bertil Hille1, Duk-Su Koh1. spectrum. Additional studies on isolated substructures of the phycobilisome, 1Department of Physiology and Biophysics, University of Washington, performed on the same instrument, facilitate the assignment of spectral states Seattle, WA, USA, 2Departamento Fisiologia e Biofisica, ICB Universidade to specific subunits of the phycobilisome and help to identify distinct blinking Federal de Minas Gerais, Belo Horizonte, Brazil. and bleaching pathways present in the phycobilisome. (This research is sup- Stability of organic dyes against photobleaching is critical in single-molecule ported by a grant from the Division of Chemical Sciences, Geosciences, and tracking and localization microscopy. Since oxygen accelerates photobleaching Biosciences, Office of Basic Energy Sciences of the U.S. Department of of most organic dyes, glucose oxidase to deplete oxygen is commonly used to Energy.) prolong dye lifetime. As demonstrated here, pyranose-2-oxidase increased the 850-Pos Board B620 lifetime of Alexa647 dye by 20-fold. However, oxygen deprivation may pose Two Photon Excitation Spectroscopy of Gold Nanorods for Bio-Sensing severe problems for live cells by reducing mitochondrial oxidative phosphory- Redmar C. Vlieg, Chris L.W. Kettenis, John van Noort. lation and ATP production. We formulate a method to sustain the intracellular Leiden University, Leiden, Netherlands. ATP level in the presence of the oxygen scavenger. Supplementation with Gold nanorods (GNRs) have unique qualities as fluorescent markers in micro- metabolic intermediates including glyceraldehyde, glutamine, and a-ketoiso- scopy. GNRs are near-infrared excitable, bright, stable, non-blinking, non- caproate, maintained the intracellular ATP level for at least 10 min via bleaching and can be easily functionalized with biomolecules - providing an increasing FADH2 despite lowered oxygen condition. Furthermore, those me- alternative for quantum dots and organic fluorophores. Besides fluorescent la- tabolites supported ATP-dependent synthesis of PIP2 and internalization of beling, spectroscopy of GNRs can be used as a highly sensitive method for bio- PAR2 receptors. Our method is potentially relevant to other circumstances sensing. Higher sensitivity and signal-to-noise ratio can be achieved using two- that involve acute fluctuation of oxygen levels, such as ischemic damage in photon microscopy, compared to single-photon excitation, due to its non-linear the brain or heart or tissues for transplantation. Our work was supported by excitation. However, typical two-photon confocal microscopes yield a low NIH grant R01-DK080840 (DSK), R01-GM083913 (BH), R37-NS08174 throughput and limit spectral accuracy. Here we present a multi-focal two- (BH), R01-GM079373 (CLA), P01-GM105537 (CLA), and 1S10RR026406 photon microscope to obtain high-accuracy, high-throughput two-photon exci- (CLA). tation spectra of single GNRs, measuring hundreds of individual rods simulta- neously. As expected, two-photon spectra of single GNRs show narrower peaks 853-Pos Board B623 compared to one-photon excitation, and can be fitted to a Lorentzian squared. Pulsed Labelling of Endogenous p53 to Dissect the Role of its Oligomeriza- Narrow spectra potentially increase the sensitivity for detecting single molecule tion and Binding in Stress Responses 1,2 1,2 2 binding events. Clustered rods would impair spectral detection, but can be dis- Alessia Loffreda , Eugenia Cammarota , Emanuela Jacchetti , 3 4 3 4 carded by looking at their spatial, spectral and light polarization dependent Serena Capozi , Samuel Zambrano , Edouard Bertrand , Marco E. Bianchi , 2,5 2 properties. In conclusion, increased spectral sensitivity by two-photon excita- Carlo Tacchetti , Davide Mazza . 1Imaging Department, European Centre for Nanomedicine, Milan, Italy, tion combined with high-throughput measurements provides a promising plat- 2 form for single-molecule sensing. Experimental Imaging Centre, San Raffaele Scientific Institute, Milan, Italy, 3RNA biogenesis, IGMM- CNRS, Montpellier, France, 4Chromatin 851-Pos Board B621 Dinamics Unit, Universita` Vita – Salute San Raffaele, Milano, Italy, Wide-Field Monitoring of Single Fluorescent Molecules and Nanoparticles 5Experimental Imaging Centre, Universita` Vita – Salute San Raffaele, Milan, without Immobilization Italy. Barak Gilboa, Bo Jing, Maabur Sow, Tao Ju Cui, Anne Plochowietz, p53 is a transcription factor (TF) that orchestrates the cellular response to gen- Achillefs N. Kapanidis. otoxic stresses. The transcriptional program activated by p53 depends on the Biological Physics Research Group, Clarendon Laboratory, Dept. of Physics, source and strength of the stimulus and lead to different cellular fates ranging University of Oxford, Oxford, United Kingdom. from DNA repair to apoptosis. Multiple regulation layers concur to the activa- Surface-based wide-field imaging (‘‘surface-WIFI’’) of single fluorescent mol- tion of p53, ranging from the modulation of its expression levels to the induc- ecules has transformed our understanding of many biological mechanisms by tion of its oligomerization and to the control of p53 binding stability to its directly accessing molecular heterogeneity and real-time reaction trajectories. binding sites on DNA, as we recently demonstrated by live-cell single-mole- By monitoring the function of hundreds of immobilised molecules simulta- cule imaging (SMI). Dissecting the role of this different mechanisms on p53 neously, surface-WIFI achieves high throughput, outperforming its confocal activity is fundamental for the design of therapies to reactivate this TF in can- microscopy counterpart that relies on point detection. However, surface- cers that express wild-type or mutated p53. WIFI relies on surface tethering of molecules, which requires the preparation Fluorescence microscopy allows to measure protein amount, oligomerization - of modified surfaces and insertion of biomolecule modifiers; further, the mol- by fluctuation spectroscopy approaches such as Number and Brightness anal- ecules of interest (e.g., proteins and their complexes) may stick to surfaces non- ysis (N&B) – and binding interactions – by SMI with single cell resolution: specifically and lose their function. These issues significantly limit the appeal to model comprehensively of the role of these phenomena on p53 activation, of surface-WIFI. it would be desirable to monitor all these properties on the same cellular pop- To address these challenges, we introduce solution-based wide-field imaging ulation. To this scope we have applied the CRISPR/Cas9 gene editing approach (‘‘solution-WIFI’’), a method that combines wide-field ratiometric imaging to label endogenous p53 with post-translational labelling systems such as and tracking of diffusing fluorescent molecules. Solution-WIFI requires no sur- HaloTag. We show that by this method we can monitor p53 abundancy, p53 face attachment, is immune to surface interactions, and also reports on molec- oligomerization kinetics and p53 binding in the same cellular population and

BPJ 8582_8585 170a Sunday, February 18, 2018 we can identify the sequence of events that lead to p53 activation in response to 856-Pos Board B626 genotoxic stress. To Flash or Not to Flash? Characterization of Fluorescein Arsenical Hairpin (FlAsH) as a Probe for Single-Molecule Fluorescence Spectros- 854-Pos Board B624 copy Deciphering the Role of ATPase Domains of CLPA using Single-Molecule Dennis D. Fernandes1, Jasbir Bamrah1, Senthilkumar Kailasam2, Optical Tweezers Gregory-Neal W. Gomes1, Yuchong Li1, Hans-Joachim Wieden2, Hema Chandra Kotamarthi, Robert Sauer, Tania Baker. Claudiu C. Gradinaru1. Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. 1 Deparment of Chemical and Physical Sciences, University of Toronto, ATP dependent molecular machines of the AAAþ family perform many 2 Mississauga, ON, Canada, RNA Research & Training Institute, Department cellular activities and are grouped into two classes depending on if they have of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, AB, one or two ATPase domains or ‘‘rings’’. E. coli ClpA, a AAAþ protein unfol- Canada. dase and part of the ClpAP protease is one of the best-characterized enzymes In recent years, new labelling strategies have been developed that involve the belonging to the two-ATPase domain class. The ClpA ATPase domains are genetic insertion of small amino-acid sequences for specific attachment of referred to as the D1 and D2 domains, with predominant ATP turnover per- small organic fluorophores. Here, we focus on the tetracysteine FCM motif formed by D2. In the current study, we have studied a Walker B mutant, (FLNCCPGCCMEP), which binds to fluorescein arsenical hairpin (FlAsH), E286Q in the ATPase active site of the D1 domain that abolishes ATP hydro- and the ybbR motif (TVLDSLEFIASKLA) which binds fluorophores conju- lysis but doesn’t impair ATP binding. Our single-molecule optical trap exper- gated to Coenzyme A (CoA) via a phosphoryl transfer reaction. We designed iments have dissected the effect of the non-hydrolyzing D1 domain on a peptide containing both motifs for orthogonal labelling with FlAsH and unfolding and translocation of substrates by ClpAP. Although in the wild- Alexa647 (AF647). Molecular dynamics simulations showed that both motifs type enzyme the D1 domain hydrolyzes only 10% of total ATP turned over remain solvent-accessible for labelling reactions. Fluorescence spectra, corre- by ClpAP, abolishing this hydrolysis reduced the unfolding rate of the model lation spectroscopy and anisotropy decay were used to characterize labelling substrate, titin I27V13P-ssrA from the C-terminal end, 3-fold and the average and to obtain photophysical parameters of free and peptide-bound FlAsH. translocation velocity by 25%. The D1 mutant had a greater effect from the The data demonstrates that FlAsH is a viable probe for single-molecule studies. N-terminus as shown by studies with a related substrate, ssrA-I27V15P; this sub- Single-molecule imaging confirmed dual labeling of the peptide with FlAsH strate was unfolded 10 times slower and the average translocation velocities and AF647. Multiparameter single-molecule Fo¨rster Resonance Energy Trans- were reduced by 60%. Slower translocation was primarily caused by an fer (smFRET) measurements were performed on freely diffusing peptides in so- increased frequency of pausing and longer pausing times compared to the lution. The smFRET histogram showed different peaks corresponding to wild-type enzyme. These results suggest that defects in ATP-hydrolysis in different backbone and dye orientations, in agreement with the molecular dy- the D1 ring cause loss of coordination between the two rings resulting in mis- namics simulations. The tandem of fluorophores and the labelling strategy firing of the D2 motor domain. Thus, our single-molecule studies provide new described here are a promising alternative to bulky fusion fluorescent proteins insight into the functional importance of the ClpA D1 ATPase domain and the for smFRET and single-molecule tracking studies of membrane proteins. role of coordination between each ClpA AAAþ ring, which were not apparent in bulk biochemical analyses. 857-Pos Board B627 855-Pos Board B625 Precise Measurement of Single-Molecule Rotational Diffusivity in Solution Measuring Oligonucleotide Hybridization Kinetics in Solution using a Hsiang-Yu Yang, W.E. Moerner. time-Resolved 3D Single-Molecule Tracking Technique Stanford University, Stanford, CA, USA. Yuan-I Chen, Cong Liu, Stephanie Phillion, Tim Yeh. Sensing the size of individual molecules in an ensemble has proven to be a power- Biomedical Engineering, The University of Texas at Austin, Austin, TX, ful tool to investigate biomolecular interactions, such as equilibrium among mul- USA. tiple oligomeric states and dynamics of association-dissociation processes. In Single-molecule measurements of DNA hybridization kinetics are mostly per- biologically-relevant solution environments, molecular size is often determined formed on a surface or inside a trap. Here we demonstrate a time-resolved, 3D by translational or rotational diffusivity. The rotational diffusivity is more sensi- single-molecule tracking (3D-SMT) method that allows us to follow a freely tive to the size and conformation of molecules as it is inversely proportional to the diffusing ssDNA molecule in solution for hundreds of milliseconds or even sec- cube of the hydrodynamic radius. The translational counterpart, however, is only onds and observe multiple annealing and melting events taking place on the inversely proportional to the hydrodynamic radius itself. Rotational diffusivity same molecule. This is achieved by combining confocal-feedback 3D-SMT has been measured, at the single-molecule level, using time-resolved fluorescence with time-domain fluorescence lifetime measurement, where fluorescence life- anisotropy decay, but with certain limitations: In single-burst-based experiments, time serves as the indicator of hybridization. With sub-diffraction-limit spatial one is only able to differentiate molecules with two orders of magnitude differ- resolution in molecular tracking and 15 ms temporal resolution in monitoring ence in rotational diffusivity, due to the limited number of photons available. In the change of reporter’s lifetime, we have demonstrated a full characterization surface-attached experiments, experiments are limited to the study of restricted 6 1 1 1 of annealing rate (kon = 5.13 10 M s ), melting rate (koff = 9.55 s ), and molecular rotation in highly confined environments. To address these limitations, 1 association constant (Ka = 0.54 mM ) of an 8 bp duplex model system we have developed a new method to measure and monitor single-molecule rota- diffusing at 4.8 mm2 s1. As our method completely eliminates the photo- tional diffusivity by combining the Anti-Brownian ELectrokinetic (ABEL) trap bleaching artifacts and diffusion interference, our kon and koff results well repre- and maximum likelihood analysis of fluorescence anisotropy decay. We demon- sent the real kinetics in solution. Our binding kinetics measurement can be strate this approach by resolving a mixture of single- and double-stranded fluores- carried out in a low signal-to-noise ratio condition (SNR z 1.4) where 130 cently-labeled DNA molecules, freely rotating in a solution-phase environment. recorded photons are sufficient for a lifetime estimation. Using a population- The rotational diffusivity, fluorescence lifetime, and initial anisotropy are simul- level analysis, we can characterize hybridization kinetics over a wide range taneously measured for each trapped single DNA molecule. The time resolution (0.5-125 s1), even beyond the reciprocals of the lifetime monitoring temporal and precision of this method were analyzed using the theory of Fisher information resolution and the average track duration. To apply this technique to study hy- and simulations. Guidelines on the choice of fluorescent label for different appli- bridization kinetics in live cells, we are testing various oxygen scavenger sys- cations are formulated based on the analysis. (Thisresearch is supported by a grant tems that are live-cell compatible. Our preliminary results have shown from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of improved dye stability in our tracking experiments. Basic Energy Sciences of the U.S. Department of Energy.)

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Posters: Optical Spectroscopy: CD, UV-VIS, resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs, fluorescent proteins (FP) that emit at longer Vibrational, Fluorescence wavelengths increased the FRET efficiency, dynamic range, and structural res- olution of high-throughput screening (HTS) fluorescence lifetime microplate 858-Pos Board B628 reader measurements. The orange FP donor’s mKO2 and mCyRFP were Measuring Structure and Disorder of (Cy3)2 Dimer Labeled DNA Fork- each paired with the far-red FP acceptor mMaroon to generate intramolecular Junctions using Two-Dimensional Fluorescence Spectroscopy (2DFS) FRET biosensors. These FPs were fused to specific sites on the cardiac calcium Amr Tamimi1, Dylan J. Heussman1, Loni M. Kringle1, Peter H. von Hippel2, pump (SERCA2a) for detection of ligand and small-molecule effector binding. Andrew H. Marcus1. FRET was evaluated using a fluorescent microplate reader capable of detecting 1 Institute of Molecular Biology and Oregon Center for Optical, Molecular, the nanosecond decay waveform in a 1536 well plate format with two-minute 2 and Quantum Science, University of Oregon, Eugene, OR, USA, Institute of scan times per plate. The mCyRFP and mMaroon SERCA FRET sensor signif- Molecular Biology, University of Oregon, Eugene, OR, USA. icantly extended the lifetime of the donor fluorophore in comparison to GFP- The structure of genomic DNA is generally pictured as the static B-form based constructs. Pulsed laser excitation at the 532 nm wavelength allowed conformation of the Watson-Crick duplex. Yet many centrally important for rapid lifetime detection with reduced background from cellular autofluores- biological processes involve dynamic molecular events requiring local re- cence and a large FRET dynamic range from known and recently discovered gions of DNA to open spontaneously allowing proteins to gain access either small-molecule effectors targeting SERCA. Global lifetime waveform analysis to single-stranded nucleotide base sequences, or to secondary structure was used to quantitate SERCA’s structural dynamics by resolving multiple motifs that depart from the stable W-C structure. The elongation events of FRET states, which directly correlate with the enzyme’s function. These DNA replication involve the binding of genome-regulatory proteins to high-content FRET methods detect minute FRET changes, with the potential non-sequence-specific positions at or near single-stranded (ss) - double- to elucidate novel structural mechanisms from small-molecules or peptide- stranded (ds) DNA junctions. To facilitate the next steps of the elongation regulators discovered through our ongoing HTS efforts. FRET sensors that process, the local conformation of DNA near ss-ds junctions likely fluctu- emit at longer wavelengths are highly attractive to the FRET biosensor commu- ates into a broad distribution of functionally relevant conformations to nity for drug discovery and structural interrogation of new therapeutic targets. permit the proper binding of replication proteins. Thus the nature and extent of conformational disorder at DNA junctions may be central to the molec- 861-Pos Board B631 ular mechanisms of the binding and the subsequent function of the FRET Based Detection of MMP-9 protein-DNA complexes involved. We have performed absorption, circular Sunil Ajit Shah1, Wlodek Mandeki2,JiLi2, Zygmunt Gryczynski3, dichroism (CD) and two-dimensional fluorescence spectroscopic (2DFS) ex- Julian Borejdo1, Ignacy Gryczynski1, Rafal Fudala1. periments to study local structural disorder at model DNA fork junctions, 1Institute of Molecular Medicine, UNT Health Science Center, Fort Worth, using DNA constructs in which dimers of the fluorescent chromophore TX, USA, 2Pharmaseq, Monmouth Junction, NJ, USA, 3Physics and Cy3 have been rigidly incorporated into the sugar-phosphate backbones of Astronomy, Texas Christian University, Fort Worth, TX, USA. the DNA chains near ss-ds DNA fork junctions. We analyze these data to Purpose: Matrix metalloproteinases (MMP’s) are a group of zinc dependent determine local backbone conformations and the extent of disorder experi- peptidases. So far, over 26 MMP’s have been identified. Out of these, MMP- enced by the (Cy3)2 dimer probes. Our analysis takes into account the 9 is of particular interest in many biomedical applications. MMP-9, also known vibrational-electronic coupling internal to each Cy3 monomer, in addition as gelatinase B, plays an important role in degrading the basement membrane to the electronic coupling between the two chromophore probes. These of the extracellular matrix (ECM). Levels of MMP-9 have been found to be up- studies suggest that this approach may be generally useful to understanding regulated in several types of cancer, including breast, bladder, colon, ovarian the local backbone conformations of DNA and RNA junctions, as well as etc. and are generally associated with poor prognosis. The overall goal of the corresponding degree of disorder, for a variety of protein-DNA interac- this project is to develop applications for detecting MMP-9 levels. This would tions relevant to DNA replication, recombination, and repair. result in rapid, non-invasive detection, and early treatment. Approach: We used Forster resonance energy transfer (FRET) to come up with a 859-Pos Board B629 custom peptide that is cleaved by MMP-9 enzyme, leading to easy detection. Photophysical Behavior of mNeonGreen, an Evolutionary Distant Green FRET is a well-known phenomenon being used today in studying molecular in- Fluorescent Protein teractions. We were able to successfully demonstrate FRET with a custom peptide Frederik Steiert, Eugene P. Petrov, Petra Schwille, Thomas Weidemann. whose partial sequence was recognized and selectively cleaved by MMP-9 Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, enzyme. The probe uses 5,6 TAMRA and HiLyte 647 as a donor and acceptor Munich, Germany. respectively. The target peptide sequence is Lys-Gly-Pro-ArgSer-Leu-Ser-Gly- Fluorescent proteins (FPs) feature complex photophysical behavior that Lys-NH2, and was optimized by Kridel et al. The fluorophores were attached must be considered when studying the dynamics of fusion-proteins in model to the peptide at the Ser-Leu bond, labeled on the ε-NH2 groups of lysine. Peptide systems and live cells. In particular, molecular brightness depends on the labelled with 5,6-TAMRA only was used as the donor control, and free HiLyte dynamics of short-lived dark states leading to flickering of the fluorescence 647 was used as the control for acceptor. The probe and donor control were dis- signal. In this work, we characterize mNeonGreen (mNG), a recently intro- solved in 10% (w/w) poly-vinyl alcohol, and dried on glass slides. This produced duced FP from Branchiostoma lanceolatum,incomparisontoEGFPand films 200 microns in thickness. Furthermore, the probe was dissolved in buffer AcGFP from Aequorea species. Fluorescence correlation spectroscopy and upon addition of MMP-9 enzyme, showed a gradual decrease in energy trans- (FCS) is used to determine flickering and molecular brightness of purified fer over time. These measurements were done by using a 1cmx1cm quartz cuvette proteins in solutions of different pH. The molecular brightness of mNG is and a square geometry set-up with 470nm as the excitation wavelength. consistently higher as compared to the traditional GFPs, but decreases slightly under acidic and basic conditions. Consistently, an advanced anal- 862-Pos Board B632 ysis of steady state spectroscopy data resolves two additional chromophore Directionality of Two-Photon Excitation in Representative Fluorescent states with reduced quantum yield that build up at low and high pH. Thus, Proteins mNG is distinguished from traditional GFPs by a two-step protonation Olga Rybakova1,2, Stepan Timr2, Josef Lazar1,2. response that reflects a chemically distinct chromophore environment. 1Center for Nanobiology and Structural Biology, Institute of Microbiology Despite the more complex pH dependence, mNG represents a superior CAS, Nove Hrady, Czech Republic, 2Institute of Organic Chemistry and GFP under a broad range of conditions. Biochemistry CAS, Prague, Czech Republic. Fluorescent proteins (FP) are the workhorses of biological molecular imaging. 860-Pos Board B630 Despite their widespread use, their optical properties are not completely under- Red-Shifted Fluorescent Proteins Improve FRET Biosensors for High- stood. This is especially true for non-linear processes, such as two-photon (2P) throughput Fluorescence Lifetime Screening excitation. We have now characterized the 2P absorptivity tensors of common Tory Schaaf1, Ang Li1, Benjamin Grant2, Prachi Bawaskar1, FPs by quantum mechanical calculations, as well as by optical experiments on Evan Kleinboehl1,JiLi1, Gregory Gillispie2, David Thomas1,3. FP crystals. Our results suggest that directionality of 2P excitation in FPs can be 1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, described by a vector (rather than a tensor), analogous to a transition dipole Minneapolis, MN, USA, 2Fluorescence Innovations, Minneapolis, MN, USA, moment. The results of our optical measurements are consistent with this 3Photonic Pharma, Minneapolis, MN, USA. notion. The combination of optical measurements with quantum mechanical We have developed fluorescence resonance energy transfer (FRET) biosensors calculations yields a vector characterizing the directionality of 2P properties with red-shifted FRET pairs which have improved characteristics for time- of representative FPs. The resulting description of non-linear properties of

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FPs will allow not only a better understanding of the process of 2P absorption in 866-Pos Board B636 an important class of fluorescent molecules, but also extraction of quantitative Using Spectral Phasor Analysis of UV-Excited Autofluorescence to Reveal structural information from 2P fluorescence microscopy experiments. Small Differences between Chemically-Induced Mitochondrial Responses Chong Kai Wong, Nazar Al Aayedi, Madhu Gaire, Martin Heidelman, 863-Pos Board B633 Paul Urayama. HomoFRET Assay for Detecting Microtubule Formation Below the Physics, Miami University, Oxford, OH, USA. Diffraction Limit of Light Spectral phasor analysis on nanosecond-gated, UV-excited autofluorescence is Joy Suh, Joseph Beggs, Malena Maxwell, Francesca Varias, being developed for the real time monitoring of cellular metabolism. Previous Keisuke Hasegawa. studies have shown that the response of autofluorescence spectrum shape to ad- Physics, Grinnell College, Grinnell, IA, USA. ditions of chemical agents (e.g., cyanide, ethanol, glucose) is due to the changing Microtubules are hollow protein filaments consisting of the alpha/beta-tubulin ensemble of cellular reduced nicotinamide adenine dinucleotide (NADH) forms. subunit, and they play important roles in various biological processes such as Here we apply the monitoring technique to investigate the autofluorescence cell division, intracellular transport, cell motility and cell morphogenesis. response of Saccharomyces cerevisiae suspensions to additions of respiratory in- The dynamics of microtubules is critical to the proper function of microtubules hibitors, including cyanide (a complex IV inhibitor) and 2-thenoyltrifluoroace- in cell division. One of the challenges in improving our understanding of micro- tone (TTFA, a complex II inhibitor). Using spectral phasor analysis to assess tubule dynamics is the small size of tubulin subunits. Because each subunit is whether observed spectral changes are consistent with a two-state model and us- only few nanometers in size and significantly smaller than the wavelength of ing nanosecond-gated spectral detection to isolate the long excited-state lifetime light, optical microscopy cannot be used to resolve the interactions of the sub- signal, we find evidence for the spectral detection of a concentration-dependent units, which lead to the formation of microtubules, in real time. We developed cyanide action and for the ability to sense complex-II inhibition despite the pres- an in vitro spectroscopy assay for detecting microtubule formation below the ence of fluorescence quenching behavior from TTFA. Results demonstrate how diffraction limit of light. The assay is based on Fo¨rster resonance energy trans- the quantification of spectrum shape can be useful in the real time, non-invasive fer between fluorescent molecules of a single type (homoFRET). Our results detection of small differences between mitochondrial responses and contribute indicate that homoFRET can be used to detect short microtubules even when to the development of NADH as a metabolic indicator and biomarker. they are diffraction limited (smaller than few hundred nanometers). We also demonstrate that when fluorophores with appropriate Fo¨rster distance are 867-Pos Board B637 used, this technique can be highly sensitive to the formation of microtubules Modulations of CA2D Sensitizers and Phosphorylation of cTnT in Dy- but less sensitive to the extent of microtubule elongation, making it suitable namic Equilibrium of cTnC N-Domain for detecting microtubule nucleation. William Schlecht, Wen-Ji Dong. 864-Pos Board B634 Voiland School of Chemical Engineering and Bioengineering, Washington Monitoring Native and Aggregate Structure of Amino Acids and Human State University, Pullman, WA, USA. An increasing body of evidence suggests cTnC exists in dynamic conformational Insulin with Blue Autofluorescence 2þ Nathan Cumberbatch1, Jillian Madine2, Heike Arnolds1. equilibrium and that Ca binding regulates this equilibrium. However, little 1Chemistry, University of Liverpool, Liverpool, United Kingdom, 2Institute direct evidence has been offered to support these claims. The present study of Integrative Biology, University of Liverpool, Liverpool, United Kingdom. aims to directly monitor dynamic conformational equilibrium of cTnC and investigate how the dynamic equilibrium is modulated by the presence of cardiac Structural characterization of proteins, peptides and amino acids is key to un- 2þ 2þ derstanding the mechanisms behind their aggregation to amyloid-like fibrillar troponin I (cTnI), cardiac troponin T (cTnT), Ca sensitizers, and a Ca desen- structures. Blue autofluorescence stems from peptide carbonyls, enhanced by sitizing phosphomimic of cTnT (cTnT(T204E). To achieve the objective, a dye þ - homodimerization approach is developed and implemented which allows for formation of zwitterionic NH3 -COO pairs. It is known to increase in intensity when proteins aggregate. Here we show that different aggregated species determination of the dynamic equilibrium between open and closed conforma- tions in cTnC’s hydrophobic cleft. Modulation of this equilibrium by other exhibit different spectral characteristics. Excitation-emission matrices (EEM) 2þ of the blue autofluorescence show distinct spectral shifts with aggregation state troponin subunits, Ca -sensitizers, and phosphomimic of cTnT(T204E) is char- acterized. Isolated cTnC contained a small open conformation population in the for both amino acids and human insulin. For cysteine and phenylalanine, we þ absence of Ca2 , which increased significantly upon addition of saturating levels uncover that with increasing aggregate size both excitation and emission max- 2þ 2þ ima shift to higher wavelengths by up to 50 nm. The tyrosine autofluorescent of Ca . This suggests the Ca induced activation of thin filament arises from an peak conversely does not shift significantly during aggregation. For human in- increase in the probability of hydrophobic cleft opening. Inclusion of cTnI increased the population of open cTnC while inclusion of cTnT had the opposite sulin, we find that fibrils formed at different pHs, which appear identical in 2þ Raman spectroscopy, exhibit distinct fluorescence EEM spectra. We suggest effect. Samples containing Ca -desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples that the spectral shifts arise from highly localized changes in the environment 2þ of the peptide group, probably related to the hydrogen bond network. Blue au- with cTnT(wt), while Ca -sensitizer treated samples generally increased open tofluorescence excitation-emission matrices are thus revealed as a useful assay conformation probability. These findings show that an equilibrium between open and closed conformations of cTnC’s hydrophobic cleft are play a significant role to monitor aggregation of both proteins and amino acids. þ in tuning the Ca2 sensitivity of the heart. 865-Pos Board B635 Concurrent Homo- and Hetero-FRET Measurements Enhance Studies of 868-Pos Board B638 Protein Interactions and Enable Development of Dual Biosensors Sedimentation Boundary Structure of Multi-Component Solutions with Tuan A. Nguyen, Grace Taumoefolau, Youngchan Kim, Henry L. Puhl III, Rapidly Reversible Interactions Steven S. Vogel. Peter Schuck, Sumit K. Chaturvedi, Huaying Zhao. SCB/LMP, NIAAA/NIH, Rockville, MD, USA. NIH, Bethesda, MD, USA. Fo¨rster Resonance Energy Transfer (FRET) has been a major non-invasive The coupled transport behavior of macromolecular mixtures with rapidly investigation method for studying protein interactions within and between com- reversible complex formation is of great interest in the study of protein interac- plexes. Unfortunately, FRET measurements have been restricted to either tions by many different methods. Complicated transport patterns arise even for homo-FRET (the donor and the acceptor fluorophores are identical) or simple bimolecular reactions, when all species exhibit different migration ve- hetero-FRET (the donor and the acceptor fluorophores are different species). locities, yet are coupled by mass action law. Although partial differential equa- Such measurements can only reveal restricted aspects of protein complex’s tions are available to describe the spatial and temporal evolution of such conformational changes, which in many cases limits the interpretation. Fluores- systems given particular initial conditions, these are often not useful to fit cent Polarization and Fluctuation Analysis (FPFA), a time-correlated single- imperfect experimental data, and do not yield any insight into the transport photon counting technique that originally combines homo-FRET and FCS, mechanism and phase behavior of the systems in parameter space. now is extended to incorporate hetero-FRET measurement to address this prob- For two-component systems, based on the limit of non-diffusing species lem. This multi-modal approach was used to observe changes in Ca2þ/calmod- migrating under constant force, we have previously derived simple algebraic ulin-dependent protein kinase II (CaMKII) holoenzyme structure in response to relationships describing the number, amplitude, and velocity of sedimentation activation and subsequent interactions with NR2B. Furthermore, dual homo- boundaries, which are consistent with the principle that the time-average veloc- and hetero-FRET was also employed in the development of Ca2þ sensors ity of all co-sedimenting components must match in each boundary. These can that can function over a wide concentration range from nano- to millimole. be considered ‘effective particles’, and their properties can be related to param- This approach opens the door for designing dual biosensors that can detect eters extracted from experimental sedimentation profiles. Here, we generalize different parameters simultaneously. this effective particle theory to N-component systems. These can have N-2

BPJ 8586_8589 Sunday, February 18, 2018 173a reaction boundaries of successively lower velocity and number of components. 871-Pos Board B641 We report algebraic relationships for velocities and composition. The concen- Single-Shot Microsecond-Resolved Spectroscopy of the Bacteriorhodopsin tration space is subdivided into regions exhibiting different boundary patterns Photocycle with Quantum Cascade Laser Frequency Combs with discontinuous transitions along characteristic subspaces. Markus Geiser1, Jessica L. Klocke2, Markus Mangold1, Pitt Allmendinger1, The extension of the effective particle theory provides physical insights into the Andreas Hugi1, Pierre Jouy1, Balint Horvath1, Jerome Faist3, coupled co-migration processes, and can be used to interpret boundary patterns Tilman Kottke2. derived from experimentally measured sedimentation coefficient distributions. 1IRsweep, Zurich,€ Switzerland, 2Bielefeld University, Bielefeld, Germany, It can be used to plan multi-component sedimentation velocity experiments to 3ETH Zurich, Zurich,€ Switzerland. determine binding constants and complex stoichiometries. Time-resolved vibrational spectroscopy is an important tool for understanding biological processes and chemical reaction pathways. Today, all available 869-Pos Board B639 methods to our knowledge require many repetitions of an experiment to acquire Accurate CD Spectrum Predictions with Sesca: Including Protein Flexi- a microsecond time-res. mid-IR spectrum. We present the IRspectrometer, a bility and Side Chains quantum cascade laser dual frequency comb spectrometer. It allows for parallel Gabor Nagy. acquisition of hundreds of mid-infrared wavelengths with microsecond time res- Biophysical Chemistry, Max Planck Institute, Go¨ttingen, Germany. olution. The formation of the light-activated L, M and N-states in bacteriorho- Electronic circular dichroism (CD) spectroscopy is a highly sensitive experi- dopsin - which only have ms to ms lifetimes - has been recorded with our mental method to probe protein structure, primarily providing information on setup: e.g. infrared response of bacteriorhodopsin to 10 ns visible light pulses the secondary structure composition. It is widely used in structural biology to with microsecond time-resolution. The different wavelengths were all measured monitor the folding and structural changes of globular proteins. Moreover, in parallel thanks to the dual-comb approach. The spectra as well as the kinetics calculating the CD spectra from model structures establishes a direct quantita- show good agreement with those from step-scan FTIR measurements. As a tive comparison between the model and a primary experimental observable and benchmark, the spectral signature of several intermediate states of the bacterio- thus allows the determination of the model’s quality and its iterative refine- rhodopsin photocycle has been recorded in a single shot measurement. This ment. Approaches including CD spectroscopy have been applied to refine pro- approach greatly reduces the complexity of time-resolved bio-spectroscopy tein structural models but rarely to improve model ensembles of intrinsically measurements in the mid-infrared which currently require many repetitions. disordered proteins (IDPs). Because of the conformational flexibility of 872-Pos Board B642 IDPs, it is often not possible to directly study their structure through conven- Using Surface Enhanced Raman Spectroscopy to Probe Single-Cell NF-kB tional crystallographic methods: ensemble refinement based on the available Dynamics during TNF-a Stimulation experimental information is the only way to study the structure of these pro- Mamadi M.S. Colley. teins. Due to their flexibility, most IDPs do not form stable secondary struc- Graduate Institute of Biomedical Materials and Tissue Engineering, Taipei tures; therefore, their ensemble refinement is more demanding and requires a Medical University, Taipei, Taiwan. higher accuracy compared to globular proteins. Here, we present SESCA: a Surface-enhanced Raman spectroscopy (SERS) is an optical device that is general method to predict the CD spectra of protein structures and protein struc- complicated but fast, that deals with high precision cells, noninvasive and ture ensembles. Test calculations and comparisons to reference spectra indi- label-free. Tumor necrosis factor-alpha (TNF-a) is primarily produced by mac- cated that purely secondary structure based methods may be insufficient for rophages an inflammatory cytokine. When TNF-a binds to TNFR, it activates IDP model refinements. Therefore, we included the conformational flexibility nuclear factor kappa B (NF-kB) transcript factors. This will lead to oscillation from molecular dynamics simulations and side chain contributions based on in nuclear NF-kB and steering gene expression by negative feedback loops. We the amino acid composition of the protein to improve the CD spectrum predic- applied biotin anti-human TNF-a antibody to bind with TNF-a proceeded by tion accuracy. Our preliminary results indicate that these corrections indeed chemical bonding for streptavidin- Phycoerythrin (PE) to detect the space yield more accurate spectrum predictions for both globular and disordered pro- structure between TNF-a and TNFR. The intracellular p65 was labeled with teins, rendering CD spectroscopy a valuable tool for the refinement of IDP GFP for NF-kB tracking. We indicated the probing of spatiotemporal interac- structural ensembles. tion between binding of TNF-a to TNF-receptor. PE interacted with TNF-a on a ratio of 1:1 and 1:4. Quantum dots (QDs) with TNF-a at 1:1. The signal 870-Pos Board B640 13 a strength had decreased in the ratio 1:4. There was low bright field in QD inter- Segmental C-Labeling for Raman Studies of -Synuclein Amyloid Struc- action to TNF-a. We used real-time imaging for single TNF-a molecule on the ture in Cells living NIH-3T3 cell surface. The result showed the ligand-induced receptor Jessica D. Flynn, Zhiping Jiang, Shannon M. Lacy, Jennifer C. Lee. dimerization and trimerization. The bioconjugation and diameter of fluorescent National Heart, Lung, and Blood Institute, National Institutes of Health, probes both affected the spatial binding of TNF-a to its receptor. Using real- Bethesda, MD, USA. time imaging of GFP-P65, we successfully visualized the intracellular NF-kB Parkinson’s disease (PD) is a prevalent age-related neurodegenerative disease nuclear translocation. Our goal is to compare the results from fluorescence im- associated with the aggregation of the neuronal protein a-synuclein (a-syn) into aging to SERS experiment. SERS has the potential to analyze NF-kB. With this b-sheet-rich fibrils, called amyloid. Natively, a-syn is an intrinsically disor- image, label-free SERS space detection and with potentially patentable novel dered protein, and it is thought conformational changes in a-syn are linked gold nano-particles (Au NPs) it can be seen NF-kB activation in two hours. to its pathogenicity. Thus, the ability to determine conformational changes of In addition, we can conduct a 96 or 384 test in parallel and increase the a-syn from soluble, unstructured monomers to b-sheet-rich aggregates is crit- throughput by two or three orders by 100-1000 faults. This will be the ultimate ical for understanding the role of amyloid fibrils in disease progression. In this goal to move this device into clinical application. work, we use Raman microspectroscopy to study the aggregation of segmen- tally 13C-labeled a-syn in real-time. Native chemical ligation is used to link 873-Pos Board B643 a uniformly 13C-labeled N-terminal portion of 1–86 to its C-terminal 12C-com- Probing Protease Active Sites with Vibrational Reporters 13 12 plement of 87–140. This ligated construct, C1-86 C87-140-a-syn, yields two Meiqi Luo, Christopher N. Eaton, Christine M. Phillips-Piro, distinct amide-I bands for observing region-specific conformational changes, Edward E. Fenlon, Scott H. Brewer. as isotopic labels shift the carbonyl stretching frequencies to two spectrally- Chemistry, Franklin and Marshall College, Lancaster, PA, USA. resolvable energies. Transmission electron microscopy shows fibril morphol- The environment of the active site of an enzyme greatly influences its activity. 13 12 ogies for C1-86 C87-140-a-syn are indistinguishable to those formed by full Here, analogues of arylsulfonyl fluoride enzyme inhibitors were modified with length 13C- and 12C-a-syn, indicating chemical ligation is not perturbative to either a nitrile or azide vibrational reporter to inhibit and probe the active site of amyloid formation. Raman kinetic data show the disordered-to-b-sheet confor- several proteases including subtilisin and trypsin. Nitrile and azide groups are mational change is first observed for the N-terminal followed by the C-terminal effective vibrational reporters due the size of the reporters and the position and 13 12 region. C1-86 C87-140-a-syn fibrils are then passively fed to cell cultures and sensitivity of the nitrile symmetric and azide asymmetric stretch frequency, probed by Raman microspectroscopy to gain structural insights into region- respectively. The sensitivity of the nitrile and azide stretch of the modified in- specific intracellular a-syn conformations. Cell viability assays are also used hibitors were first measured in a range of solvents designed to mimic various to understand how amyloid structures influence cell health. This work demon- biological environments to assess the sensitivity of these observables to local strates coupling strategic isotopic-labeling with Raman microspectroscopy can environment. This initial work permitted the observed nitrile and azide stretch reveal both molecular and mechanistic insights into a-syn amyloid formation in frequencies of the inhibitors bound to the proteins to be correlated to the local vitro and in cellular environments. We anticipate this strategy will be broadly hydration state of the active sites. These infrared spectroscopic results of the applicable to other studies of amyloid formation. inhibitor and protein-inhibitor complexes will be presented in addition to the

BPJ 8586_8589 174a Sunday, February 18, 2018 synthesis of the modified inhibitors and relevant protein crystal structures of approximate CD spectrum. Our computed spectra are found to be in good these protein-inhibitor complexes. agreement with their experimental counterparts. As a result, this model could be utilized to describe conformational changes of a given protein or peptide. 874-Pos Board B644 Study of Redox Process of Cytochrome C in Yeast under Cold Plasma 877-Pos Board B647 Irradiation through Raman Micro-spectroscopy Probing Local Protein Environments with a Vibrational Reporter Unnat- Zhu Chen, Jinghua Liu, Qing Huang. ural Amino Acid Physical Biology, Chinese Academy of Sciences, Hefei, China. Gwendolyn Fowler, Caroline Kearney, Trexler Hirn, Lukasz Olenginski, The production of reactive oxygen species (ROS) through external factors can Daniyal Tariq, Christine M. Phillips-Piro, Scott H. Brewer. lead to various microbial processes. As a physical means, cold plasma has a Chemistry, Franklin and Marshall College, Lancaster, PA, USA. wide range of applications in the areas of bio-engineering and bio-medicine. Vibrational reporter unnatural amino acids have the potential to serve as sensi- It can produce ROS and affect microorganisms effectively, resulting in the ef- tive, site-specific probes of local protein environments. Here the vibrational re- fects of stimulation, inactivation, apoptosis and necrosis of cells. Cytochrome c porter unnatural amino acid 4-cyano-L-phenylalanine (pCNF) was utilized to is a common globular protein located in the inner mitochondrial membrane of probe multiple distinct sites in the Heme Nitric Oxide and/or Oxygen cells, and its function is to transfer electrons in the mitochondrial respiratory (H-NOX) binding domain from Caldanaerobacter subterraneus. pCNF is an chain between cytochrome c reductase (complex III) and cytochrome c oxidase effective reporter of local environment due to the position and sensitivity of (complex IV). Under oxidative stress, cytochrome c can be released from the the nitrile stretch frequency. pCNF was genetically incorporated into H-NOX inner mitochondrial membrane into the cytoplasm so that it is also regarded into three distinct sites using the amber codon suppression methodology. The as a pro-apoptotic factor. In this work we employed atmospheric room- sites were selected to represent a myriad of local protein environments. The fre- temperature plasma (ARTP) to treat yeast, and employed Raman micro- quency of the nitrile symmetric stretch of pCNF and the temperature depen- spectroscopy to monitor the state changes of yeast cells under plasma irradia- dence of this frequency was utilized to assess the solvation state of the nitrile tion. As a result, we found that the Raman signals attributable to the reduced group in each of these sites in the protein. This analysis was aided by measuring yeast cytochrome c at 750 cm1, 1128 cm1, 1310 cm1, 1585 cm1 decreased the frequency and temperature dependence of the nitrile stretch vibration of with the increase of discharge time, and at a certain time after the plasma irra- pCNF in a variety of solvents to mimic different protein environments. These diation, the Raman peak at 1636 cm1 which is attributed to oxidation state of results probing the local environments in H-NOX will be presented. cytochrome c became prominent and enhanced. We also examined the apoptosis of plasma irradiated yeast cells by Annexin V-FITC/PI, and found 878-Pos Board B648 that the number apoptotic cells increased gradually with the increase of plasma Micro- and Nanostructured Surface Architectures for Label-Free Interro- irradiation time. This work thus demonstrates that we can utilize Raman micro- gation of Protein Structure and Function 1 1 2 spectroscopy to track the vital redox processes of some important biomolecules Julia Flesch , Tabea Brodo¨l , Maximilian Bettenhausen , 3 3 1 such as cytochrome c in single yeast cells, which thus may help us to under- Marcin Kazmierczak , Subhajit Guha , Changjiang You , 2 3 1 stand the cold plasma irradiation induced biological effects in the microbes. Bernd Witzigmann , Thomas Schroeder , Jacob Piehler . 1University of Osnabrueck, Osnabrueck, Germany, 2University of Kassel, 875-Pos Board B645 Kassel, Germany, 3IHP-Microelectronics, Frankfurt Oder, Germany. Protein Fold Recognition by Circular Dichroism Spectroscopy Most of the complex functions of proteins in cells result from their ability to spe- Andra´s Micsonai1, Frank Wien2, Judit Kun1, Henrietta Vada´szi1, cifically recognize other biomolecules in a highly regulated manner and to prop- Matthieu Refregiers2,Jo´zsef Kardos1. agate this information via structural reorganization. While screening for drugs 1ELTE Eo¨tvo¨s Lora´nd University, Budapest, Hungary, 2Synchrotron that efficiently block binding sites of proteins is well established, probing confor- SOLEIL, Gif-sur-Yvette, France. mational changes involved in downstream propagation remains challenging. We Experimental determination of protein structure has great importance in protein here aimed to develop surface architectures that enable efficient interrogation of science. CD spectroscopy is a classical method for the study of the secondary protein interaction and conformational organization by label-free surface- structure of polypeptides, especially in the lack of high resolution structure. We enhanced spectroscopy techniques. To ensure structural integrity and full func- constructed a program package based on our recently published CD spectrum tionality of immobilized proteins, we developed surface biofunctionalization for analysis algorithm, named Beta Structure Selection (http://bestsel.elte.hu)(Mic- site-specific protein capturing into micro- and nanostructured sensor surfaces sonai et al., PNAS 112:E3095, 2015). BeStSel takes into account the parallel- both in vitro and in live cells. Two label-free sensing approaches were explored: antiparallel orientation and twist of b-sheets providing more detailed information Silicon micropillar structures combined with orthogonal surface chemistry were on the secondary structure. Using the extra structural information, we have devel- used for high density in situ protein capturing from cells cultivated on-chip. Pro- oped a new algorithm, which is capable of characterizing protein folds using the tein interrogation was carried out by FTIR spectroscopy which enables optimal latest fold database and modern mathematics. Based on this fold prediction from a interrogation of IR-active secondary and tertiary protein structures by IR field single CD spectrum at the CATH (Sillitoe et al., Nucleic Acids Res 43:D376, enhancement via the silicon pillar sidewalls. As a second approach gold nanopar- 2015) Homology level, for the first time, a reasonable 3D homology model can ticles (AuNP) immobilized onto a glass surface were employed for LSPR-based be built for the proteinstructure. This fast and cheap fold determination is valuable reflection spectroscopy and microscopy. Surface functionalization of immobi- for recombinant protein production, functional genomics, protein therapeutics lized AuNP with tris-(nitrilotiacetic acid) (trisNTA) yielded efficient site- and rational protein design. This work was supported by the Hungarian National specific capturing of his-tagged proteins that allowed real-time monitoring of Research, Development and Innovation Office (grants K_120391, KH_125597 proteins interaction by LSPR detection. By micropatterning of gold nanopar- and TE´T_16-1-2016-0134) and the Ja´nos Bolyai Research Scholarship program ticles for LSPR-based reflection microscopy, we aim to achieve label-free of the Hungarian Academy of Sciences (to A. M.). LSPR detection of interactions with membrane proteins in the plasma membrane of living cells. Moreover, integrating gold nanoparticles on silicon micropillars 876-Pos Board B646 will allow highly sensitive interrogation of protein conformations by surface- A Coarse-Grained Model of Circular Dichroism of Proteins enhanced infrared absorption spectroscopy (SEIRAS). Mauricio D. Carbajal-Tinoco1, Carmen G. Granados-Ramı´rez2, Claudia G. Benı´tez-Cardoza3. 879-Pos Board B649 1Physics, Cinvestav-IPN, Mexico City, Mexico, 2Grupo de Inv. Bioquı´mica Probing Local Solvation Environments in H-NOX Proteins using 4-Cyano- & LIBBIQ-UN, Universidad Nacional de Colombia, Bogota´, Colombia, L-Phenylalanine 3Laboratorio de Investigacio´n Bioquı´mica, ENMyH-IPN, Mexico City, Trexler D. Hirn, Caroline Kearney, Gwendolyn D. Fowler, Mexico. Lukasz T. Olenginski, Daniyal Tariq, Scott H. Brewer, Circular dichroism (CD) is a useful technique to investigate the secondary Christine M. Phillips-Piro. structure of proteins and some other biomolecules like RNA. There are various Franklin and Marshall College, Lancaster, PA, USA. theoretical approaches intended to correlate the three-dimensional structure to Heme Nitric Oxide and/or Oxygen (H-NOX) binding domains are gas-sensing the corresponding CD spectrum and some of them depend on accurate quantum domains found in both eukaryotic and prokaryotic cells and are involved in a mechanics calculations. Such approaches, however, require an important variety of functions in the cell. The heme pocket in Caldanaerobacter subter- computational effort. In this work, we present a computationally tractable raneus H-NOX binds diatomic molecules such as O2, NO, and CO. Crystal model that is based on the classical theory of optical activity. In first stage, structures of Cs H-NOX have provided static structural images of the protein we estimate a mean polarizability per residue from experiments of molar ab- that suggest solvent is inaccessible to the heme pocket. The present study fo- sorptivity. Then, we determine the complex polarizability that is used together cuses on site-specifically genetically incorporating the unnatural amino acid with a protein structure obtained from the Protein Data Bank to calculate the (UAA) 4-cyano-L-phenylalanine (pCNF) in various distinct sites within the

BPJ 8586_8589 Sunday, February 18, 2018 175a protein. Specifically, pCNF was individually incorporated at sites 5, 36 and 78. This technology empowers biomedical research in neurobiology, immunology, Site 36 is a surface site while sites 5 and 78 are in the heme pocket. UV/Vis stem cell biology, oncology and tissue engineering. spectroscopy was utilized to confirm successful heme incorporation, and heme oxidation and ligation state of each construct. FTIR spectral analysis 882-Pos Board B652 of the pCNF containing protein constructs were recorded for the unligated pro- Electronic Tongue Development using Dielectric Spectroscopy Christopher E. Bassey, Mary C. Bassey. tein, NO bound, CO bound and O2 bound proteins revealing a distinct blue shift at each ligation state for the 36 site in comparison to the 5 and 78 sites indic- Mathematics and Physics, Azusa Pacific University, Azusa, CA, USA. ative of differing solvation states. This data will be presented illustrating the The tongue is an organ in the mouth of humans and animals used to detect taste ability of 4-cyano-L-phenylalanine to be an effective probe of local solvation in foods and drinks. It possesses the unique ability to determine degrees of environments in a biologically relevant protein. sweetness, bitterness, and saltiness. The objective of this work was to use the dielectric spectroscopy technique, to quantify sweetness in sugar solutions 880-Pos Board B650 and beverages. We used Agilent E5071C Automatic Network Analyzer Effect UV- and Gamma Radiation on Human Hair (ANA) in conjunction with a dielectric probe, to measure the dielectric proper- Ervin Palma1, Yuri V. Griko2. ties of these liquids in the microwave frequencies, between 10 MHz and 3.0 5 1California State University, East Bay, Hayward, CA, USA, 2Life Sciences, GHz, and at an average room temperature of 21.5 0.5 degrees Celsius. Re- NASA Ames Research Center, Mountain View, CA, USA. sults generally showed a decrease in dielectric constant with frequency for all The effect of UV- and 137Cs gamma radiation on the structural and chemical samples. Results also showed a linear increase in the dielectric loss factor with integrity of human hair was studied to determine the feasibility of using hu- sweetness. We also recorded a linear decrease in the dielectric constant with man hair as a non-invasive biomarker of radiation exposure to ionized sweetness. These results indicate that dielectric spectroscopy technique pro- gamma- and non-ionized UV-radiation. Steady state tryptophan (Trp) fluo- vides a reliable technique to quantify the degree of sweetness of beverages. rescence and chemical analytical methods were used to evaluate the molec- This promises to be a valuable tool in breweries and wineries. Future research ular integrity of Trp fluorophores and SH-groups in hair proteins and to will explore the quantification of bitterness and saltiness. assess the radiation induced damage quantitatively. It was found that human 883-Pos Board B653 hair fibers were progressively damaged by exposure to both UV- and ionized Minibrains on Chip for Neurological Disorder Investigation gamma radiation. Damage to the hair was evidenced by a decrease in the SoonGweon Hong1, Minsun Song1, Philip Lee1, Luke P. Lee1,2. fluorescence intensity as a result of observed depletion of the amino acid 1BioEngineering, University of California, Berkeley, Berkeley, CA, USA, tryptophan as well as significant reduction in a number of free SH-groups 2School of Medicine and Faculty of Engineering, Singapore, Singapore. in hair proteins. Hair damage was dose-dependent for exposures between Neurological malfunction or degeneration has detrimental effects of quality of life 0 and 10.0 Gy and 0 - 20 J/cm2 of UV-radiation. Additional results due to the essential role of the brain in our bodies’ functioning, memory and demonstrate that hair-fibers exposed to gamma rays, with much higher communication. Studying the development of neuropathogenesis allows us to quantum energy than UV, undergo a smaller extent of changes in Trp fluo- find possible solutions. Current research is limited as animal models cannot accu- rescence than when exposed to lower or equal energy of UV-irradiation. rately replicate human-specific features and the existing alternative is using a stat- The stable Trp fluorophore appears to be extremely sensitive to UV- iccell culture method Herein, we present a minibrain model recapitulating cellular radiation in contrast to the ionized gamma radiation whose damage is orig- organization, harmonic electrical activities and early development maturing in a inated from the reaction of free radicals and direct deposition of energy. physiologically relevant condition. Our integrated microfluidic platform, mini- We conclude that fluorescence spectroscopy represents a useful tool in the brains on chip is designed to support an array of 3-dimensional spherical tissues quantitative evaluation of the radiation exposure and could also be used within an invivo like interstitial fluid via artificial endothelium-like barrier and for the rapid and non-invasive assessment of radiation dose i.e. biodosimeter. spacious hemispherical chambers. On top of the minibrain, the surface electrodes The approach is simple, non-invasive and appears to have considerable po- are integrated to continuously monitor EEG-like electrical activities of brain tis- tential that enables quantitative evaluation of radiation dose exposure in a sues. Our minibrain model successfully captures pathological developments of single hair fiber. the brain tissue in defined etiological factors, potentially addressing a central chal- lenge of animal models. New dynamic in vitro minibrain culture will accelerate Posters: Bioengineering I prevailing research from brain modelling to fundamental neuroscience, neuropa- thogenesis, and pharmaceutical applications. 881-Pos Board B651 Maskless Quantitative Multi-protein Photopatterning to Orchestrate 884-Pos Board B654 Cellular Microenvironment Ultrafast Photonic PCR-based Precision Molecular Diagnostics for Dengue Pierre-Olivier Strale, Louise Bonnemay, Nadia Ziane, Matthieu Opitz, Jonghwan Lee1, SoonGweon Hong1, Luke P. Lee1,2. Josselin Ruaudel. 1Bioengineering, University of California, Berkeley, Berkeley, CA, USA, Alveole, Paris, France. 2Biomedical Institute for Global Healthcare Research & Technology Cell biology is faced with significant challenges when attempting to create (BIGHEART), National University of Singapore, Singapore, Singapore. complex microenvironments to unravel intricate mechanisms involved in Dengue is an endemic viral disease that affects tropical and subtropical areas. . It cell adhesion, cell polarity, cell migration etc... These challenges can be over- is estimated that more than 50 million infections occur worldwide per year. Due come by molecular printing which involves the controlled deposition of mol- to its lack of pathognomonic clinical features, dengue is often mistakenly diag- ecules on a substrate at the micrometer scale. These approaches have nosed as other febrile diseases, which thus leads to ineffective and costly over- developed tremendously in the past few years and micropatterned substrates treatment. Here we report an integrated microfluidic chip capable of on-site are now routinely used for biological research. To yield biologically relevant quantitative nucleic acids and proteins directly without separate sample prepara- data, printed biomolecules should mimic the complexity of the in vivo micro- tion steps. We integrated bothPreviously developed diagnostic tests for dengue environment. Micrometer-scale gradients of multiple proteins are thus highly can only detect a single biomarker (or two to three kinds of targets) at a time and desirable. they also lack comprehensive and syntagmatic analysis between various dengue- Here we present a maskless quantitative multi-protein photopatterning solution specific biomarkers. For an effective and precision diagnostics of dengue infec- which is based on the light-induced molecular adsorption of proteins (LIMAP) tion, a diagnostic test should not only be highly sensitive and specific, but also technology. This system combines a UV illumination module and a specific determine dengue virus serotype and distinguish between primary and secondary photoactivatable reagent (PLPP). infection. This can only be accomplished by developing a multiplexed test that The combined action of UV-light and PLPP locally degrades antifouling covers multiple targets. Here we present an integrated multiplexed optical mi- polymer brushes allowing for the adsorption of proteins in a well-defined crofluidic system (iMOMs) withultrafast PCR and immune-PCR, which allows area. the detection capability of four different nucleic acids biomarkers and five PRIMO relies on a wide-field DMD-based projection system coupled to an epi- different protein biomarkers (i.e. NS-1, IgM, IgG, IgA, and IgE) on chip. We fluorescence microscope to project custom-defined patterns of UV light onto a accomplished a single-step sample preparation module on the chip by We cell culture surface. As a result, micrometer scale patterns are generated within design, simulate, and fabricate the iMOMs using polymer microfluidic sub- seconds. The remaining background allows for the sequential patterning of strates. We integratinge biological printing technology with the microfluidic de- multiple proteins. Controlled protein gradients of custom-defined shape can vice technology. We demonstrate multiplexed dengue specific-nucleic acid also be patterned. In addition, PRIMO technology allows micro- amplifications and enhanced immunoassays. The iMOMsThis platform will be manufacturing by photopolymerization of UV-sensitive materials and protein an ideal dengue diagnostic platform for both developed and developing coun- patterning onto pre-existing 3D surfaces. tries and can be applied to give accurate and ultra-sensitive point-of-care

BPJ 8586_8589 176a Sunday, February 18, 2018 diagnoses for other intractable diseases as well.provide promising foundations metic platform will be developed. Essentially, the BBB will be recreated within for future digital healthcare. the microfluidic device, into which SPMNs will be introduced. Microcoils incorporated into the device will elicit EMF of varying strengths in order to in- 885-Pos Board B655 fluence the movement of the SPMNs across the BBB, and sensors placed at the A Handheld Optical Coherence Tomography System for Cosmetic Medi- bottom of the device will measure the resulting electrochemical signals of the cine Research BBB tissue. Due to the optical transparency of the overall system, microscopic 1 2 1 Chih-Ming Cheng , Yu-Fen Chang , Hung-Chih Chiang , observations will be made, allowing for the analysis of this technique at the Chir-Weei Chang1. 1 2 cellular level. By using this multi-disciplinary platform, we offer an innovative Industrial Technology Research Institute, Hsinchu County, Taiwan, Noble approach for in vitro studies of the SPMN-EMF technique. Furthermore, due to Medical Cosmetic Center, Tao Shin Hospital, Taoyuan City, Taiwan. the flexibility in the design of the platform, the effects of different drugs in The optical coherence tomography (OCT) has been developed for about two various tissues can be studied using the device. Hence, the proposed biomi- decades and it is a tomography technique using non-ionizing radiation sour- metic device is a highly versatile tool to use for in vitro studies. ces. The OCT system is really fast and its resolution is excellent through continuous improvement. A handheld B-scan spectra domain OCT system 888-Pos Board B658 has been successfully developed by our team and this system can acquire Interactions of Engineered Silica Nanoparticles with Lipid Monolayers cross-sectional skin images with 1 mm depth and 10 mm resolution. Therefore, and Bilayers we can identify epidermis, dermo-epidermal junction, pores, dermis, vessels Ali Asghari Adib1, Saeed Nazemidashtarjandi1, Alexander Kelly2, and collagen. In this study, we collect skin data from different ages. The items Adelaide Kruse3, Katherine Cimatu3, Allan David2, Amir Farnoud1. include Physician Skin Assessment Scale, skin elasticity and oil measurement 1Chemical and Biomolecular Engineering, Ohio University, Athens, OH, by MPA system and skin structure measurement by handheld skin OCT sys- USA, 2Chemical Engineering, Auburn University, Auburn, AL, USA, tem. The information collected in this study will be used as a statistical anal- 3Chemistry and Biochemistry, Ohio University, Athens, OH, USA. ysis basis to analyze the relationship between OCT skin structure images and The widespread use of engineered nanomaterials has led to significant interest skin texture. Thus, we can develop OCT skin detection methods for cosmetic in studying their interactions with the cell plasma membrane. Mechanistic medicine research. studies of nanoparticle-membrane interactions have generally used lipid mono- 886-Pos Board B656 layers and lipid vesicles as membrane models. However, it is unclear whether Magnetomotive Optical Coherence Tomography as New Method for differences in membrane models might affect their interactions with nanopar- Endogenous Magnetite Detection ticles. The current study aimed to elucidate the interactions of engineered silica Jessica Barrick1, Amy L. Oldenburg1, Kenneth J. Lohmann2, nanoparticles (105 nm) with lipid monolayers and vesicles in order to provide David A. Ernst2. information on the potential differences between these commonly used models 1Physics & Astronomy, UNC-Chapel Hill, Chapel Hill, NC, USA, 2Biology, in their interactions with nanomaterials. UNC-Chapel Hill, Chapel Hill, NC, USA. Lipid monolayers and vesicles, both comprised of equimolar concentrations of Endogenous magnetic iron oxides (MIOs) are of growing interest in under- sphingomyelin, cholesterol, and dioleoylphosphatidylcholine, were used as standing geomagnetic navigation. However, locating MIOs within an animal membrane models. Silica nanoparticles, surface-modified with hydroxyl, amine, is technically challenging. Methods exist for imaging MIOs in single cells and polyethylene glycol (PEG) with molecular weights of 2K, 5K, and 20K Da, and small tissue samples (SQUID, TEM), while methods such as MRI and mag- were used as particle model. A Langmuir-Wilhelmy apparatus was used to study netic particle imaging may not offer sufficient sensitivity. We propose magne- nanoparticle effects on lipid monolayer interfacial properties. Atomic force mi- tomotive optical coherence tomography (MMOCT) to quickly and non- croscopy (AFM) was used to examine nanoparticle effects on lipid topography. invasively scan large volumes of tissue for MIOs. OCT is a non-invasive, Nanoparticle effects on vesicle integrity were monitored using a vesicle leakage real-time, micrometer-scale resolution biological imaging modality which pro- assay with carboxylfluorescein (CF) as the fluorescent probe. duces cross-sectional images from back-scattered light. In MMOCT, a sinusoi- Nanoparticle effects on lipid monolayers were dependent on particle surface dally varying magnetic field produced by an electromagnet induces axial properties. Hydroxyl- and amine-modified nanoparticles did not affect the vibration of the MIOs. The vibration couples to the highly-scattering surround- interfacial properties of lipid monolayers while PEGylated nanoparticles ing tissue and is detected as a periodic optical phase shift. induced a reduction in surface tension. AFM experiments demonstrated that We characterized our MMOCT system by the equivalent MIO diameter (the only PEGylated nanoparticles penetrated into the lipid monolayer. Nanopar- minimum MIO diameter that can be detected within one resolution volume). ticles effects on lipid bilayers were different from monolayers. Hydroxyl-, amine-, and PEG 20K-modified nanoparticles disrupted the vesicles, causing We embedded varying concentrations of Fe3O4 in silicone samples with elastic and optical properties mimicking biological tissue. We measured an Fe sensi- significant CF leakage, while PEG 2K- and PEG 5K-modified nanoparticles tivity of 30 mg Fe/g, with a resolution ellipsoid of 3 mmx12mmx12mm, did not cause vesicle disruption. These results demonstrate that lipid mono- yielding an equivalent MIO diameter of 125 nm. layers and vesicles interact differently with engineered nanoparticles, suggest- As a first step, we attempted to detect MIOs found in magnetotactic bacteria ing that it might not be possible to translate the results from one model to (Magnetospirillum magneticum strain AMB-1) which form chains hundreds another. of nanometers in length and thus should be detectible by our MMOCT system. Bacteria were fixed and embedded in agarose with added light scattering par- 889-Pos Board B659 ticles for MMOCT imaging. Preliminary results (N = 4) are statistically incon- The Role of Membrane Asymmetry in Nanoparticle-Induced Plasma clusive. We expect our results to show statistical significance with larger N, and Membrane Damage 1 2 2 for MMOCT signals to positively correlate with bacteria concentration in Saeed Nazemidashtarjandi , Alexander Kelly , Allan David , Amir Farnoud3. future experiments. This will position the MMOCT technology for studies of 1 2 larger animals, where locating endogenous MIOs are difficult. Ohio University, Athens, OH, USA, Aubrun University, Auburn, AL, USA, 3Chemical and Biomolecular Engineering, Ohio University, Athens, 887-Pos Board B657 OH, USA. A Blood Brain Barrier Biomimetic Platform to Study the use of Electromag- Over the past decade, there has been increasing interest in understanding the in- netic Force on Superparamagnetic Nanoparticles for Drug Delivery Purposes teractions between nanomaterials and the cell plasma membrane. However, Reema Rahman. despite a number of studies, the structural complexities of the plasma membrane York University, Toronto, ON, Canada. including lipid asymmetry between the cytofacial and exofacial leaflets have The blood-brain barrier (BBB) has proven to be a difficult obstacle to overcome been largely overlooked. The present study focused on the role of individual for the treatment of neurological diseases and disorders due to its highly restric- membrane leaflets in regulating nanoparticle-plasma membrane interactions. tive nature. Therapeutics larger than 500 Da are prevented from traversing Specifically, the effects of amine, hydroxyl, and polyethylene glycol(PEG)- across the BBB, and into the brain, which introduces the need to develop a modified silica nanoparticles (105 5 4 nm) on the integrity of vesicles method to bypass it. One method that does so involves the use of superparamag- mimicking the exofacial (Vexo) and cytofacial leaflets (Vcyto) of the plasma netic nanoparticles (SPMNs) as drug delivery vehicles. Under the influence of membrane of erythrocytes was examined and compared with nanoparticle- an external electromagnetic field (EMF), drug-loaded SPMNs are guided to induced hemolysis in erythrocytes. Vesicle integrity was studied by encapsu- physically move across the BBB, and into the brain, thus being a non- lating the self-quenching fluorescent probe, carboxyfluorescein, in vesicles invasive therapeutic method. Though promising in theory, an in vitro proof- and studying its leakage after exposure to 0.0001-0.01 g/L of nanoparticles at of-concept study must be done in order to determine the viability of this 37 C. Confocal microscopy on giant unilamellar vesicles was used to monitor method. To test this SPMN-EMF method, a novel microfluidic-based biomi- nanoparticle effects on vesicle morphology. Nanoparticle-induced hemolysis

BPJ 8586_8589 Sunday, February 18, 2018 177a was evaluated by measuring the absorbance of hemoglobin released from eryth- Bacteriorhodopsin was encapsulated within amorphous titanium dioxide gel in rocytes after incubation with 0.01 g/L of nanoparticles at 37 C. Nanoparticle in- order to sensitize the bulk material to visible light and promote mass transfer of teractions with Vexo and Vcyto vesicles were drastically different. Vexo vesicles protons within the pore of the gel for the purpose of hydrogen production showed significant leakage after exposure to hydroxyl- and amine-modified through photocatalytic-driven water-splitting. The dynamics of the bacterio- nanoparticles, but were not disrupted by PEGylated nanoparticles. Conversely, rhodopsin within the gel matrix were investigated through spectroscopic tech- none of the particles caused significant leakage in Vcyto vesicles. GUV images niques to determine the stability and functionality of the protein under indicated visual disruption of Vexo vesicles by nanoparticles. Interestingly, confinement and in the presence of ethanol. Ethanol concentrations within hemolysis of erythrocytes by nanoparticles was consistent with leakage assays the gel pores varied up to 11.4 M with evidence of the presence of properly using Vexo vesicles. Hemolysis was observed after erythrocyte incubation folded bacteriorhodopsin in gels with ethanol concentrations up to 6.4 M with hydroxyl- and amine-modified particles, but not after incubation with with no indications of protein loss over the course of two weeks. Under visible PEGylated particles. In conclusion, these results suggest that artificial vesicles light irradiation, entrapped bacteriorhodopsin retained the ability to make mimicking the lipid composition of the exofacial leaflet of the cell plasma mem- reversible conformational changes associated with the initiation of the protein’s brane might be a useful tool in predicting nanoparticle-induced membrane dam- photocycle necessary to perform as a proton pump. In response to increasing age in cells. temperatures, the bacteriorhodopsin was also able to achieve the same level of secondary structure shifts in the gel as in solution. The amorphous titanium 890-Pos Board B660 dioxide without encapsulated protein was shown to achieve a comparable level Biomimetic Aquaporin Membrane Fabrication using Electrokinetic Inter- of photocatalytic activity to commercial crystalline nanoparticles under UV- actions only irradiation and the inclusion of bacteriorhodopsin to increase this activity 1 2 1 2 Ahmed Fuwad , Hyunil Ryu , Sun Min Kim , Tae Joon Jeon . and make the material more applicable for solar-driven photocatalysis is prom- 1Mechanical Engineering, Inha University, Incheon, Republic of Korea, 2 ising. This sets the stage for the development of a bio-nanocomposite material Biological Engineering, Inha University, Incheon, Republic of Korea. for renewable hydrogen production. Nature has a very sophisticated water transportation system via water channels called aquaporin proteins. Therefore, biomimetic aquaporin membranes 893-Pos Board B663 emerge as the next generation water purification platform having high water Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Non- flux and salt rejection as compared to conventional technologies. Despite of canonical Amino Acids into Photosynthetic Reaction Centers its substantial advantages the major barrier in using these membranes is the Jared B. Weaver, Steven G. Boxer. fragility and uniform selective coating of membrane substrate. To address Chemistry, Stanford University, Stanford, CA, USA. above issues, we presented an electrokinetic approach, which coated the sup- Bacterial reaction centers (RCs) are the membrane proteins responsible for the porter surface uniformly. The charged liposomes under the influence of electro- initial charge separation steps central to photosynthesis. They capture solar en- kinetic force block the substrate pore selectively. While to ensure their ergy in a highly efficient combination of energy and electron transfer steps. RCs mechanical stability, a protective coating of polydopamine and histidine was possess two branches of chromophores arranged in nearly identical paths, L and performed in a layer by layer scheme. These coating layers protect the aqua- M. Following photoexcitation, however, electrons are only transferred down porin membrane from the harsh environment and ensure their stability during the L branch of chromophores. Despite intense effort, the origin(s) of this uni- operation. As compared to control sample our membrane shows two times wa- directional electron transfer is not fully understood, and even the role of inter- ter flux and salt rejection in a forward osmosis system. mediary electron acceptors, is unclear. To better study these issues, we have 891-Pos Board B661 recently transferred an amber codon suppression system from E. coli into the Laurdan Imaging and Spectral Phasor Analysis Reveals Increased Mem- bacteria needed to produce RCs, R. sphaeroides. Numerous challenges were brane Fluidity in Huntington Disease overcome to implement amber suppression in R. sphaeroides; they further Sara Sameni. inform on the often-unforeseeable hurdles that can be involved in host transfer University of California, Irvine, Irvine, CA, USA. of amber suppression from E. coli. Huntington disease (HD) is a late-onset genetic neurodegenerative disorder With this system in hand, we can more finely dissect the role of key amino acids caused by the expansion of the CAG trinucleotide repeats in exon 1 of the in a way previously inaccessible to photosynthetic research using non- gene encoding polyglutamine (polyQ). This is an incurable neurodegenerative canonical amino acids. Initial work focuses on the -OH dipole strength in tyro- disorder with complex pathogenies including protein aggregation and dysregu- sine M210, believed to play a key role in modulating primary charge separa- lation of lipid homeostasis. Lipids are vital for brain function. Altered lipid tion. This is accomplished by using differently halogenated tyrosine variants metabolism has been linked to dysfunction in Huntington disease. In particular, (e.g. mono-Cl, -Br and -I) to inspect the gradual effect of these structural cholesterol metabolism appears to be disturbed in patients with HD and in an- changes on RC function. Detailed characterization of RC mutants incorporating imal models. Here we used a biophysical approach to examine the effect of HD these variants will be described. Non-canonical amino acids in the form of in the cell plasma membrane fluidity influenced by the pathogenic protein site-specific probes are also being pursued. These tools greatly broaden the (97QmRuby) compared to non-pathogenic protein (25QmRuby) and non- scope of site-directed mutagenesis in this complex membrane protein. Their labeled cells. To characterize membrane fluidity and hydration in HD, we additional insight may help us better understand protein design in this model used the fluorescent membrane probe LAURDAN that is sensitive to physical for electron transfer. states of phospholipids. Using LAURDAN emission we characterized mem- brane fluidity. We further validated our results using secondary fluorescent 894-Pos Board B664 membrane probe Nile red (NR). Our investigations indicate alteration in plasma Photoactive Split Green Fluorescent Protein: Engineering a New Optoge- membrane homeostasis that is relevant as it plays a role in cell death, and also netic and Imaging System can alter ions and molecular transport. We observed a significant shift toward Matthew G. Romei, Chelsea K. Longwell, Jennifer R. Cochran, increased polar relaxation indicating increased membrane fluidity in both HEK Steven G. Boxer. 293 cells and differentiated PC12 cells expressing 97QP. Such alteration in Stanford University, Stanford, CA, USA. membrane fluidity can be used as a biomarker for HD and also for future Visible light is a non-invasive reagent with the potential to control molecular drug discovery. This work is supported by National Institutes of Health processes with high spatiotemporal resolution, offering a unique approach to (NIH) grant (P41-GM103540 and P50-GM076516). SS is also supported by biotechnology. Many light-sensitive small molecules and proteins have been National Science Foundation (NSF) in the Biophotonics across Energy, Space used to photochemically control, or ‘‘cage’’, biologically active molecules. and Time (BEST) Integrative Graduate Education and Research Traineeship Initially suppressed, the molecule’s activity is recovered with light. As versatile (IGERT) Program and President’s Dissertation Year Fellowship. This work in vivo tools, these photocaged molecules have applications in drug delivery, is also currently under review on Nature Scientific Reprots journal. gene regulation, biosensing, and imaging. However, current photocaging sys- tems are limited by large size, restrictive cellular localization, toxic excitation 892-Pos Board B662 wavelengths, and the need for exogenous chromophores. While green fluores- Investigation of Stability and Dynamics of Gel-Encapsulated Bacteriorho- cent proteins (GFPs) are well-established imaging tools, recent findings indicate dopsin split GFPs have the potential to function as genetically-encoded photocaging re- Kaitlin E. Johnson1, Sukriti Gakhar1, Subhash H. Risbud2, agents without the limitations of other systems [1]. To optimize the light sensi- Marjorie L. Longo1. tivity of split GFP photocaging with directed evolution, we designed and tested a 1Chemical Engineering, University of California, Davis, Davis, CA, USA, functional assay that directly links split GFP’s photoresponse to transcription of 2Materials Science & Engineering, University of California, Davis, Davis, a reporter gene. We then developed a complex, two-part high-throughput screen CA, USA. based on reporter gene expression to select for improved light sensitivity among

BPJ 8586_8589 178a Sunday, February 18, 2018 split GFP variants. A new multipurpose protein engineering platform called 897-Pos Board B667 mSCALE (microcapillary single-cell analysis and laser extraction) allowed for Photo Control of Small G Protein RAS using the Synthetic Peptide Modi- the rapid visualization of mutant libraries and subsequent isolation of variants fied with Water Soluble Azobenzene with the desired phenotype [2]. In addition to the practical applications of an Nobuyuki Nishibe1, Masahiro Kuboyama1, Kenichi Taii2, improved photocage, characterization and mutational analysis of the evolved, Toshio Nagashima3, Toshio Yamazaki3, Shinsaku Maruta1. light-activated split GFP variants provide insight into the coupling between 1Department of Bioinformatics, Soka University Guraduate School of the chromophore excited state and protein dynamics, which broadly impacts Engineering, Hachioji, Japan, 2Department of Bioinformatics, Soka our understanding of diverse photobiological systems. University Faculty of Science and Engineering, Hachioji, Japan, 3Center for 1. Lin, C.-Y. et al. Proc. Nat. Acad. Sci. 2017, 114 (11), 2146-2155. Life Science Technologies, RIKEN, Yokohama, Japan. 2. Chen, B. et al. Nat. Chem. Biol. 2016, 12 (2), 76–81. The small G protein Ras is a central regulator of cellular signal transduction processes, functioning as a molecular switch. Switch mechanisms utilizing 895-Pos Board B665 conformational changes in nucleotide-binding motifs have been well studied Engineering a Cytochrome with Tunable Bandgap Potentials at the molecular level. Previously we have shown that guanine nucleotide ex- Coleman Swaim1, P. Raj Pokkuluri2, Oleksandr Kokhan1. change activity of SOS for Ras is photoreversibly regulated by the peptide 1Chemistry and Biochemistry, James Madison University, Harrisonburg, VA, mimicking SOSaH helix region, which is modified with hydrophobic USA, 2Biosciences Division, Argonne National Lab, Lemont, IL, USA. azobenzene-di-maleimide (ABDM). However, the efficiency of photoregula- For bio-hybrid mimics of natural photosynthetic systems to be efficient solu- tion was not sufficient to utilize as a photo-switching. In this study, we em- tions to current energy challenges, the relative bandgap potentials of compo- ployed water soluble bifunctional azobenzene 2,2’-bis(sulfonate)-4,4’- nent energy transfer structures must be optimized. To this end, we developed bis(chloroacetamide)azobenzene (BSBCA22) to introduce photo-switching and extensively characterized 12 point mutations of PpcA, a 3-heme member into SOSaH peptide, FCGIYRLEALKACEAN-NH2. BSBCA22 was incorpo- of the cytochrome c family native to Geobacter sulfurreducens. These muta- 7 rated into the peptide stoichimetrically. The SOSaH peptide modified with tions were engineered to influence the redox potential (E ) of the middle heme m BSBCA22 showed absorption spectral change accompanied by UV and (heme III) in PpcA by using four different strategies: performing charge visible light irradiations, reflecting cis-trans isomerization. CD spectral anal- reversal mutations, decreasing solvent access to the heme plane with bulky res- ysis indicated that the unmodified peptide showed randum secondary struc- idues, altering the native bis-histidine axial ligation of the heme, and by at- ture. NMR spectra of the peptide modified with BSBCA22 clearly revealed tempting to form hydrogen bonds with the propionates of the heme. The the two cysteine residues in the peptide intramoleculary cross-linked. CD latter strategy is expected not only to increase E but also to introduce a redox m and NMR analysis suggested BSBCA22 modified peptide formed partial- Bohr effect. Out of 12 mutants, 11 were expressed in E.coli in sufficient quan- lya-helix structure and UV and visible light irradiations induced the change tities and show thermal stability in temperature-dependent CD experiments of secondary structure of the modified peptide. Other aH helix analog pep- comparable to wild-type protein (T > 90C). HPLC-ESI-MS was used to m tides which have slightly different amino acid composition were also exam- confirm both the purity and the mass of the expressed mutants. Small-angle ined. Moreover, Photoreversible control of GDP-GTP exchange of Ras X-ray scattering confirmed that the mutant proteins were folded correctly using SOSaH peptide modified with BSBCA22 was also studied using fluo- and formed the expected compact globular structures while high resolution rescent GDP analog. crystallographic data that has been obtained for A23R and K14E shows unex- pected structures. Optical redox titrations have shown our ability to obtain reli- able and reproducible data thereby allowing us to measure the effect of the 898-Pos Board B668 mutations on the electrochemical properties of all 3 hemes and to understand Conformational Change of HVR Domain of Small GTPase Ras Reflecting the underlying principles and viable approaches in tuning relative heme redox Physiological Function 1 2 3 potentials. Successful development of this project may lead to biological semi- Takashi Hashimoto , Nobuhisa Umeki , Yasunobu Sugimoto , Shinsaku Maruta1. conductors with much smaller footprints and selectively tunable bandgap 1 properties. Department of Bioinformatics, Graduate School of Engineering, Soka University, Tokyo, Japan, 2Cellular Informatics Laboratory, RIKEN, 896-Pos Board B666 Saitama, Japan, 3Synchrotron Radiation Research Center, Nagoya Photo Regulation of Small G-Protein RAS using Photochromic Peptide University, Aichi, Japan. Masahiro Kuboyama, Nobuyuki Nishibe, Kazuo Fujiwara, The lipid-anchored small G protein Ras is a central regulator of cellular signal Kazunori Kondo, Shinsaku Maruta. transduction processes, functioning as a molecular switch. Switching mecha- Soka university, Hachioji, Japan. nisms utilizing conformational changes in the nucleotide-binding motifs have The small guanine nucleotide binding proteins (G-protein), which are known been well studied at the molecular level. H-Ras has the hypervariable region as a molecular switch is central regulator of cellular signaling pathway. The (HVR) at the C-terminal. It is known that HVR is modified by farnesylation regulation mechanism of G-protein is well studied at molecular level. Ras is and palmitoylation. The lipid modification enable H-Ras bind to membrane one of the G-protein which have essential role in cellular signal is regulated to act physiological role. We have previously shown that chemical modification by guanine nucleotide exchange factor (GEF) and GTPase activating protein of the cysteine residues in the HVR of Ras with bulky hydrophobic SH group (GAP). GTP-Ras induced by GEF is active state, whereas GDP-Ras induced specific reagents induced multimerization of Ras. The HVR domain is believed by GAP is inactive state. The conformational change induced by GTP bind- to perform physiologically important roles. Therefore, the multimerization phe- ing enables Ras to transduce a signal into downstream through direct inter- nomenon may reflect the physiological function of Ras. In this study, we action with its effectors. Also, mutant Ras known as constitutive active analyzed conformation of the HVR domain in the Ras multimer by synchrotron cause excess signaling transduction to downstream effectors, which is path- X ray scattering and fluorescent probes. Small angle X ray scattering analysis ogenesis of canceration. In this study, we focused on the interaction between suggested that the Ras modified with fluorescent probes forms pentamer. Inter- Ras and its GEF, Son of Sevenless (SOS) to control Ras function using faces between subunits of the pentamer was examined by the fluorescence photochromic compounds. Previously it was shown that the peptide quenching method. The results suggested that HVR region is located in the mimicking the aH-helix, Ras binding region of SOS effectively inhibits interface. Furthermore, we also analyzed the conformation of HVR domain Ras function competing with native SOS. Therefore, it is expected that by FRET. The FRET efficiencies from the tryptophan in HVR domain to the photo-induced structural change of the peptide conjugated with photo- fluorophore of NBD-GTP or NBD-GDP trapped in the GTP binding site indi- chromic compound enable us to control Ras activity indirectly. We designed cated that HVR domain of Ras-GDP locates closer to catalytic domain than and synthesized several SOS aH-Helix peptides which contains two cysteine Ras-GTP. residues in order to cross-link with bi-functional photochromic molecule of azobenzene derivative, ABDM which change its structure reversibly by UV- 899-Pos Board B669 VIS irradiation. CD and NMR analysis revealed that the modification of the Utilization of Single-Chain Antibody for Drug Discovery Application peptide with ABDM stabilizes secondary structure and UV and VIS light ir- Hiroaki Matsukawa, Shinji Kakuda, Asaka Kikuchi, Takashi Yamamoto. radiations induce secondary structural change reversibly. SOS dependent Teijin Institute for Bio-Medical Research, Teijin Pharma Limited, Tokyo, nucleotide exchange reaction of Ras in the presence of ABDM-SOS peptide Japan. were also altered photo-reversibly. Spontaneous cellular uptake of FITC Antibodies are very useful in basic research and drug discovery because of their conjugated peptide was observed by confocal microscopy. We also exam- high antigen specificity. However, antibodies with high molecular weights have ined the photoreversible ERK pathway inhibition with ABDM-peptide in disadvantages such as poor tissue permeability and difficulty in producing and HeLa cell. handling antibodies. Fragmentation of antibodies to obtain products such as

BPJ 8586_8589 Sunday, February 18, 2018 179a single-chain variable fragments (scFvs) can overcome the abovementioned disad- of SynNotch receptors can be used to enable or disable their signaling capacity vantages. Here we focused on cytokine receptor A and enzyme B to advance drug in response to cells bearing biotin-binding proteins (such as streptavidin and target. To improve the expression, stability, and affinity for antigens, we reduced anti-biotin antibodies). Additionally, we describe a method of controlling Syn- the molecular weight of the antibodies by modifying them to scFvs, with both an- Notch ligands through a protease-mediated cleavage event, which can be pre- tibodies for new drug discovery and tools. The cytokine receptor A and enzyme B vented in the presence of certain small molecule drugs. Finally, we show that are related to several types of autoimmune diseases and are expected to be new these ligand-expressing cells are able to induce SynNotch signaling only drug discovery targets. However, useful inhibitors have not yet been discovered. when the drug is present. Together, the described methods of controlling Syn- In this study, based on biochemical analysis and structural biology, we de- Notch receptors and ligands enables the ability to study cellular patterning signed scFvs to recognize each target to promote drug discovery with the cyto- based on post-translational changes in receptor/ligand binding. kine receptor A and enzyme B. In addition, on the basis of full-body antibody structure and sequence, several types of scFvs with different linkers were ex- Posters: Micro- and Nanotechnology I pressed by E. coli and animal cells. The binding affinity of scFvs to each target was confirmed using bio-layer interferometry and by evaluating enzyme activ- 902-Pos Board B672 ity. Furthermore, structural analysis was conducted and superposed into a full- Nanopore Fabrication in Ultrathin HFO2 Membranes for Nanopore-Based body IgG structure to compare conformational changes. DNA Sequencing We discussed the property and structure changes based on the function and Yinghua Qiu1, Christopher Arcadia2, Mohammad Amin Alibakhshi1, structure data obtained. In future, we aim to introduce a method for drug dis- Jacob Rosenstein2, Meni Wanunu1. covery or a tool for clearing molecular functions by enhancing the use of these 1Department of Physics, Northeastern University, Boston, MA, USA, obtained scFvs. 2School of Engineering, Brown University, Providence, MA, USA. Hafnium dioxide (HfO2) nanopores can serve as a perfect candidate for 900-Pos Board B670 nanopore-based-DNA sequencing, due to their superior resilience and Development of Novel Protein-Capture Reagents against Epidermal chemically resistant properties, as compared with silicon nitride of similar Growth Factor as Potential Anticancer Agents geometry. Another attractive feature of HfO2 is the slower speed by which Hariharan Parameswaran1, Sangama Vemulapally1, Claudia Santillan1, DNA molecules pass through the pore, due to strong interactions between 1 1 1 2 Tyler Helten , Elena Tikhonova , Manuel Ramos , Tong-Chuan He , the pore surface and nucleic acids. While traditional HfO2 nanopores Lan Guan1. have been fabricated using a transmission electron microscope, (TEM), is- 1Cell Physiology & Molecular Biophysics, Center for Membrane Protein sues with TEM-based fabrication, including long fabrication time and Research, School of Medicine, Texas Tech University Health Sciences e-beam induced crystallization, often compromise the pore stability. In Center, Lubbock, TX, USA, 2Molecular Oncology Laboratory, The this work, we explore the use of dielectric breakdown as a means to produce University of Chicago Medical Center, Chicago, IL, USA. small nanopores in freestanding HfO2 membranes that are sub-5-nm thick. Ankyrin repeat combinatorial library is a useful tool for the development of We have produced amorphous and crystallized HfO2 membranes using specific protein capture reagents (or blockers). To establish an in vivo selection atomic layer deposition and high-temperature annealing following deposi- method, we developed a high-throughput selection based on a bacterial two- tion, respectively. We further investigate the ability of such nanopores to hybrid system. Functional reconstitution of adenylate cyclase (CyaA) activity detect DNA. Successful implementation of this approach may lead to a from two fusions based on T18 and T25 fragments of the catalytic domain of solid-state platform for DNA sequencing and other high-resolution single- a bacterial toxin is used for the detection. A E. coli strain with deletion of its molecule detection applications. single cyaA gene is created to be the reporter strain. A pair of compatible plas- mids has been engineered for the generation of fusions with a target gene or a 903-Pos Board B673 ankyrin repeat combinatorial library by a fragment exchange cloning method. Towards High Accuracy De Novo Nanopore Sequencing The epidermal growth factor (EGF) induces mitogenic activities by binding Matthew T. Noakes, Henry Brinkerhoff, Andrew H. Laszlo, exclusively to the extracellular domain of EGF receptor (EGFR) to activate Ian M. Derrington, Kyle W. Langford, Jonathan W. Mount, its intracellular tyrosine kinase, and elevated activations have been demon- Jasmine Bowman, Kenji M. Doering, Benjamin I. Tickman, strated to be involved in cancer development. To develop novel binders against Hugh H. Higinbotham, Katherine S. Baker, Jens H. Gundlach. EGF, we used a directed-evolution approach based on ankyrin repeat combina- University of Washington, Seattle, WA, USA. torial library and high throughput selections by the CyaA-based two-hybrid Nanopore DNA sequencing is a next generation technology providing long read system, as well as a ribosome display technique. We obtained few hits that lengths and direct detection of epigenetic base modifications. The high error grew on the selective minimal media supplemented with maltose as the sole rate for single-read de novo sequencing is the main obstacle to realizing nano- carbon source and formed red colonies on MacConkey agar containing maltose pore sequencing as a stand-alone sequencing platform. We address the primary as the sole carbohydrate source. Maltose fermentation depends on cAMP, the sources of base calling errors by using a DNA helicase with desirable properties CyaA product, so the data suggest that the CyaA activity is reconstituted, which and applying a time-varying voltage to jointly control DNA motion through the may result from the interaction of a ankyrin fusion with the EGF fusion. The pore, and report an improved de novo sequencing accuracy for nanopore biophysical characterizations of purified ankyrin repeats and its functional ef- experiments. fect on EGF will be discussed. 904-Pos Board B674 901-Pos Board B671 Controlling the Conformation of Double-Stranded DNA during Transloca- Post-translational Control of Synthetic Notch Receptors and Ligands tion through a Glass Nanocapillary Jeffrey B. McMahan, John T. Ngo. Niklas Ermann, Nikita Hanikel, Ulrich F. Keyser. Biomedical Engineering, Boston University, Boston, MA, USA. Cavendish Laboratory, University of Cambridge, Cambridge, United Notch receptors regulate a variety of cell fate decisions and are activated by the Kingdom. binding and endocytosis of ligands presented on the cell surface of neighboring Nanopores have emerged as powerful single-molecule sensors, enabling the cells. Synthetic Notch (SynNotch) receptors are engineered version of the pro- detection of analytes in a simple system purely based on the modulation of tein that can be programmed to yield user-specified input and output relation- an ion current. Nanopore diameters of roughly 15 nanometres offer advan- ships, and such receptors have been used to reprogram T cells to detect cancer tages in terms of fabrication, a higher frequency of translocation and reduced antigens and induce cell-killing transcriptional programs. In natural Notch non-specific interaction with the pore wall. They also permit non-linear con- signaling, various mechanisms are used to fine-tune the activity of the receptor formations of double-stranded DNA during translocation, such as a single in order to achieve diverse context-dependent signaling outcomes. For fold which wraps a section of the strand back onto itself. We demonstrate example, Notch is fucosylated in the endoplasmic reticulum (ER) and further that such folding can be controlled through the salt concentration of the mea- modified in the Golgi apparatus to adjust its ability to recognize specific li- surement solution. The share of unfolded, linear translocations increases gands, thus controlling its perception of certain cells. In contrast, the ligand from below 50% to above 90% as the electrolyte concentration is reduced. sensing control mechanisms of SynNotch receptors have mostly been through Further measurements suggest this is due to electro-osmotic outflow from substitution of the receptor’s ligand-binding region and intracellular transcrip- the pore set up by the charged nanochannel walls, which creates a barrier tional effector domain. Here, we describe a method for controlling SynNotch to entry for high-drag DNA conformations. The ability to control conforma- receptors through an analogous post-translational mechanism using a chimera tions is critical for sensing techniques which rely on unfolded, linear trans- containing biotin accepting peptide, which can be enzymatically biotinylated in locations to read information from the strand, such as DNA-carrier based the ER by an E. coli-derivedbiotin ligase. We show that regulated biotinylation protein sensing. In addition, the results show that nanofluidic flows through

BPJ 8586_8589 180a Sunday, February 18, 2018 nanopores can be used to study polymer dynamics in fluid flow on the single- 908-Pos Board B678 molecule level. Transverse Detection of DNA in a MoS2 Nanopore Michael Graf1, Ke Liu1, Aditya Sarathy2, Jean-Pierre Leburton2, 905-Pos Board B675 Aleksandra Radenovic1. Slowing Down DNA Translocation Speed thorough a Nanopore by a 1Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Nanofibre Meshed Layer Engineering, EPFL, Lausanne, Switzerland, 2Beckman Institute for Daming Zhou, Yue Zhao, Enling Tian, Deqiang Wang. Advanced Science and Technology, University of Illinois, Urbana, IL, USA. Chongqing Insitute, Chongqing, China. Solid-state nanopores can be used for single-molecule detection and potentially Nanopore sequencing is regarded to be one of the most promising sequencing provide a platform for rapid DNA sequencing. DNA molecules along with wa- technologies to realize the destination of ‘‘$1000 Genome’’. Both of protein ter and ions are electrophoretically driven through a small orifice in a mem- and solid-state nanopores have been extensively investigated in the last de- brane. The ionic current blockade is measured and provides information cades. However, due to the weak interaction between DNA molecules and about the size and the length of the molecule. Typically, solid-state nanopores the inner surface of nanopore, DNA translocation is such fast, just leaving are fabricated in relatively thick material (20nm), thus not providing single- a short current drop without sufficient information to recognize the nucleotide base resolution. Recently, monolayer materials such as MoS2 have proven sequence in the strand. In this paper, we propose a nanopore-nanofiber mesh very useful in detecting and differentiating single nucleotides. Compared to (NP-NFM) hybridized structure to decelerate DNA translocation speeds. e.g. graphene, monolayer MoS2 is an intrinsic semiconductor with a bandgap Experimental results reveal that due to hydrophobic interaction between of 1.8eV and can thus be used to fabricate a field effect transistor. Here we the DNA fragments and the nanofibers, the DNA moving speed can be show that a nanopore in monolayer MoS2 can be integrated with a two- retarded by two orders of magnitude slower. Furthermore, according to the dimensional field-effect transistor. We report simultaneous and correlated theory simulations, the additional fiber layer will reduce the electric field detection of the ionic current trace and the transverse sheet current through sus- in the channel but elongate the capture region at the pore orifice, that will pended MoS2 during DNA translocation. Signal-to-noise ratios in the sheet cur- be helpful to increase the capture rate and extend the DNA dwelling time rent are superior to ionic current and seem to follow a different sensing in the meanwhile. principle. During translocation through the nanopore, the negative charge of 906-Pos Board B676 DNA decreases the drain-source conductance of the n-type semiconductor, Investigation of Compacted DNA Structures Induced by NaD and KD effectively gating the semiconductor. Such a device configuration might over- Monovalent Cations using Biological Nanopore come resolution limitations due to the dominating access-resistance in ionic Trang A. Vu, Shanna-Leigh Davidson, Julia Borgesi, Jiwook Shim. current through ultra-thin membranes. Furthermore, this technology provides Biomedical Engineering, Rowan University, Glassboro, NJ, USA. a simple way of parallelization: An array of nanopores can be created on the In aqueous solutions, an elongated, negatively charged DNA chain can same membrane without the need of fabricating individually addressable mi- quickly change its conformation into a compacted globule in the presence crofluidic chambers. The ionic voltage would then just act as the driving force of positively charged molecules, or cations. This well-known process, called of bringing the molecules to the field-effect transistor and all detection is done DNA compaction, is a highly potential method for gene therapy and deliv- on the transistors. Further work is required in order to better understand the ery. Experimental conditions to induce these compacted DNA structures are sensing principle and establish the resolution of these new devices. often limited to the use of common compacting agents, such as cationic sur- factants, polymers, and multivalent cations. In this study, we show that in 909-Pos Board B679 highly concentrated buffers of 1M monovalent cation solutions at pH 7.2 Sensitive Detection and Identification of Nucleic Acid Nanoparticles in and 10, biological nanopores allow real-time sensing of individual com- Solid-State Nanopores 1 2 3 pacted structures induced by Kþ and Naþ, the most abundant monovalent Mohammad Amin Alibakhshi , Justin R. Halman , James Wilson , 3 2,4 1,5 cations in human bodies. Herein, we study the ratio between compacted Aleksei Aksimentiev , Kirill A. Afonin , Meni Wanunu . 1Department of Physics, Northeastern University, Boston, MA, USA, and linear structures for 15- mer single-stranded DNA molecules containing 2 only cytosine nucleotides, optimizing the probability of linear DNA chains Department of Chemistry, University of North Carolina at Charlotte, Charlotte, NC, USA, 3Department of Physics, University of Illinois at being compacted. Since the binding affinities of each nucleotide to cations is 4 different, the ability of DNA to fold and compact depends highly on the type Urbana–Champaign, Urbana, IL, USA, The Center for Biomedical Engineering and Science, University of North Carolina at Charlotte, of cations and nucleotides present. Our experimental results compare favor- 5 ably with findings from previous molecular dynamics simulations for DNA Charlotte, NC, USA, Department of Chemistry and Chemical Biology, compacting potential of Kþ and Naþ monovalent cations. We estimate that Northeastern University, Boston, MA, USA. the majority of single-stranded DNA molecules in our experiment are com- Nucleic acid nanoparticles (NANPs) are programmable self-assembled nano- pacted. From the current traces of nanopores, the ratio of compacted DNA to structures composed of multiple RNA or DNA molecules. These nanoparticles linear DNA molecules is about 30:1 and 15:1, at a pH of 7.2 and pH of 10 have found applications in medicine and biotechnology, including their uses respectively. Our comparative studies reveal that Naþ monovalent cations as carriers for RNA interference inducers, aptamers, fluorophores, and as cus- have a greater compacting potential on the 15C-ssDNA than a Kþ cation tomizable materials. This study investigates transport dynamics of different does. NANPs through solid-state nanopores, and aims at evaluating nanopores for label-free analysis of these soft nanoparticles, as well as exploiting NANPS 907-Pos Board B677 for biosensing applications. We observe that the fractional current blockade Large Scale Parallel DNA Detection by 2D Solid-State Multi-pore System monotonically increases with the applied bias which is consistent with deforma- Nagendra Bala Murali Athreya, Aditya Sarathy, Jean-Pierre Leburton. tion of the nanoparticles as they traverse the pore. This deformation-governed ECE, University of Illinois, Urbana-Champaign, Urbana, IL, USA. transport—which is further verified by the anomalously long dwell times of We propose a new scalable device design based on the use of nanoscale semi- NANPs in relatively large nanopores—allows their efficient detection using a conductor materials to detect and identify translocations of many biomole- high applied bias at picomolar concentrations. We further demonstrate the differ- cules in a massively parallel detection scheme. These devices hold the entiation between ring-shaped and cube-shaped nanoparticles with excellent ef- potential for utilization in the risk assessment of early diagnosis as well as ficiency. Our findings have been verified against molecular dynamics seamless integration with semiconductor electronics. In our scenario, DNA simulations, which point to the nanopore serving as a mechanical barrier to molecules are treaded through nanopores drilled in electrically active two- free translocation of the particles and confirm the difference in the translocation dimensional graphene to be sensed by the changes produced on an electronic signal amplitudes observed for the two types of nanoparticles. This study ad- current flowing through the membrane. We use molecular dynamics coupled vances our understanding of translocation characteristics of NANPs and opens to nanoscale device simulations to explore the ability of this device setup to an avenue for devising new nanopore-based biosensing modalities. uniquely recognize parallel DNA translocation. In particular, we show that electronic sensing offers a higher detection resolution compared to ionic cur- 910-Pos Board B680 rent blocking technique, since simultaneous translocation of DNA through Muitilpe Nanopores Fabrication in a SiNX Membrane via Controlled multiple pores can result in crosstalk between the ionic currents while trans- Breakdown verse sheet currents are sufficiently insulated due to their ability to sense the Yunlong Wang, Cuifeng Ying, Wenyuan Zhou, Zhibo Liu, Jianguo Tian. local potential changes only. Our device design paves the way towards devel- NanKai University, TianJin, China. oping large scale solid-state biomolecular integrated circuits (ICs) with differ- Solid-state nanopores are widely used in single-molecule detection and bio- ential control over individual pore sensing geometries as well as detection molecule analysis, i.e. DNA sensing, DNA - protein and protein- protein inter- sensitivities. action. Recently, the Tabard-Cossa group reported a low-cost and broadly

BPJ 8586_8589 Sunday, February 18, 2018 181a accessible synthetic nanopore fabrication approach, controlled breakdown Solid-state nanopores are versatile tools for single molecule sensing. Because (CDB). Since CDB uses conductance feedback to monitor the nanopore fabri- their sizes can be tuned from few nm until hundreds nanometers, they are partic- cation, it cannot tell whether there is a large single nanopore or multiple small ularly promise to characterize the biomacromolecule assembly. We were inter- nanopores in the membrane. In this work, we found that despite the stochastic ested by the detection of amyloid fibril obtained from model proteins. To reach process during the breakdown, nanopores created via breakdown in a SiNx this goal three main problems have to be tackle. First, the protein unfolding usu- membrane tend to have the same scale. We proposed a resistance model to ally promotes the adsorption at solid/liquid interfaces especially by non- govern the multiple nanopores formation by the conductance feedback - the electrostatic interactions inducing the nanopore fouling. Second, the protein am- number of nanopores in the membrane was determined by the membrane resis- yloid fibrils are more rigid than polymer chain. Thus their translocations require tance and the nanopore sizes were controlled by the enlargement electric field. an important energy overcome to barrier of entrance and the one to escape. This is We further characterized our multiple nanopores by transmission electron mi- amplified by the adsorption. Finally, the characterization of the fibrillation kinetic croscopy (TEM) imaging and the fluorescence of Ca2þ-activated dyes. We imposes to work with very stable nanopores. We have produced different nano- anticipate that by combining with optical measurements, this fabrication pores to evaluate their potentiality for amyloid fibril detection. Then, these nano- approach could accelerate the process of nanopore sensing towards a high- pores were functionalized with PEG chain, in order to prevent the unspecific throughput and multichannel technique. adsorption and improve their lifetime. We have studied the translocation of small protein aggregate as well as large fibrils through these nanopores. The SiN nano- 911-Pos Board B681 pore coated with PEG is limited to the detection of small protein aggregate. How- Slowed Down Double-Stranded DNA Transport through Solid-State Nano- ever, it allows the relative current blockade is related to the morphology of the pores by using a Lithium Chloride Concentration Gradient aggregate. The conical nanopore on PET performed by track-etched technique Julian Bello, Maksudul Mowla, Nicholas Troise, Jiwook Shim. is the most suitable to follow the growing of amyloid fibril due to a longer lifetime Biomedical Engineering, Rowan University, Glassboro, NJ, USA. and an optimum geometry which allow to decrease the energy barrier of entrance. Nanopore biosensors represent the future of diagnostic devices as they are low- cost, high-throughput engines for single molecule detection. Solid-state nano- 914-Pos Board B684 pores are particularly attractive since their size can be controlled to detect a Nanopore-Based, Multi-Parametric Characterization of Single, Unlabeled wide range of analytes in solution. However, progress in the field is impeded Proteins in Solution by inadequate sensitivity of data acquisition systems in detection of fast Jared Houghtaling1,2, Olivia M. Eggenberger2, Cuifeng Ying2, DNA translocations through the pore. One way to overcome this is by slowing Michael Mayer2. translocation of DNA through the nanopore by use of various media or by 1Department of Biomedical Engineering, University of Michigan, Ann altering experimental parameters. Applying a concentration gradient of KCl Arbor, MI, USA, 2Adolphe Merkle Institute, University of Fribourg, in the experimental buffer has been shown to effectively prolong dwell times Fribourg, Switzerland. by creating a free-energy barrier that DNA molecules have to overcome. In Most methods for characterizing proteins - including electron microscopy, NMR addition to this, the concentration gradient enhances the magnitude of the local spectroscopy, and X-Ray crystallography - require either some sort of physical potential, Vr, increasing the capture rate of DNA and thereby increasing event modification such as purifying, drying, freezing, or crystallizing proteins, or frequency. Our previous work has demonstrated the ability of LiCl buffer to they require chemical modification such as fluorescently labeling or immobilizing slow down the transport of dsDNA through the nanopore by up to 10-fold proteins. Resistive pulse-based nanopore sensing of proteins is emerging as a through cation/DNA interactions. However, this drastically reduced the event powerful analysis technique; it has the potential to overcome these requirements frequency, which can affect the efficacy of this system as a reliable biosensor while characterizing individual, unlabeled proteins in aqueous solution. We downstream. Here, we present the use of a concentration gradient of Lithium recently used lipid-coated synthetic nanopores to determine the volume, shape, Chloride buffer to increase the event frequency of dsDNA translocation and charge, dipole moment, and rotational diffusion coefficient of single proteins further increase dwell time to enhance detection of single molecules through that we tethered to the fluid lipid bilayer to slow their passage through the pore. a solid state nanopore. By using 0.5M/3M LiCl on the cis/trans chambers Here, we compare two different non-adsorptive nanopore coatings combined respectively, average dwell times experienced up to a 3-fold increase when with high bandwidth recordings to determine the shape and volume of freely compared to experiments run in symmetric 1M LiCl. Additionally, experiments translocating proteins in solution. The difficulty of this approach lies in the lateral using the 0.5M/3M displayed a greater than 10-fold increase in event fre- diffusion of untethered proteins transiting a nanopore, which facilitates position- quency, confirming the capture propensity of the asymmetric conditions. dependent, asymmetric disruptions in the electric field as proteins approach the nanopore walls. These ‘off-axis’ effects pose a challenge to multi-parametric pro- 912-Pos Board B682 tein characterization because they distort the observed resistive pulse magnitudes, High throughput Characterization of Dielectric Breakdown Nanopore leading to analysis artifacts. Using lipid-coated nanopores to minimize non- Sensors in a Meniscus Contact Platform specific protein adhesion, we mitigated these off-axis effects by reducing the resi- Christopher E. Arcadia, Rukshan T. Perera, Jacob K. Rosenstein. dence time of proteins at the pore wall. This approach made it possible to estimate Engineering, Brown University, Providence, RI, USA. shape and volume values for nine different native proteins free in solution. In Solid-state nanopores have shown high promise as electrochemical sensors of contrast, pores coated with the detergent Tween-20 were not suitable for protein single biomolecules. However, experiments with these devices can be chal- shape and volume characterization likely because of significant adhesion to the lenging due to the limited number of pores that can be fabricated and tested pore wall and concomitant off-axis effects. By examining unlabeled proteins in per day. Recently, we introduced a new platform for automated in-situ fabrica- an aqueous sample on a single molecule level in lipid bilayer-coated nanopores, tion and testing of solid-state nanopores. The system utilizes dielectric break- we enable new applications for nanopore-based protein sensing, especially those down to form a nanopore in a thin insulating membrane which is contacted by involving transient protein complexes or macromolecules. a micron-scale liquid meniscus. The confined liquid contact area results in a nanopore interface with less than 0.2pF capacitance, and allows for arrays of more than 100 nanopores to be sequentially fabricated and measured on a single 915-Pos Board B685 membrane in one day. Taking advantage of the system’s high throughput, we Off-Axis Effect on the Determination of Nanoparticle Volume and Shape have performed a series of systematic studies to explore the effects of different by Resistive-Pulse Based Nanopore Sensing chemical conditions and electrical protocols on pore formation and performance. Cuifeng Ying, Jared Houghtaling, Michael Mayer. Within these datasets, we assess the sensing viability of each individual pore by Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland. monitoring the baseline current stability and I-V characteristics. Understanding Resistive pulse-based nanopore sensing has become a powerful single-molecule the factors which lead to consistent and high-quality nanopores is of utmost characterization technique. It relies on the Coulter counting principle, where importance, as the dielectric breakdown method has the potential to usher in each particle transiting a nanopore displaces a quantity of current-conducting the broad adoption of low-cost solid-state nanopore sensing technology. ions proportional to its shape and volume, and this transient ion displacement can be precisely measured as a resistive pulse. Most approaches to analyze these 913-Pos Board B683 pulses assume that the particle translocation occurs along the central pore axis - Amyloid Fibril Analysis using Single Nanopore i.e. equidistant from all pore walls. In reality, particles freely transiting the pore Nicoletta Giamblanco1, Diego Coglitore1, Tianji Ma1, also diffuse laterally, and as they approach the pore walls they generate non- Pierre Eugene Coulon2, Emmanuel Balanzat3, Mikhael Bechelany1, uniform distortions in the electric field, resulting in larger-than-expected resis- Jean-Marc Janot1, Sebastien Balme1. tive pulses. In this work, we studied these ‘off-axis effects’ for nanoparticle 1universite de Montpellier, Montpellier, France, 2Laboratoire des Solides translocation through a cylindrical nanopore using finite element simulations. Irradies, E´ cole polytechnique, Palaiseau, France, 3Centre de recherche sur les We varied the size, shape, and axial position of individual particles, as well as Ions, les Materiaux et la Photonique, Caen, France. the diameter of the nanopore. We demonstrate that randomly sampling a

BPJ 8586_8589 182a Sunday, February 18, 2018 translocating nanoparticle at different distances from the central axis leads to tons dissolved in water were forced through a sub-nanometer-diameter pore overestimates of that particle’s volume. Additionally, we show that accounting spanning an ultra-thin silicon nitride membrane and the current was for off-axis effects or minimizing them is critical for accurate determination a measured. Except for protons, the measurements revealed a conductance se- particle’s shape since estimates of ellipsoidal shape can deviate by up to 200% lective to positive ions that vanished when extrapolated to pores smaller than deviation due to off-axis effects. To minimize off-axis effects, we have deter- about 0.25 nm in diameter, which was comparable to the diameter of a water mined optimal diameter ratios of nanopore to nanoparticle for an experiment molecule (0.28 nm). On the other hand, the proton conductance persisted, with freely translocating particles, and highlight the importance of tethering extrapolating to zero only when the pore diameter was about 0.15 nm. nanoparticles to the pore walls to mitigate these unwanted effects on the nano- Furthermore, an analysis of the low frequency (pink) noise power spectral particle’s shape determination. By considering off-axis effects in nanopore density exposed a threshold, below which the noise was independent of sensing, and possibly correcting for them, we are further improving the ability the current, and beyond which it increased quadratically. This dependence to accurately characterize individual nanometer-sized particles in solution. on current proved that the spectral density components of the noise, which were uncorrelated below threshold, became nearly perfectly correlated above 916-Pos Board B686 it. Coincidently, the onset of correlations in the noise current for Liþ, Mg2þ, Translocation of Sequence-Controlled Synthetic Polymers through Biolog- Naþand Kþ ions extrapolates to pore diameters of 0.12 5 0.11 nm, 0.12 5 ical Nanopores 0.11 nm, 0.21 5 0.11 nm and 0.23 5 0.11 nm, respectively. Altogether, 1 2 2 Mordjane Boukhet , Niklas F. Ko¨nig , Abdelaziz Al Ouahabi , these data were consistent with the correlated motion of (at least partially) Gerhard Baaken1, Jean-Franc¸ois Lutz2, Jan C. Behrends3. 1 2 unscreened metal ions with a grossly distorted hydration shell permeating Ionera Technologies GmbH, Freiburg, Germany, Institut Charles Sadron, the smallest pores at high current. Strasbourg, France, 3Physiology, University of Freiburg, Freiburg, Germany. Precision synthesis of sequence-encoded heteropolymers opens the possibility 919-Pos Board B689 of information storage on molecular length scales as a sequence of monomers, Broadband Amplifier for Nanopore-Based Biomolecular Analysis akin to information storage on DNA. Nanopore sequencing, as shown for DNA, Frank Tsang, Michael Goryll. is an attractive proposition also for read-out of sequence information of syn- School of Electrical, Energy and Computer Engineering, Arizona State thetic polymers. In fact, synthetic polymers can be designed so that they are University, Tempe, AZ, USA. especially well adapted to be read by nanopore assays. Using monomeric spe- Nanopore sensors can provide information about proteins, molecules and ions. cies with strongly different structures and block copolymer sequences, it may One problem, however, is whether the electronics providing the ion current be possible to circumvent the problems associated with low monomeric transduction can support the speed at which the translocation events occur. contrast as well as fast and poorly controlled threading that impose the highly Traditionally, electronics for nanopore sensors focused on the lowest noise complex molecular setting required for DNA-sequencing. Here, we show nano- possible. Only recently, high-bandwidth electronics have been developed which pore detection of relatively short model poly(phosphodiester)s (56 and 104 r.u.) reveal translocation events at time scales that were previously inaccessible. This synthetized by automated iterative phosphoramidite chemistry.1 Our data show study presents an amplifier which allows an approximately ten times wider band- evidence for voltage-dependent interaction and threading and underline the in- width than previous designs, enabling the study of translocation events faster fluence of the molecular structure and orientation of the precision polymers on than one microsecond. The amplifier features frequency modulation of the input the observed residual current signal as well as on the translocation dynamics. In signal, coupled with synchronous demodulation to reject out-of-band noise con- particular, they suggest a strong entropic contribution due to the high flexibility tributions. The integrated oscillator enables the use of the amplifier in a lock-in of the phosphodiester chain. (1) Ouahabi, A. A. et al. ACS Macro Lett. 2015, configuration with a modulated stimulus signal. The modular design of the front- 1077-1080. end allows studying the effect of the particular design on the speed, noise and stability of the amplifier. Comparison will be drawn between the classical ampli- 917-Pos Board B687 fier and the wide-bandwidth design. This study will give an insight on the optimal Analyte Properties Determining the Position and Spacing of Maxima in electronic equipment for nanopore sensing in terms of balancing lowest possible Residual Current Spectra Obtained by Single Molecule Nanopore Analysis noise and largest possible bandwidth. of Polymer Samples Monasadat Talarimoghari1, Gerhard Baaken2, Jan C. Behrends1. 920-Pos Board B690 1Physiology, University of Freiburg, Freiburg, Germany, 2Ionera Membrane Arrays for Single-Channel Recordings Technologies GmbH, Freiburg, Germany. Ekaterina Zaitseva1,So¨nke Petersen1, Juan Del Rio Martinez2, Polymer interaction with nanopores results in transient blocks of ionic current Ibrahim Halimeh2, Jan C. Behrends2, Gerhard Baaken1. called resistive pulses. For aqueous solutions of polydisperse poly(ethylenegly- 1Ionera Technologies GmbH, Freiburg, Germany, 2Laboratory for Membrane col) (PEG) of degree of polymerization (d.p.) in the range 20-50 (Mn=1000-2000 Physiology & Technology, University of Freiburg, Freiburg, Germany. g/mol), it has been shown that the depth of resistive pulses is related to the d.p. of Single-channel recording using artificial lipid bilayers is a very powerful tech- individual molecules, yielding histograms of residual current as a fraction of nique not only for physiological and pharmacological studies of ion channels, open pore current (I/Io) which can be compared to mass spectra obtained by but for analytical applications with biological nanopores. However for efficient MALDI-TOF and show single monomer resolution. However, despite a decade single-channel analysis an automated chip-based parallel measurement platform of research, it is still unclear which physical dimension related to d.p. is reflected is required. Here we describe formation and characterization of versatile mem- in the degree of ionic current block. We here report on experiments with poly brane arrays suitable for single-channel studies on the surface of different micro (dimethylacrylamide) (PDMAA), a neutral polymer which, like PEG, generates electrode cavity array (MECA) chips. The new chip design allows simultaneous resistive pulses when interacting with the alpha-hemolysin pore. We report optical monitoring and electrical recordings using MECA-4-Opto technology. detection of single oligomers in aqueous solutions of polydisperse PDMAA of Developed techniques allow for rapid formation of reproducible membrane ar- Mn=1000-2000. In histograms of I/Io, maxima are observed at much higher rays from different substances including natural or synthetic lipids and their values (0.4-0.9) than for PEGs of comparable mass (<0.4). In addition, spacing mixtures, artificial diaminophospholipids and polyoxazoline based triblock co- between maxima was found to be in the range of 0.1 I/Io as opposed to 0.01. This polymers both manually and in an automated fashion. indicates that one monomer of PDMAA (MW=99 g/mol) adds ten times more Formation, electrical properties and stability of membranes made from resistance than one monomer of PEG (MW=44 g/mol), while simultaneously different solutions on the cavities of different sizes were extensively character- a given PDMAA molecule blocks the pore 50% less than a PEG of similar mo- ized. Diverse protein nanopores and ion channels were tested for insertion ef- lecular mass. We conclude that neither the depth of block nor the spacing be- ficiency and performance. tween maxima in the I/Io-histogram is related to molecular mass. Instead, Limitations such as appearance of stable pores in membranes containing other physical parameters like monomer bulkiness, hydrophobicity and polymer oxidized lipids as well as lipid mixtures close to the phase transition tempera- coil density must be invoked to explain these findings. ture of one of the components are shown and discussed. 918-Pos Board B688 921-Pos Board B691 Direct Measurements of the Size and Correlations between Single Ions Ionic Transport through 1.5 NM Diameter Carbon Nanotube Porins Impelled through a Sub-Nanometer-Diameter Pore Yun-Chiao Yao1,2, Robert Henley3, Ramya Tunuguntla2, Meni Wanunu3, Gregory Timp. Aleksandr Noy1,2. Biolgical Science and Electrical Engineering, University of Notre Dame, 1UC Merced, Merced, CA, USA, 2Lawrence Livermore National Lab, Notre Dame, IN, USA. Livermore, CA, USA, 3Northeastern University, Boston, MA, USA. The size and charge of an ion affects everything from the structure of water Artificial membrane channels can have similar functions to channel proteins: they to life itself. To gauge their size, metal alkali and alkali earth ions, and pro- can span cell membranes, create pathways for passive diffusion, and transfer

BPJ 8586_8589 Sunday, February 18, 2018 183a water, ions, and a variety of chemicals across the lipid barrier. Comparing to pro- biophysics experts and casual programmers, who all created a variety of exper- tein channels, synthetic channels have longer lifetime and can survive harsh or iments. Hence this toolkit can be used to quickly automate biophysics experi- even extreme conditions. Our group has developed a new type of synthetic mem- ments with Euglena, thereby lowering access barriers for interdisciplinary brane channel: carbon nanotube porins (CNTPs), which are ultrashort single- education, i.e. students studying biology, physics, and computer science in walled carbon nanotubes (ca. 10 nm in length) and have similar properties to bio- tandem. logical porins such as comparable pore diameter and the ability to self-insert into the lipid bilayer. In this study, we used a modified planar lipid bilayer platform to 925-Pos Board B695 quantify ion conductance and selectivity of a larger 1.5 nm diameter CNTPs. We ‘‘Eating DNA’’ a Way to Motivate Girls to Get into Biophysics also report the effect of divalent cations on ionic transport through these pores. Yuly E. Sa´nchez1, Maria Helena Ramirez2, Elsa J. Gomez3. These findings can expand our understanding of transport in nanotube pores. 1Physics, National University of Colombia, Bogota, Colombia, 2Biology, National University of Colombia, Bogota, Colombia, 3National University of 922-Pos Board B692 Colombia, Bogota, Colombia. Carbon Nanotube Porins: A Versatile Synthetic Biomimetic Membrane Participation of woman in science and engineering is growing even if recent data Channel Platform suggest that is some STEAM fields more gender balanced than others. Just fewer Aleksandr Noy. women than men pursue majors in science. Among first-year college students, Biology and Biotechnology Division, Lawrence Livermore National women are much less likely than men to say that they intend to major in science, Laboratory, Livermore, CA, USA. technology, engineering, or math(STEM). By graduation, men outnumber women Living systems control transport of ions or small molecules across biological in nearly every science and engineering field, and in some, such as physics, engi- membranes using protein channels that form exquisitely-defined pores in lipid neering, and computer science, the difference is dramatic, with women earning bilayers. Synthetic channels that replicate this functionality have the potential only 20 percent of bachelor’s degrees. Women’s representation in science and en- of delivering comparable performance in a robust, tailorable, and scalable gineering declines further at the graduate level and yet again in the transition to the platform. We have recently developed a new type of synthetic membrane chan- workplace. Additionally, women in Colombia have been victims of both armed nel - carbon nanotube porins (CNTPs) - which consist of ultrashort single-walled conflict and a society run by men and her role in scientific areas is not increasing. carbon nanotubes (ca. 10 nm in length) the replicate the main geometry and func- A way to reduce this gap we develop an outreach project under the name ‘‘Scien- tionality of a biological porin. Moreover, membrane pores formed these ultra- tific Women and Girls building peace ’’ (Spanish: ‘‘Mujeres y Nin˜as Cientı´ficas short nanotubes have transport properties that come remarkably close and often Construyendo Paz’’), aimed at recruiting high school girls from armed conflict exceed those of their biological analogs. I will discuss the performance of several areas in Colombia get them into Biophysics and STEAM areas in general. One types of CNTPs in water, ion, and proton transport experiments. I will specif- of the activities was to build a DNA model with candies, these activities were per- ically focus on the role of molecular-scale confinement and solvation interac- formed with 4 different groups of 25-30 girls each and as a complement they ex- tions in enabling fast and selective transport through these pores, and different tracted their own DNA with low cost materials. We found that after finish these physical mechanisms of ion selectivity. Overall, CNTPs represent a simplified activities; they were able to conclude that was much more than a science experi- nanofluidic platform that is ideal for studying fundamental biophysics of trans- ment, and they become more interested to learn more about this topic. This pro- port in biological channels, and for engineering versatile bionanostructures. posal incorporating ‘‘Eatable DNA’’ as educational choice into today’s school 923-Pos Board B693 curriculum is a great way to encourage girl’s participation in STEAM areas. Enhanced Fluidic Transport through CNT Membrane Based Platforms Steven F. Buchsbaum, Eric Meshot, Owen Chiatai Chen, Anh Pham, 926-Pos Board B696 Shirui Guo, Ngoc Bui, Viktor Rozsa, Francesco Fornasiero. Excitable Membrane, Ion Channel, Electrostatics: A Historical Sketch Physical Life Sciences, Lawrence Livermore National Laboratory, H. Richard Leuchtag. Livermore, CA, USA. Retired, Kerrville, TX, USA. Carbon nanotubes (CNTs) exhibit truly nm-scale size, easily functionalized open- The roughly one-tenth of a volt across an excitable membrane may not seem ings, uniquely smooth walls, and a well-defined structure which sets them apart large, but across a membrane two lipid molecules thick it yields an electric field from other types of synthetic nanopores in the literature. As a result, they show comparable to one causing atmospheric lightning. Early attempts to explain the remarkable fluidic properties which make them very attractive building blocks physical basis of the nerve impulse modeled its ion currents as stemming from for next-generation biomimetic materials. Towards this end, we have developed two processes: the movement of ionic charges by the electric field and the ion a fabrication process to generate free-standing and flexible single-walled CNT diffusion due to ion-concentration differences across the membrane. Hampered based membranes with well-aligned, high-density tubes. Through the use of these by its assumptions of constant properties, this classical electrodiffusion model membranes we have studied the unique fluidic transport behavior seen in CNTs couldn’t explain axonal membrane data. An eclectic approach combining an under multiple driving forces, including pressure, concentration, and voltage gra- equation from electrodiffusion theory with elements of cable theory, wave the- dients. In some cases, remarkable transport rates were seen that are several orders ory and circuit theory—capacitor, batteries, variable conductors—transformed of magnitude above typical continuum models. In addition, we have combined our biophysical science with its successful fitting of the action potential’s time CNT membrane fabrication with focused ion beam (FIB) nanomachining to create course. The development of glass pipette electrodes that restrict ion flow to a platform that allows for activation of individual (or a few) tubes. By employing microscopic membrane patches led to the isolation of ion channels, glycopro- this platform, we analyzed voltage-driven ionic transport through CNTs and show tein macromolecules that selectively conduct ion currents. Because it’s easy to giant ionic currents and a power-law increase in conductance with KCl concentra- visualize water-filled pores across these macromolecules, fitted with gates that tion, a behavior that seems unique to CNTs. Moreover, we demonstrated that this block or allow ion currents across the membrane, they were named voltage- power-lawrelationshipcan be pHdependent, whichhelps explain a possible origin gated ion channels. This approach is challenged because of its: simplistic of the large electroosmotic flow thought to be present in the tubes. models of everyday devices, including screws and paddles; assumption of con- stant channel capacitance; and disregard of electrostatic repulsions between Posters: Biophysics Education same-charge residues. Genetic engineering techniques brought the discovery of structural features of many such ion channels. One universal feature is a 924-Pos Board B694 membrane-spanning segment with a regular array of positively charged A Programming Toolkit for Automating Biophysics Experiments with amino-acid residues. All also contain multiple amino acids whose sidechains Microorganism Swarms fork into two branches. An electrostatic model based on the ion channel’s Peter Washington, Karina Samuel-Gama, Ingmar Riedel-Kruse. strong similarities to ferroelectric liquid crystals containing branched chains Bioengineering, Stanford University, Palo Alto, CA, USA. helps explain the depolarization-driven activation of voltage-sensitive ion Collecting data for a biophysics experiment is currently challenging and time channels. [See Leuchtag H R (2008) Voltage-Sensitive Ion Channels, Springer; consuming even for experts in the field, as setting up a robust experiment (2017) Biophys. J. 112(3):544a; 112(3):543a-544a; 112(3):464a.] can require knowledge of biotechnology, hardware design, and software devel- opment in addition to continuous access to reliable and responsive biological 927-Pos Board B697 materials. We have therefore developed a JavaScript-based web toolkit Use of Interactive Graphical Tools to Demonstrate Changes in Time- enabling rapid programming of biophysical experiments with microorganism Resolved Fluorescence Intensity Decays swarms. The toolkit allows users to script experiments with swarms on Euglena Sarthak Arora1, Rajaram Swaminathan2. gracilis housed in a remote wet lab. Users can program event-based manipula- 1Physics, Indian Institute of Technology Guwahati, Guwahati, India, tions of organism stimuli and track position, velocity, acceleration, and rotation 2Biosciences and Bioengineering, Indian Institute of Technology Guwahati, for all Euglena in the microfluidic chamber. We evaluated the tool with both Guwahati, India.

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Time-resolved fluorescence intensity decays measured today, comprise of several molecular modeling and simulation to high school and undergraduate college stu- thousands of data points, often plotted on a logarithmic intensity scale with a linear dents. Furthermore, to aid in introducing students to UNIX, we have included a time axis. Demonstrating changes in the intensity decay curves arising from varied web interface to a virtual machine running the Ubuntu operating system. Topics causes like convolution with instrument response function, quenching or altered include: the UNIX operating system, text editors, scientific programming using rotational motion are not easily accomplished using static teaching tools like the Python programming language, PDB basics, molecule drawing, all-atom en- blackboards/printed handouts/slides. A dynamic and interactive graphical inter- ergy function & force fields, coarse-grained force fields, boundary conditions, en- face, where consequences of altering any single interaction parameter is instantly sembles, particle mesh Ewald methods, minimization, normal mode analysis, reflected on the plotted intensity decay is needed.Here in this work we employ monte Carlo, dynamics (molecular dynamics, Langevin dynamics, and Brownian graphical interactive manipulation tools available in Mathematica (version 11.0) dynamics), introduction to complex systems (biological and other complex fluids), to illustrate the following concepts in time-resolved fluorescence: A. Effect of implicit solvents, replica exchange methods, alchemical free energy calculations, mono-exponential, multi-exponential and non-exponential decay kinetics on the umbrella sampling free energy calculations, pathsampling, QM/MM, introduction fluorescence intensity decay curve; B. Effect of using incorrect geometry factor to protein structure prediction (homology modeling, sequence alignment, ab initio (G-factor) on the time-resolved fluorescence anisotropy decay; C. Effect of the modeling), and introduction to protein-ligand docking (scoring function, sampling magnitudes of individual rotational correlation times and fluorescence lifetimes method, virtual screening). Many of these modules are under development, but in a mixture of fluorophores displaying associated anisotropy decay; D. Effect most of the basic modules are already available. of changing Forster resonance energy transfer efficiency or orientation factor (kappa-squared) on the donor fluorescence intensity decay. We hope that the 930-Pos Board B700 graphical tools will make it easier for biology students to understand and appre- In Silico Structural Studies of a Tripeptide Building Block ciate the concepts related to time-resolved fluorescence measurements. Subhasish Chatterjee, Katherine Liu. Chemistry, Reed College, Portland, OR, USA. 928-Pos Board B698 Using Computational and Wet-Lab Methods to Determine Enzyme Func- Molecular self-assembly of peptide building blocks into nanostructures provides tion in an Undergraduate Biochemistry Lab Course minimalistic avenues for developing biologically inspired, ‘‘smart’’ materials with numerous applications in biomedical sciences, photonics, and nanotech- Julia R. Koeppe1, Webe C. Kadima1, Rebecca Roberts2, Paul A. Craig3. 1Chemistry, State University of New York at Oswego, Oswego, NY, USA, nology. Structural investigations of peptide building blocks with a wide range 2Biology, Ursinus College, Collegeville, PA, USA, 3Chemistry, Rochester of chemical functionality also hold much promise to decipher their molecular evo- lution toward the chemical origin of living systems. Our work delves into the Institute of Technology, Rochester, NY, USA. We are developing an undergraduate biochemistry lab curriculum based on structure design and dynamics of a unique tri-peptide unit, which contains three authentic inquiry. Over 3800 structures in the Protein Data Bank have unknown amino acids such as phenylalanine, tyrosine, lysine, and promotes the formation function. Students use structural bioinformatics tools to narrow the list of of nanostructures via offsetting the effects of its hydrophobic and hydrophilic possible functions for a given structure. Students then assess these functions in components. The results of our computer simulation studies reveal structural the wet lab. The in silico modules include protein visualization with PyMOL, cues, demonstrating preferential intra-residue interactions for the favored confor- mations that could facilitate the self-assembling process. In addition, open-source structural alignment using Dali and ProMOL, sequence exploration with BLAST software and computer simulation methodologies offer attractive opportunities to and Pfam, and ligand docking with PyRX and Autodock Vina. The goal is to pre- dict the function of the enzymes and to identify promising substrates for the foster the connection between biophysics and nanotechnology applications and to active sites. In the wet lab, students express and purify the target enzymes and pursue this interdisciplinary research theme in the liberal arts college setting. perform kinetic assays with substrates selected from their docking studies. We are assessing their learning as students and their growth as scientists in terms 931-Pos Board B701 of research methods, visualization, biological context, and mechanism of protein Virtual Reality Environment to Visualize and Manipulate Molecular function. The lab course is an extension of successful undergraduate research ef- Structures 1 2 3 3 forts at RIT and Dowling College. We have disseminated our instructional mod- Jordan McGraw , Wei Zhang , Amanda D. Luginbuhl , George Takahashi , 2 2 ules to the scientific community via a web site (promol.org), including both Roy F. Tasker , Gaurav Chopra . 1Computer Graphics Technology, Purdue University, West Lafayette, IN, protocols and captioned video instructions for the techniques involved. Over 2 3 the course of the project, we have followed changes in faculty and teaching as- USA, Chemistry, Purdue University, West Lafayette, IN, USA, Envision sistant competence, in addition to measuring student outcomes. We are also Center, Purdue University, West Lafayette, IN, USA. analyzing the flexibility of the curriculum for adoption as a whole in a single Molecular structure and interactions that determine the nature of biomolecules course or in parts across multiple courses. We are optimizing the curriculum are difficult to imagine, even with the aid of graphics in textbooks. Several for wider dissemination, and we seek input from additional potential adopters computational tools have been developed to visualize molecular structures, about their level of interest and the barriers that they anticipate on their cam- such as, PyMol, Chimera, JMol, Odyssey, etc. These are typically command puses. This project is supported in part by NSF IUSE 1709355. line or standard GUI based applications that require moderate to advanced knowledge to be used effectively. To understand biomolecular structure and in- 929-Pos Board B699 teractions in detail requires a 3D, dynamic perspective, and educational research CHARMM-GUI Lecture Series on Molecular Modeling and Simulation indicates an inquiry-led approach is more effective than a didactic approach. Nathan R. Kern1, Jumin Lee2, Morgan Fine-Morris1, Jeffery B. Klauda3, Motivated by the need to enhance education and visual learning experience of Krzysztof Kuczera4, Patrick Fleming5, Wonpil Im2. students, we have developed a virtual reality (VR) educational ‘‘game’’ for visu- 1Computer Science and Engineering, Lehigh University, Bethlehem, PA, alization and manipulation of biomolecules based on concepts of molecular in- USA, 2Biological Sciences, Lehigh University, Bethlehem, PA, USA, teractions that are taught in a dynamic immersive environment. Our VR platform 3Chemical and Biomolecular Engineering, University of Maryland, College educates students about the principles of intermolecular forces and structural Park, MD, USA, 4Chemistry, University of Kansas, Lawrence, KS, USA, manipulation. A series of video tutorials have been developed to teach bond 5Biophysics, Johns Hopkins University, Baltimore, MD, USA. strength, angle, torsion, inter- and intra- molecular hydrogen bonding in a class- Over the past 10 years, CHARMM-GUI (http://www.charmm-gui.org) has been room setting at low cost to meet key learning objectives. Our VR platform can developed to provide a user-friendly interface with various modules to read and load any Protein Data Bank (PDB) structure, visualize it with different structural manipulate PDB structure files (both in PDB and PDBx/mmCIF formats) and to representations, select atoms and bonds for manipulation by mutating amino build complex biomolecular systems reliably. It also provides various force field acids, breaking bonds, rotations and torsional movements to discover important topology and parameter information for most commonly used molecules, so that biomolecular interactions in a complex environment (e.g. protein binding site). users can almost always find what they need for their biomolecular systems. To Using our immersive VR game, the students are able to manipulate a molecule in support broader simulation communities, CHARMM-GUI now provides 3D to optimize its interactions to discover the factors responsible for optimum CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, and binding, guided by a live score based on our CANDOCK software indicating OpenMM inputs with well-validated, optimized protocols. To supplement the tools the strength of interaction in real-time. We believe this platform will enhance on CHARMM-GUI, we have designed a series of online tutorials (http://www. student learning experience through exploration in an immersive environment charmm-gui.org/lecture). These tutorials introduce useful tools and concepts in by repurposing a well-adopted gaming technology for teaching.

BPJ 8586_8589